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Sample records for gnnqqny heptapeptide decamer

  1. The DECam Legacy Survey

    NASA Astrophysics Data System (ADS)

    Blum, Robert D.; Burleigh, Kaylan; Dey, Arjun; Schlegel, David J.; Meisner, Aaron M.; Levi, Michael; Myers, Adam D.; Lang, Dustin; Moustakas, John; Patej, Anna; Valdes, Francisco; Kneib, Jean-Paul; Huanyuan, Shan; Nord, Brian; Olsen, Knut A.; Delubac, Timothée; Saha, Abi; James, David; Walker, Alistair R.; DECaLS Team

    2016-06-01

    The DECam Legacy Survey (DECaLS) is conducting a 3-band imaging survey of the Sloan Digital Sky Survey (SDSS) extragalactic footprint as part of the Legacy Survey, which is associated with the Dark Energy Spectroscopic Instrument (DESI) redshift survey. The Legacy Survey covers 14000 square degrees in the g, r, and z bands and is being executed on the Blanco 4-m, Mayall 4-m, and Bok 2.3-m telescopes. For DECaLS, the Dark Energy Camera (DECam) will image the footprint overlapping SDSS in the region -20 < Dec < +30 deg, to depths of g=24.7, r=23.9, z=23.0 and will eventually cover a total of 7500 square degrees. The survey began in 2014 and will run through Spring 2017. Images and catalogs were introduced in Public Data Release 2 (DR2), which occurred in January 2016. The algorithm "Tractor" applies multi-wavelength forced photometry to DECam and WISE data to produce galaxy (and star) magnitudes (as well as shape and other information) for the catalogs. In total, the optical data in DR2 cover a disjoint footprint in 2078, 2141 and 5322 square degrees in g, r, and z bands, respectively; 1807 square degrees has been observed in all three optical filters. There are approximately 260 million unique sources in DR2 spread over 97,554 0.25 x 0.25 square degree bricks.The Dark Energy Spectroscopic Instrument (DESI) will observe 30+ million galaxies and quasars in a 14,000 square degree extragalactic footprint. The targeting in that footprint will be provided by a combination of these DECam data, the MOSAIC camera on the Mayall 4-meter, and the 90Prime camera on the Bok telescope.

  2. On cooperative effects and aggregation of GNNQQNY and NNQQNY peptides

    NASA Astrophysics Data System (ADS)

    Nochebuena, Jorge; Ireta, Joel

    2015-10-01

    Some health disturbances like neurodegenerative diseases are associated to the presence of amyloids. GNNQQNY and NNQQNY peptides are considered as prototypical examples for studying the formation of amyloids. These exhibit quite different aggregation behaviors despite they solely differ in size by one residue. To get insight into the reasons for such difference, we have examined association energies of aggregates (parallel β-sheets, fibril-spines, and crystal structures) from GNNQQNY and NNQQY using density functional theory. As we found that GNNQQNY tends to form a zwitterion in the crystal structure, we have investigated the energetics of parallel β-sheets and fibril-spines in the canonical and zwitterionic states. We found that the formation of GNNQQNY aggregates is energetically more favored than the formation of the NNQQNY ones. We show that the latter is connected to the network of hydrogen bonds formed by each aggregate. Moreover, we found that the formation of some NNQQNY aggregates is anticooperative, whereas cooperative with GNNQQNY. These results have interesting implications for deciphering the factors determining peptide aggregation propensities.

  3. Characterization of DECam focal plane detectors

    SciTech Connect

    Diehl, H.Thomas; Angstadt, Robert; Campa, Julia; Cease, Herman; Derylo, Greg; Emes, John H.; Estrada, Juan; Kibik, Donna; Flaugher, Brenna L.; Holland, Steve E.; Jonas, Michelle; /Fermilab /Madrid, CIEMAT /LBL, Berkeley /Argonne /Pennsylvania U.

    2008-06-01

    DECam is a 520 Mpix, 3 square-deg FOV imager being built for the Blanco 4m Telescope at CTIO. This facility instrument will be used for the 'Dark Energy Survey' of the southern galactic cap. DECam has chosen 250 ?m thick CCDs, developed at LBNL, with good QE in the near IR for the focal plane. In this work we present the characterization of these detectors done by the DES team, and compare it to the DECam technical requirements. The results demonstrate that the detectors satisfy the needs for instrument.

  4. Molecular Dynamics Simulations on the Oligomer-Formation Process of the GNNQQNY Peptide from Yeast Prion Protein Sup35

    PubMed Central

    Zhang, Zhuqing; Chen, Hao; Bai, Hongjun; Lai, Luhua

    2007-01-01

    Oligomeric intermediates are possible cytotoxic species in diseases associated with amyloid deposits. Understanding the early steps of fibril formation at atomic details may provide useful information for the rational therapeutic design. In this study, using the heptapeptide GNNQQNY from the yeast prion-like protein Sup35 as a model system, for which a detailed atomic structure of the fibril formed has been determined by x-ray microcrystallography, we investigated its oligomer-formation process from monomer to tetramer at the atomistic level by means of a molecular dynamics simulation with explicit water. Although the number of simulations was limited, the qualitative statistical data gave some interesting results, which indicated that the oligomer formation might start from antiparallel β-sheet-like dimers. When a new single peptide strand was added to the preformed dimers to form trimers and then tetramers, the transition time from disorder aggregates to regular ones for the parallel alignment was found to be obviously much less than for the antiparallel one. Moreover, the parallel pattern also statistically stayed longer, providing more chances for oligomer extending, although the number of parallel stack events was almost equal to antiparallel ones. Therefore, our simulations showed that new strands might prefer to extend in a parallel arrangement to form oligomers, which agrees with the microcrystal structure of the amyloid fibril formed by this peptide. In addition, analysis of the π-π stacking of aromatic residues showed that this type of interaction did not play an important role in giving directionality for β-strand alignment but played a great influence on stabilizing the structures formed in the oligomer-formation process. PMID:17483185

  5. Stability of single sheet GNNQQNY aggregates analyzed by replica exchange molecular dynamics: Antiparallel versus parallel association

    SciTech Connect

    Vitagliano, Luigi; Esposito, Luciana; Pedone, Carlo; De Simone, Alfonso

    2008-12-26

    Protein and peptide aggregation into amyloid plaques is associated with a large variety of neurodegenerative diseases. The definition of the molecular bases of these pathologies is hampered by the transient nature of pre-fibrillar small-oligomers that are considered the toxic species. The ability of the peptide GNNQQNY to form amyloid-like structures makes it a good model to investigate the complex processes involved into amyloid fiber formation. By employing full atomistic replica exchange molecular dynamics simulations, we constructed the free energy surface of small assemblies of GNNQQNY to gain novel insights into the fiber formation process. The calculations suggest that the peptide exhibits a remarkable tendency to form both parallel and antiparallel {beta}-sheets. The data show that GNNQQNY preference for parallel or antiparallel {beta}-sheets is governed by a subtle balance of factors including assemblies' size, sidechain-sidechain interactions and pH. The samplings analysis provides a rationale to the observed trends.

  6. The Dark Energy Survey Camera (DECam)

    SciTech Connect

    Diehl, H.Thomas; /Fermilab

    2011-09-09

    The Dark Energy Survey (DES) is a next generation optical survey aimed at understanding the expansion rate of the Universe using four complementary methods: weak gravitational lensing, galaxy cluster counts, baryon acoustic oscillations, and Type Ia supernovae. To perform the survey, the DES Collaboration is building the Dark Energy Camera (DECam), a 3 square degree, 570 Megapixel CCD camera that will be mounted at the prime focus of the Blanco 4-meter telescope at the Cerro Tololo Inter-American Observatory. CCD production has finished, yielding roughly twice the required 62 2k x 4k detectors. The construction of DECam is nearly finished. Integration and commissioning on a 'telescope simulator' of the major hardware and software components, except for the optics, recently concluded at Fermilab. Final assembly of the optical corrector has started at University College, London. Some components have already been received at CTIO. 'First-light' will be sometime in 2012. This oral presentation concentrates on the technical challenges involved in building DECam (and how we overcame them), and the present status of the instrument.

  7. DECam Magellanic Clouds Survey Pilot Program

    NASA Astrophysics Data System (ADS)

    Nidever, David; Olsen, Knut; Saha, Abhijit; Olszewski, Ed; Gallart, Carme; Monelli, Matteo; Kunder, Andrea; Gruendl, Robert; Chu, You-Hua; Walker, Alistair; Zaritsky, Dennis; Martin, Nicolas; Stringfellow, Guy; Munoz, Ricardo; Conn, Blair; Noel, Noelia; Blum, Robert; Majewski, Steven; van der Marel, Roeland

    2012-08-01

    As two of our closest galactic neighbors, the Magellanic Clouds (MCs) have long been enticing targets for investigating galaxy formation and evolution, especially of interacting systems. The commissioning of the wide-field Dark Energy Camera (DECam) on the CTIO-4m will enable the next generation of large-scale and deep photometric surveys of the Magellanic Clouds. We aim to propose for a community DECam survey of the Clouds covering hundreds of deg^2 to ~25th mag in multiple filters that will complement the Dark Energy Survey's partial coverage of the Magellanic periphery. Such a survey would make possible a number of important scientific inquiries: (1) Mapping the stellar periphery of the MCs with old main-sequence turnoff stars that should reveal relics of their formation and past interactions. (2) Spatially-resolved star formation histories covering all ages of the MCs that will further complement our understanding of their past evolution. (3) 3D mapping of the complex geometries of the MCs with RR Lyrae variable stars. And, finally, (4) use the large-scale stellar and HI distributions to probe the nature of the hot gaseous Galactic halo. We propose here a pilot program using shared risk time to study and evaluate the best observing and data strategy for a future DECam Magellanic Clouds survey.

  8. Status of the Dark Energy Survey Camera (DECam) Project

    SciTech Connect

    Flaugher, Brenna L.; Abbott, Timothy M.C.; Angstadt, Robert; Annis, Jim; Antonik, Michelle, L.; Bailey, Jim; Ballester, Otger.; Bernstein, Joseph P.; Bernstein, Rebbeca; Bonati, Marco; Bremer, Gale; /Fermilab /Cerro-Tololo InterAmerican Obs. /ANL /Texas A-M /Michigan U. /Illinois U., Urbana /Ohio State U. /University Coll. London /LBNL /SLAC /IFAE

    2012-06-29

    The Dark Energy Survey Collaboration has completed construction of the Dark Energy Camera (DECam), a 3 square degree, 570 Megapixel CCD camera which will be mounted on the Blanco 4-meter telescope at CTIO. DECam will be used to perform the 5000 sq. deg. Dark Energy Survey with 30% of the telescope time over a 5 year period. During the remainder of the time, and after the survey, DECam will be available as a community instrument. All components of DECam have been shipped to Chile and post-shipping checkout finished in Jan. 2012. Installation is in progress. A summary of lessons learned and an update of the performance of DECam and the status of the DECam installation and commissioning will be presented.

  9. Serinocyclins A and B, Cyclic Heptapeptides from Metarhizium anisopliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two new cyclic heptapeptides, serinocyclins A (1) and B (2), were isolated from conidia of the entomopathogenic fungus Metarhizium anisopliae. Structures were elucidated by a combination of mass spectrometric, NMR, and X-ray diffraction techniques. Serinocyclin A (1) contains three serine units, a...

  10. Structural Characterization of GNNQQNY Amyloid Fibrils by Magic Angle Spinning NMR. †

    PubMed Central

    van der Wel, Patrick C.A.; Lewandowski, Józef R.; Griffin, Robert G.

    2010-01-01

    Several human diseases are associated with the formation of amyloid aggregates, but experimental characterization of these amyloid fibrils and their oligomeric precursors has remained challenging. Experimental and computational analysis of simpler model systems has therefore been necessary, for instance on the peptide fragment GNNQQNY7-13 of yeast prion protein Sup35p. Expanding on a previous publication, we report here a detailed structural characterization of GNNQQNY fibrils using magic angle spinning (MAS) NMR. Based on additional chemical shift assignments we confirm the coexistence of three distinct peptide conformations within the fibrillar samples, as reflected in substantial chemical shift differences. Backbone torsion angle measurements indicate that the basic structure of these co-existing conformers is an extended β-sheet. We structurally characterize a previously identified localized distortion of the β-strand backbone specific to one of the conformers. Intermolecular contacts are consistent with each of the conformers being present in its own parallel and in-register sheet. Overall the MAS NMR data indicate a substantial difference between the structure of the fibrillar and crystalline forms of these peptides, with a clear increased complexity in the GNNQQNY fibril structure. These experimental data can provide guidance for future work, both experimental and theoretical, and provide insights into the distinction between fibril growth and crystal formation. PMID:20695483

  11. A Pilot DECam Time-Domain Survey

    NASA Astrophysics Data System (ADS)

    Bloom, Joshua; Matheson, Tom; Ridgway, Steve; Miller, Adam; Klein, Christopher; Walkowicz, Lucianne; Nugent, Peter; Shivvers, Isaac; Smith, Chris; Olsen, Knut; Becker, Andrew; Norman, Dara; Simcoe, Rob; Oluseyi, Hakeem; Ridgway, Susan; Saha, Abi; Richards, Joey; Cenko, S. Bradley; Lauer, Tod R.

    2013-02-01

    We propose a SSIZE sq. degree time-domain imaging survey, in z and Y-bands, focused on discovery and characterization of short timescale (< 2 day) Galactic variables. The systematic discovery and classification of sources across the variable star taxonomy at 3 different Galactic latitudes would be highly complementary to existing efforts (generally blue-focused, over longer timescales, and extragalactic oriented) and would directly impact the planning and scheduling of LSST. We estimate that there are 3times10^5 variables at >0.05 mag rms at the single-epoch limiting depth of the proposed survey. With an areal coverage more than SDSSREL times that of SDSS/Stripe 82 and a depth 10 times fainter per epoch, the proposed DECam survey should uncover more than 1000 RR Lyrae and, through their 3- d clustering, could reveal new galactic halo substructure in the Southern sky out to 100 kpc. The resultant variability and probabilistic (machine-learned) classification catalogs of sources found this semester will be made public; rarities and novel sources would be prime candidates for community follow-up in subsequent semesters. We see the proposed survey as establishing a new concept of ``variability fields'' (akin to extragalactic fields such as COSMOS) in the Southern hemisphere: these may be well-studied for years at a variety of timescales across the electromagnetic spectrum. it Though highly ranked for 2012B, the approved observations are unlikely to occur given the scheduling constraints during shared risk time of 2012B.

  12. The DECam minute cadence survey - I

    NASA Astrophysics Data System (ADS)

    Belardi, Claudia; Kilic, Mukremin; Munn, Jeffrey A.; Gianninas, A.; Barber, Sara D.; Dey, Arjun; Stetson, Peter B.

    2016-11-01

    We present the first results from a minute cadence survey of a 3 deg2 field obtained with the Dark Energy Camera. We imaged part of the Canada-France-Hawaii Telescope Legacy Survey area over eight half-nights. We use the stacked images to identify 111 high proper motion white dwarf candidates with g ≤ 24.5 mag and search for eclipse-like events and other sources of variability. We find a new g = 20.64 mag pulsating ZZ Ceti star with pulsation periods of 11-13 min. However, we do not find any transiting planetary companions in the habitable zone of our target white dwarfs. Given the probability of eclipses of 1 per cent and our observing window from the ground, the non-detection of such companions in this first field is not surprising. Minute cadence DECam observations of additional fields will provide stringent constraints on the frequency of planets in the white dwarf habitable zone.

  13. DECam Observations of the Tidal Shells Around NGC 3923

    NASA Astrophysics Data System (ADS)

    Miller, Bryan; Grooms, Connor; Puzia, Thomas H.; Matthew, Taylor; Graeme, Candlish; McGaugh, Stacy S.; Mihos, Chris; Smith, Rory; Schirmer, Mischa

    2016-01-01

    Stellar shells around elliptical galaxies are thought to be the results of near-radial mergers with low mass companions. Thus, the shell systems contain information about the merger history and gravitational potential of the elliptical galaxy. We present a preliminary census of the shell system of NGC 3923 from extremely deep g and i-band DECam imaging. NGC 3923 has the largest know shell system, with different studies finding between 27 and 42 shells. We present an overview of the DECam data reduction and an initial analysis of the shell system.

  14. Characterizing the assembly of the Sup35 yeast prion fragment, GNNQQNY: structural changes accompany a fiber-to-crystal switch.

    PubMed

    Marshall, Karen E; Hicks, Matthew R; Williams, Thomas L; Hoffmann, Søren Vrønning; Rodger, Alison; Dafforn, Timothy R; Serpell, Louise C

    2010-01-20

    Amyloid-like fibrils can be formed by many different proteins and peptides. The structural characteristics of these fibers are very similar to those of amyloid fibrils that are deposited in a number of protein misfolding diseases, including Alzheimer's disease and the transmissible spongiform encephalopathies. The elucidation of two crystal structures from an amyloid-like fibril-forming fragment of the yeast prion, Sup35, with sequence GNNQQNY, has contributed to knowledge regarding side-chain packing of amyloid-forming peptides. Both structures share a cross-beta steric zipper arrangement but vary in the packing of the peptide, particularly in terms of the tyrosine residue. We investigated the fibrillar and crystalline structure and assembly of the GNNQQNY peptide using x-ray fiber diffraction, electron microscopy, intrinsic and quenched tyrosine fluorescence, and linear dichroism. Electron micrographs reveal that at concentrations between 0.5 and 10 mg/mL, fibers form initially, followed by crystals. Fluorescence studies suggest that the environment of the tyrosine residue changes as crystals form. This is corroborated by linear dichroism experiments that indicate a change in the orientation of the tyrosine residue over time, which suggests that a structural rearrangement occurs as the crystals form. Experimental x-ray diffraction patterns from fibers and crystals also suggest that these species are structurally distinct. A comparison of experimental and calculated diffraction patterns contributes to an understanding of the different arrangements accessed by the peptide.

  15. Identifying Electromagnetic Counterparts to Gravitational Wave Triggers With DECam

    NASA Astrophysics Data System (ADS)

    Cowperthwaite, Philip

    2016-03-01

    Identifying the electromagnetic counterpart to a gravitational wave (GW) event is one of the great observational challenges in modern astronomy. We report on our work to overcome this challenge by investigating the theoretical and practical issues associated with optical follow-up of a GW event. This includes a systematic study of the potential contaminant population and their impact on counterpart detectability in simulated observations. Additionally, we utilize data taken with the Dark Energy Camera (DECam) on the Blanco 4-m telescope at CTIO. These data serve as a mock follow-up to a GW event and assist in the characterization of contamination not captured in simulations. P.S.C. is grateful for support provided by the NSF through the Graduate Research Fellowship Program, Grant DGE1144152.

  16. The Dark Energy Spectroscopic Instrument (DESI): The NOAO DECam Legacy Imaging Survey and DESI Target Selection

    NASA Astrophysics Data System (ADS)

    Schlegel, David J.; Blum, Robert D.; Castander, Francisco Javier; Dey, Arjun; Finkbeiner, Douglas P.; Foucaud, Sebastien; Honscheid, Klaus; James, David; Lang, Dustin; Levi, Michael; Moustakas, John; Myers, Adam D.; Newman, Jeffrey; Nord, Brian; Nugent, Peter E.; Patej, Anna; Reil, Kevin; Rudnick, Gregory; Rykoff, Eli S.; Ford Schlafly, Eddie; Stark, Casey; Valdes, Francisco; Walker, Alistair R.; Weaver, Benjamin; DECam Legacy Survey Collaboration

    2015-01-01

    The DECam Legacy Survey will conduct a 3-band imaging survey of the Sloan Digital Sky Survey (SDSS) extragalactic footprint. The Dark Energy Camera (DECam) will be used to image the 6700 square degree footprint overlapping SDSS in the region -20 < Dec < +30 deg, to depths of g=24.7, r=23.9, z=23.0. The survey will be conducted from Fall 2014 through Spring 2017, with periodic data releases beginning in March 2015. These releases will include catalogs constructed with the Tractor-based multi-wavelength forced photometry applied to the DECam and WISE satellite data.The Dark Energy Spectroscopic Instrument (DESI) will observe 24 million galaxies and quasars in a 14,000 square degree extragalactic footprint. The targeting in that footprint will be provided by a combination of these DECam data, the MOSAIC camera on the Mayall 4-meter, and the 90Prime camera on the Bok Telescope.

  17. A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad of biochemical changes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The active site motif of proteins belonging to ‘Histidine Acid Phosphatase’ (HAP) contains a hepta-peptide region, RHGXRXP. A close comparison among fungal and yeast HAPs has revealed the fourth residue of the hepta-peptide to be E instead of A, which is the case with A. niger phyA phytase. However,...

  18. Stabilized Heptapeptide A7R for Enhanced Multifunctional Liposome-Based Tumor-Targeted Drug Delivery.

    PubMed

    Ying, Man; Shen, Qing; Liu, Yu; Yan, Zhiqiang; Wei, Xiaoli; Zhan, Changyou; Gao, Jie; Xie, Cao; Yao, Bingxin; Lu, Weiyue

    2016-06-01

    (L)A7R (ATWLPPR) is a heptapeptide with high binding affinity in vitro to vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) overexpressed on glioma, glioma vasculogenic mimicry and neovasculature. However, its tumor targeting efficacy is significantly reduced in vivo due to proteolysis in blood circulation. To improve the in vivo stability and targeting efficacy, the retro inverso isomer of (L)A7R ((D)A7R) was developed for glioma-targeted drug delivery. (D)A7R was expected to have a similar binding affinity to its receptors in vitro (VEGFR2 and NRP-1), which was experimentally confirmed. In vivo, (D)A7R-modified liposomes achieved improved glioma-targeted efficiency than did (L)A7R-modified liposomes. After loading a chemotherapeutic agent (doxorubicin), (D)A7R-modified liposomes significantly inhibited subcutaneous model tumor in comparison to free doxorubicin, plain liposomes and (L)A7R-modified liposomes. In summary, the present study presented the potential of a proteolytically stable d-peptide ligand for in vivo tumor-targeted drug delivery. PMID:27195531

  19. Front-End Electronics for the Dark Energy Survey Camera (DECam)

    NASA Astrophysics Data System (ADS)

    Shaw, Theresa M.; Huffman, D.; Kozlovsky, M.; Olsen, J.; Stuermer, W.; Barcelo, M.; Cardiel, L.; Castilla, J.; DeVicente, J.; Martinez, G.; Moore, P.; Schmidt, R.

    2006-12-01

    The front-end electronics design for the Dark Energy Survey Camera (DECam) is based on the MONSOON Image Acquisition System that was developed by the National Optical Astronomy Observatory (NOAO). MONSOON systems are currently being used to test and characterize CCDs. The Dark Energy Survey group both in the U.S. and Spain will produce custom versions of these systems for use in the production readout that will better match the requirements of a large focal plane of 70+ CCDs and the tight space constraints of a prime focus instrument. The customization of the MONSOON boards and the electronics path will be presented.

  20. Characterization and correction of charge-induced pixel shifts in DECam

    DOE PAGES

    Gruen, D.; Bernstein, G. M.; Jarvis, M.; Rowe, B.; Vikram, V.; Plazas, A. A.; Seitz, S.

    2015-05-28

    Interaction of charges in CCDs with the already accumulated charge distribution causes both a flux dependence of the point-spread function (an increase of observed size with flux, also known as the brighter/fatter effect) and pixel-to-pixel correlations of the Poissonian noise in flat fields. We describe these effects in the Dark Energy Camera (DECam) with charge dependent shifts of effective pixel borders, i.e. the Antilogus et al. (2014) model, which we fit to measurements of flat-field Poissonian noise correlations. The latter fall off approximately as a power-law r-2.5 with pixel separation r, are isotropic except for an asymmetry in the directmore » neighbors along rows and columns, are stable in time, and are weakly dependent on wavelength. They show variations from chip to chip at the 20% level that correlate with the silicon resistivity. The charge shifts predicted by the model cause biased shape measurements, primarily due to their effect on bright stars, at levels exceeding weak lensing science requirements. We measure the flux dependence of star images and show that the effect can be mitigated by applying the reverse charge shifts at the pixel level during image processing. Differences in stellar size, however, remain significant due to residuals at larger distance from the centroid.« less

  1. Characterization and correction of charge-induced pixel shifts in DECam

    SciTech Connect

    Gruen, D.; Bernstein, G. M.; Jarvis, M.; Rowe, B.; Vikram, V.; Plazas, A. A.; Seitz, S.

    2015-05-01

    Interaction of charges in CCDs with the already accumulated charge distribution causes both a flux dependence of the point-spread function (an increase of observed size with flux, also known as the brighter/fatter effect) and pixel-to-pixel correlations of the {Poissonian} noise in flat fields. We describe these effects in the Dark Energy Camera (DECam) with charge dependent shifts of effective pixel borders, i.e. the Antilogus et al. (2014) model, which we fit to measurements of flat-field {Poissonian} noise correlations. The latter fall off approximately as a power-law r(-)(2.5) with pixel separation r, are isotropic except for an asymmetry in the direct neighbors along rows and columns, are stable in time, and are weakly dependent on wavelength. They show variations from chip to chip at the 20% level that correlate with the silicon resistivity. The charge shifts predicted by the model cause biased shape measurements, primarily due to their effect on bright stars, at levels exceeding weak lensing science requirements. We measure the flux dependence of star images and show that the effect can be mitigated by applying the reverse charge shifts at the pixel level during image processing. Differences in stellar size, however, remain significant due to residuals at larger distance from the centroid.

  2. Characterization and correction of charge-induced pixel shifts in DECam

    SciTech Connect

    Gruen, D.; Bernstein, G. M.; Jarvis, M.; Rowe, B.; Vikram, V.; Plazas, A. A.; Seitz, S.

    2015-05-28

    Interaction of charges in CCDs with the already accumulated charge distribution causes both a flux dependence of the point-spread function (an increase of observed size with flux, also known as the brighter/fatter effect) and pixel-to-pixel correlations of the Poissonian noise in flat fields. We describe these effects in the Dark Energy Camera (DECam) with charge dependent shifts of effective pixel borders, i.e. the Antilogus et al. (2014) model, which we fit to measurements of flat-field Poissonian noise correlations. The latter fall off approximately as a power-law r-2.5 with pixel separation r, are isotropic except for an asymmetry in the direct neighbors along rows and columns, are stable in time, and are weakly dependent on wavelength. They show variations from chip to chip at the 20% level that correlate with the silicon resistivity. The charge shifts predicted by the model cause biased shape measurements, primarily due to their effect on bright stars, at levels exceeding weak lensing science requirements. We measure the flux dependence of star images and show that the effect can be mitigated by applying the reverse charge shifts at the pixel level during image processing. Differences in stellar size, however, remain significant due to residuals at larger distance from the centroid.

  3. The size distribution of Near-Earth Asteroids from the DECam NEO Survey

    NASA Astrophysics Data System (ADS)

    Allen, Lori; Valdes, Francisco; Trilling, David; James, David; Herrera, David; Fuentes, Cesar; Axelrod, Tim; Rajagopal, Jayadev; IAU Minor Planet Center, Cerro Tololo Inter-American Observatory

    2016-10-01

    We analyzed data from the first year of a survey for Near Earth Objects (NEOs) that we are carrying out with the Dark Energy Camera (DECam) on the 4 meter Blanco telescope at the Cerro Tololo Inter-American Observatory. We implanted synthetic NEOs into the data stream to derive our nightly detection efficiency as a function of magnitude and rate of motion. Using these measured efficiencies and the Solar System absolute magnitudes derived by the Minor Planet Center for the 1377 measurements of 235 unique NEOs detected, we directly derive, for the first time from a single observational data set, the NEO size distribution from 1 km down to 10 meters. We find that there are 106.6 NEOs larger than 10 meters. This result implies a factor of ten fewer small NEOs than some previous results (e.g., Harris & D'Abramo 2015, Boslough et al. 2015) but a factor of ten more than Tricarico (2016). This result also implies that the impact risk for small- and medium-sized NEOs is less than previously thought.

  4. Intragenic and Extragenic Suppressors of Mutations in the Heptapeptide Repeat Domain of Saccharomyces Cerevisiae RNA Polymerase II

    PubMed Central

    Nonet, M. L.; Young, R. A.

    1989-01-01

    The largest subunit of RNA polymerase II contains a repeated heptapeptide sequence at its carboxy terminus. Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs. Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain. Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants. Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence. Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit. An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations. The SRB2 gene was isolated and mapped, and an SRB2 partial deletion mutation (srb2Δ10) was constructed. The srb2Δ10 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs. These phenotypes are characteristic of mutations in genes encoding components of the transcription apparatus. We propose that the SRB2 gene encodes a factor that is involved in RNA synthesis and may interact with the CTR domain of the large subunit of RNA polymerase II. PMID:2693207

  5. Spatial structure of heptapeptide Glu-Ile-Leu-Asn-His-Met-Lys, a fragment of the HIV enhancer prostatic acid phosphatase, in aqueous and SDS micelle solutions

    NASA Astrophysics Data System (ADS)

    Bloсhin, Dmitri S.; Aganova, Oksana V.; Yulmetov, Aidar R.; Filippov, Andrei V.; Gizatullin, Bulat I.; Afonin, Sergii; Antzutkin, Oleg N.; Klochkov, Vladimir V.

    2013-02-01

    Prostatic acid phosphatase (PAP) is a protein abundantly present in human seminal fluid. PAP plays important role in fertilization. Its 39-amino-acid fragment, PAP(248-286), is effective in enhancing infectivity of HIV virus. In this work, we determined the spatial structure in aqueous solution of a heptapeptide within the PAP fragment, containing amino acid residues 266-272 (Glu-Ile-Leu-Asn-His-Met-Lys). We also report the structure of the complex formed by this heptapeptide with sodium dodecyl sulfate micelles, a model of a biological membrane, as determined by 1H NMR spectroscopy and 2D NMR (TOCSY, HSQC-HECADE, NOESY) spectroscopy. Complex formation was confirmed by chemical shift alterations in the 1H NMR spectra of the heptapeptide, as well as by the signs and values of NOE effects. We also present a comparison of the spatial structure of Glu-Ile-Leu-Asn-His-Met-Lys in water and in complex with sodium dodecyl sulfate.

  6. The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2Cys-peroxiredoxins.

    PubMed

    Alphey, M S; Bond, C S; Tetaud, E; Fairlamb, A H; Hunter, W N

    2000-07-21

    Tryparedoxin peroxidase (TryP) is a recently discovered 2Cys-peroxiredoxin involved in defence against oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata TryP, in the reduced state, has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises a decamer with 52 symmetry, ten chloride ions with 23 water molecules and has been refined, using data to 3.2 A resolution (1 A=0.1 nm), to an R-factor and R(free) of 27.3 and 28.6 %, respectively. Secondary structure topology places TryP along with tryparedoxin and glutathione peroxidase in a distinct subgroup of the thioredoxin super-family. The molecular details at the active site support ideas about the enzyme mechanism and comparisons with an oxidised 2Cys-peroxiredoxin reveal structural alterations induced by the change in oxidation state. These include a difference in quaternary structure from dimer (oxidised form) to decamer (reduced form). The 2Cys-peroxiredoxin assembly may prevent indiscriminate oligomerisation, localise ten peroxidase active sites and contribute to both the specificity of reduction by the redox partner tryparedoxin and attraction of peroxides into the active site.

  7. Incorporation of local structure into kriging models for the prediction of atomistic properties in the water decamer.

    PubMed

    Davie, Stuart J; Di Pasquale, Nicodemo; Popelier, Paul L A

    2016-10-15

    Machine learning algorithms have been demonstrated to predict atomistic properties approaching the accuracy of quantum chemical calculations at significantly less computational cost. Difficulties arise, however, when attempting to apply these techniques to large systems, or systems possessing excessive conformational freedom. In this article, the machine learning method kriging is applied to predict both the intra-atomic and interatomic energies, as well as the electrostatic multipole moments, of the atoms of a water molecule at the center of a 10 water molecule (decamer) cluster. Unlike previous work, where the properties of small water clusters were predicted using a molecular local frame, and where training set inputs (features) were based on atomic index, a variety of feature definitions and coordinate frames are considered here to increase prediction accuracy. It is shown that, for a water molecule at the center of a decamer, no single method of defining features or coordinate schemes is optimal for every property. However, explicitly accounting for the structure of the first solvation shell in the definition of the features of the kriging training set, and centring the coordinate frame on the atom-of-interest will, in general, return better predictions than models that apply the standard methods of feature definition, or a molecular coordinate frame. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  8. Incorporation of local structure into kriging models for the prediction of atomistic properties in the water decamer.

    PubMed

    Davie, Stuart J; Di Pasquale, Nicodemo; Popelier, Paul L A

    2016-10-15

    Machine learning algorithms have been demonstrated to predict atomistic properties approaching the accuracy of quantum chemical calculations at significantly less computational cost. Difficulties arise, however, when attempting to apply these techniques to large systems, or systems possessing excessive conformational freedom. In this article, the machine learning method kriging is applied to predict both the intra-atomic and interatomic energies, as well as the electrostatic multipole moments, of the atoms of a water molecule at the center of a 10 water molecule (decamer) cluster. Unlike previous work, where the properties of small water clusters were predicted using a molecular local frame, and where training set inputs (features) were based on atomic index, a variety of feature definitions and coordinate frames are considered here to increase prediction accuracy. It is shown that, for a water molecule at the center of a decamer, no single method of defining features or coordinate schemes is optimal for every property. However, explicitly accounting for the structure of the first solvation shell in the definition of the features of the kriging training set, and centring the coordinate frame on the atom-of-interest will, in general, return better predictions than models that apply the standard methods of feature definition, or a molecular coordinate frame. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. PMID:27535711

  9. Incorporation of local structure into kriging models for the prediction of atomistic properties in the water decamer

    PubMed Central

    Davie, Stuart J; Di Pasquale, Nicodemo

    2016-01-01

    Machine learning algorithms have been demonstrated to predict atomistic properties approaching the accuracy of quantum chemical calculations at significantly less computational cost. Difficulties arise, however, when attempting to apply these techniques to large systems, or systems possessing excessive conformational freedom. In this article, the machine learning method kriging is applied to predict both the intra‐atomic and interatomic energies, as well as the electrostatic multipole moments, of the atoms of a water molecule at the center of a 10 water molecule (decamer) cluster. Unlike previous work, where the properties of small water clusters were predicted using a molecular local frame, and where training set inputs (features) were based on atomic index, a variety of feature definitions and coordinate frames are considered here to increase prediction accuracy. It is shown that, for a water molecule at the center of a decamer, no single method of defining features or coordinate schemes is optimal for every property. However, explicitly accounting for the structure of the first solvation shell in the definition of the features of the kriging training set, and centring the coordinate frame on the atom‐of‐interest will, in general, return better predictions than models that apply the standard methods of feature definition, or a molecular coordinate frame. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. PMID:27535711

  10. Peroxiredoxin-1 from the human hookworm Ancylostoma ceylanicum forms a stable oxidized decamer and is covalently inhibited by conoidin A

    PubMed Central

    Nguyen, Jennifer B.; Pool, Christopher D.; Wong, Christina Y.B.; Treger, Rebecca S.; Williams, David L.; Cappello, Michael; Lea, Wendy A.; Simeonov, Anton; Vermeire, Jon J.; Modis, Yorgo

    2013-01-01

    Summary Hookworms are parasitic nematodes that have a devastating impact on global health, particularly in developing countries. We report a biochemical and structural analysis of a peroxiredoxin from the hookworm Ancylostoma ceylanicum, AcePrx-1. Peroxiredoxins provide antioxidant protection and act as signaling molecules and chaperones. AcePrx-1 is expressed in adult hookworms and can be inactivated by 2,3-bis(bromomethyl)quinoxaline-1,4-dioxide (conoidin A). Conoidin A inactivates AcePrx-1 by alkylating or crosslinking the catalytic cysteines, while maintaining the enzyme in the “locally unfolded” conformation. Irreversible oxidation of the resolving cysteine may contribute additional inhibitory activity. A crystal structure of oxidized AcePrx-1 reveals a disulfide-linked decamer. A helix macrodipole near the active site increases the reactivity of the catalytic cysteines to conoidin A. This work demonstrates the promise of conoidin compounds as probes to evaluate peroxiredoxins as drug targets in human parasites. PMID:23891152

  11. How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

    PubMed

    Morais, Mariana A B; Giuseppe, Priscila O; Souza, Tatiana A C B; Alegria, Thiago G P; Oliveira, Marcos A; Netto, Luis E S; Murakami, Mario T

    2015-03-27

    2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.

  12. How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

    PubMed

    Morais, Mariana A B; Giuseppe, Priscila O; Souza, Tatiana A C B; Alegria, Thiago G P; Oliveira, Marcos A; Netto, Luis E S; Murakami, Mario T

    2015-03-27

    2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress. PMID:25666622

  13. How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily*

    PubMed Central

    Morais, Mariana A. B.; Giuseppe, Priscila O.; Souza, Tatiana A. C. B.; Alegria, Thiago G. P.; Oliveira, Marcos A.; Netto, Luis E. S.; Murakami, Mario T.

    2015-01-01

    2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His113) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp76 from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress. PMID:25666622

  14. Structure-Based Design of a Br Halogen Bond at the Complex Interface of the Human Placental HtrA1 PDZ Domain with Its Heptapeptide Ligand.

    PubMed

    Dou, Shuo-Fen; Liu, Hong; Cao, Tong-Mei; Wen, Qing-Li; Li, Jie; Shao, Qing-Chun

    2016-04-01

    The shock-induced serine protease HtrA1 is a potential regulator of human placenta development during pregnancy. The protein contains a functional PDZ domain that has been solved in complex with a phage display-derived heptapeptide: Asp-6 Ser-5 Arg-4 Ile-3 Trp-2 Trp-1 Val0 . In this study, a rationally designed halogen bond was introduced to the domain-peptide complex based on its NMR structure in solution. We computationally compared the stabilization energies and hindrance effects due to the presence of different halogens X (X = F, Cl, Br, or I), using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach, and found that the Br atom could considerably promote the peptide binding free energy (ΔΔG = -5.2 kcal/mol). Fluorescence assays confirmed that the peptide affinity to the HtrA1 PDZ domain was improved by approximately sevenfold upon bromination. Structural analysis identified a geometrically perfect halogen bond between the Br atom of the peptide Trp-1 residue and the carbonyl O atom of the HtrA1 Ile385 residue, with a bond length and an interaction energy of d = 3.20 Å and ΔE = -3.7 kcal/mol, respectively. PMID:26972470

  15. Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry.

    PubMed

    Ortea, Paloma Muñiz; Allis, Orla; Healy, Brendan M; Lehane, Mary; Ní Shuilleabháin, Aine; Furey, Ambrose; James, Kevin J

    2004-06-01

    Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.

  16. Heat Shock Protein 27-Targeted Heptapeptide of the PKC{Delta} Catalytic V5 Region Sensitizes Tumors With Radio- and Chemoresistance

    SciTech Connect

    Lee, Hae-June; Kim, Eun-Ho; Seo, Woo Duck; Choi, Tae Hyun; Cheon, Gi-Jeong; Lee, Yoon-Jin; Lee, Yun-Sil

    2011-05-01

    Purpose: Previous data suggest that the PKC{delta} catalytic V5 (PKC{delta}-V5) heptapeptide (HEPT) (FEQFLDI) binds HSP27 and blocks HSP27-mediated radio- or chemoresistance. Here we investigated further the in vivo function of the PKC{delta}-V5 HEPT. Methods and Materials: Labeling of HEPT with Cy5.5 or fluorescein isothiocyanate was performed to evaluate in vitro or in vivo distribution of HEPT. A clonogenic survival assay, flow cytometry, and Western blotting of cleaved caspase-3 were performed to determine in vitro sensitization effects of HEPT plus ionizing radiation (IR) versus IR alone or those of HEPT plus cisplatin(Cis) versus Cis alone. A nude mouse xenografting system was also applied to detect in vivo sensitizing effects of HEPT. Results: HEPT efficiently bound to HSP27 and showed sensitization after combined treatment with IR versus treatment with Cis alone in NCI-H1299 lung carcinoma cells, with higher HSP27 expression, which was similar to that of combined treatment with IR or with Cis alone in NCI-H460 lung carcinoma cells with lower HSP27 expression. In vivo image analysis using Cy5.5-labeled HEPT showed that HEPT was retained in HSP27-overexpressing cancer cells after xenografting to nude mice. Combined treatment of HEPT with IR versus that with Cis alone in xenografted mice showed that HEPT increased radio- or chemosensitization in NCI-H1299 cells compared to that in mice xenografted with NCI-H460 cells. Conclusions: The novel PKC{delta}-V5 HEPT may help overcome HSP27-mediated radio- or chemoresistance.

  17. Highly stable, fluorescence-labeled heptapeptides substituted with a D-amino acid for the specific detection of oxidized low-density lipoprotein in plasma.

    PubMed

    Sato, Akira; Yamanaka, Hikaru; Oe, Keitaro; Yokoyama, Izumi; Yamazaki, Yoji; Ebina, Keiichi

    2015-03-01

    Probes that can detect oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques can be useful for the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that two heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled to fluorescein isothiocyanate (FITC) through the ε-amino group of N-terminus Lys in the absence/presence of 6-amino-n-caproic acid (AC) linker to FITC-(FITC)KP6 and (FITC-AC)KP6-can be useful as fluorescent probes for the specific detection of ox-LDL. In this study, to develop the fluorescent peptides with high plasma stability for the specific detection of ox-LDL, we investigated the interaction of (FITC)KP6 and (FITC-AC)KP6 substituted with D-Lys at the N-terminus-(FITC)dKP6 and (FITC-AC)dKP6-with ox-LDL, and the in vitro stability of these peptides in mouse plasma. (FITC)dKP6 and (FITC-AC)dKP6 bound with high specificity to ox-LDL in a dose-dependent manner, and also to ox-LDL in the mouse plasma. Furthermore, (FITC)dKP6 was more stable than (FITC)KP6 in mouse plasma (102.1% versus 69.0% remained after 1 h). These findings strongly suggest that (FITC)dKP6 and (FITC-AC)dKP6 may be effective fluorescent probes with higher plasma stability than (FITC)KP6 and (FITC-AC)KP6 for the specific detection of ox-LDL.

  18. Structure determination and analysis of helix parameters in the DNA decamer d(CATGGCCATG)2 comparison of results from NMR and crystallography.

    PubMed

    Dornberger, U; Flemming, J; Fritzsche, H

    1998-12-18

    The solution structure of the DNA decamer (CATGGCCATG)2 has been determined by NMR spectroscopy and restrained molecular dynamic and distance geometry calculations. The restrainted data set includes interproton distances and torsion angles for the deoxyribose sugar ring which were obtained by nuclear Overhauser enhancement intensities and quantitative simulation of cross-peaks from double quantum filtered correlation spectroscopy. The backbone torsion angles were constrained using experimental data from NOE cross-peaks, 1H-1H and 1H-31P-coupling constants. The NMR structure and the crystal structure of the DNA decamer deviates from the structure of the canonical form of B-DNA in a number of observable characteristics. Particularly, both structures display a specific pattern of stacking interaction in the central GGC base triplet. Furthermore, a specific local conformation of the TG/CA base-pair step is present in NMR and crystal structure, highlighting the unusually high flexibility of this DNA duplex part. The solution structure of the TG/CA base-pair step obtained by our high resolution NMR study is characterized by a positive roll angle, whereas in crystal this base-pair step tends to adopt remarkably high twist angles.

  19. MicroRNA Profiling of the Effect of the Heptapeptide Angiotensin-(1-7) in A549 Lung Tumor Cells Reveals a Role for miRNA149-3p in Cellular Migration Processes.

    PubMed

    Silva, Brenda de Oliveira da; Lima, Kelvin Furtado; Gonçalves, Letícia Rocha; Silveira, Marina Bonfogo da; Moraes, Karen C M

    2016-01-01

    Lung cancer is one of the most frequent types of cancer in humans and a leading cause of death worldwide. The high mortality rates are correlated with late diagnosis, which leads to high rates of metastasis found in patients. Thus, despite all the improvement in therapeutic approaches, the development of new drugs that control cancer cell migration and metastasis are required. The heptapeptide angiotensin-(1-7) [ang-(1-7)] has demonstrated the ability to control the growth rates of human lung cancer cells in vitro and in vivo, and the elucidation of central elements that control the fine-tuning of cancer cells migration in the presence of the ang-(1-7), will support the development of new therapeutic approaches. Ang-(1-7) is a peptide hormone of the renin-angiotensin system (RAS) and this study investigates the modulatory effect of the heptapeptide on the expression pattern of microRNAs (miRNAs) in lung tumor cells, to elucidate mechanistic concerns about the effect of the peptide in the control of tumor migratory processes. Our primary aim was to compare the miRNA profiling between treated and untreated-heptapeptide cells to characterize the relevant molecule that modulates cellular migration rates. The analyses selected twenty one miRNAs, which are differentially expressed between the groups; however, statistical analyses indicated miRNA-149-3p as a relevant molecule. Once functional analyses were performed, we demonstrated that miRNA-149-3p plays a role in the cellular migration processes. This information could be useful for future investigations on drug development. PMID:27598578

  20. MicroRNA Profiling of the Effect of the Heptapeptide Angiotensin-(1-7) in A549 Lung Tumor Cells Reveals a Role for miRNA149-3p in Cellular Migration Processes

    PubMed Central

    da Silva, Brenda de Oliveira; Lima, Kelvin Furtado; Gonçalves, Letícia Rocha; da Silveira, Marina Bonfogo; Moraes, Karen C. M.

    2016-01-01

    Lung cancer is one of the most frequent types of cancer in humans and a leading cause of death worldwide. The high mortality rates are correlated with late diagnosis, which leads to high rates of metastasis found in patients. Thus, despite all the improvement in therapeutic approaches, the development of new drugs that control cancer cell migration and metastasis are required. The heptapeptide angiotensin-(1–7) [ang-(1–7)] has demonstrated the ability to control the growth rates of human lung cancer cells in vitro and in vivo, and the elucidation of central elements that control the fine-tuning of cancer cells migration in the presence of the ang-(1–7), will support the development of new therapeutic approaches. Ang-(1–7) is a peptide hormone of the renin-angiotensin system (RAS) and this study investigates the modulatory effect of the heptapeptide on the expression pattern of microRNAs (miRNAs) in lung tumor cells, to elucidate mechanistic concerns about the effect of the peptide in the control of tumor migratory processes. Our primary aim was to compare the miRNA profiling between treated and untreated-heptapeptide cells to characterize the relevant molecule that modulates cellular migration rates. The analyses selected twenty one miRNAs, which are differentially expressed between the groups; however, statistical analyses indicated miRNA-149-3p as a relevant molecule. Once functional analyses were performed, we demonstrated that miRNA-149-3p plays a role in the cellular migration processes. This information could be useful for future investigations on drug development. PMID:27598578

  1. Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.

    PubMed

    Foti, M; Marshalko, S; Schurter, E; Kumar, S; Beardsley, G P; Schweitzer, B I

    1997-05-01

    The nucleoside analog 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir, DHPG) is an antiviral drug that is used in the treatment of a variety of herpes viruses in immunocompromised patients and in a gene therapy protocol that has shown promising activity for the treatment of cancer. To probe the structural effects of ganciclovir when incorporated into DNA, we determined and compared the solution structure of a modified ganciclovir-containing decamer duplex [d(CTG)(ganciclovir)d(ATCCAG)]2 and a control duplex d[(CTGGATCCAG)]2 using nuclear magnetic resonance techniques. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H and 31P NMR experiments. Proton-proton distances determined from NOESY data and dihedral angles determined from DQF-COSY data were used in restrained molecular dynamics simulations starting from canonical A- and B-form DNA models. Both the control and ganciclovir sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures that were in excellent agreement with the observed NOE intensities. Examination of the final ganciclovir-containing structures reveals that the base of the ganciclovir residue is hydrogen bonded to its complementary dC and is stacked in the helix; in fact, the base of ganciclovir exhibits increased stacking with the 5' base relative to the control. Interestingly, some of the most significant distortions in the structures occur 3' to the lesion site, including a noticeable kink in the sugar-phosphate backbone at this position. Further examination reveals that the backbone conformation, sugar pucker, and glycosidic torsion angle of the residue 3' to the lesion site all indicate an A-type conformation at this position. A possible correlation of these structural findings with results obtained from earlier biochemical studies will be discussed.

  2. Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.

    PubMed

    Foti, M; Marshalko, S; Schurter, E; Kumar, S; Beardsley, G P; Schweitzer, B I

    1997-05-01

    The nucleoside analog 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir, DHPG) is an antiviral drug that is used in the treatment of a variety of herpes viruses in immunocompromised patients and in a gene therapy protocol that has shown promising activity for the treatment of cancer. To probe the structural effects of ganciclovir when incorporated into DNA, we determined and compared the solution structure of a modified ganciclovir-containing decamer duplex [d(CTG)(ganciclovir)d(ATCCAG)]2 and a control duplex d[(CTGGATCCAG)]2 using nuclear magnetic resonance techniques. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H and 31P NMR experiments. Proton-proton distances determined from NOESY data and dihedral angles determined from DQF-COSY data were used in restrained molecular dynamics simulations starting from canonical A- and B-form DNA models. Both the control and ganciclovir sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures that were in excellent agreement with the observed NOE intensities. Examination of the final ganciclovir-containing structures reveals that the base of the ganciclovir residue is hydrogen bonded to its complementary dC and is stacked in the helix; in fact, the base of ganciclovir exhibits increased stacking with the 5' base relative to the control. Interestingly, some of the most significant distortions in the structures occur 3' to the lesion site, including a noticeable kink in the sugar-phosphate backbone at this position. Further examination reveals that the backbone conformation, sugar pucker, and glycosidic torsion angle of the residue 3' to the lesion site all indicate an A-type conformation at this position. A possible correlation of these structural findings with results obtained from earlier biochemical studies will be discussed. PMID:9154915

  3. Molecular structures of two crystalline forms of the cyclic heptapeptide antibiotic ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala-].

    PubMed

    Miller, R; Galitsky, N M; Duax, W L; Langs, D A; Pletnev, V Z; Ivanov, V T

    1993-12-01

    The crystal structures of two solvated forms of ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala-] are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P2(1)2(1)2(1), a = 11.563(1), b = 21.863(2), c = 36.330(4) A. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P2(1)2(1)2(1), a = 14.067(2), b = 16.695(1), c = 36.824(6) A. N-Methylation of four of the seven residues of ternatin appears to reduce the number of low-energy conformations the molecule can assume. The same H-bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino-acid side chains, with the exception of D-Ile2, have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala3 and (NMe)Leu4, (ii) a type II beta-bend, involving residues Leu5-(NMe)Ala6-D-(NMe)Ala7-beta-OH-D-Leu1, stabilized by two H-bonds, N1-->O5 and N5-->O1, between Leu5 and beta-OH-D-Leu1 residues, (iii) a third intramolecular H-bond, observed in each of the four molecules, between the hydroxyl group of beta-OH-D-Leu1 and the carbonyl oxygen of D-Ile2.

  4. The crystal structure of the C45S mutant of annelid Arenicola marina peroxiredoxin 6 supports its assignment to the mechanistically typical 2-Cys subfamily without any formation of toroid-shaped decamers

    PubMed Central

    Smeets, Aude; Loumaye, Eléonore; Clippe, André; Rees, Jean-François; Knoops, Bernard; Declercq, Jean-Paul

    2008-01-01

    The peroxiredoxins (PRDXs) define a superfamily of thiol-dependent peroxidases able to reduce hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. Besides their cytoprotective antioxidant function, PRDXs have been implicated in redox signaling and chaperone activity, the latter depending on the formation of decameric high-molecular-weight structures. PRDXs have been mechanistically divided into three major subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs, based on the number and position of cysteines involved in the catalysis. We report the structure of the C45S mutant of annelid worm Arenicola marina PRDX6 in three different crystal forms determined at 1.6, 2.0, and 2.4 Å resolution. Although A. marina PRDX6 was cloned during the search of annelid homologs of mammalian 1-Cys PRDX6s, the crystal structures support its assignment to the mechanistically typical 2-Cys PRDX subfamily. The protein is composed of two distinct domains: a C-terminal domain and an N-terminal domain exhibiting a thioredoxin fold. The subunits are associated in dimers compatible with the formation of intersubunit disulfide bonds between the peroxidatic and the resolving cysteine residues in the wild-type enzyme. The packing of two crystal forms is very similar, with pairs of dimers associated as tetramers. The toroid-shaped decamers formed by dimer association and observed in most typical 2-Cys PRDXs is not present. Thus, A. marina PRDX6 presents structural features of typical 2-Cys PRDXs without any formation of toroid-shaped decamers, suggesting that it should function more like a cytoprotective antioxidant enzyme or a modulator of peroxide-dependent cell signaling rather than a molecular chaperone. PMID:18359859

  5. Partial characterization of cyclic AMP-dependent protein kinases in guinea-pig lung employing the synthetic heptapeptide substrate, kemptide. In vitro sensitivity of the soluble enzyme to isoprenaline, forskolin, methacholine and leukotriene D4.

    PubMed

    Giembycz, M A; Diamond, J

    1990-04-15

    This paper describes the partial characterization of soluble cyclic AMP-dependent protein kinase (A-kinase) in guinea-pig lung using Kemptide, a synthetic serine-containing heptapeptide, and examines the sensitivity of this enzyme to drugs which are reported to increase and to decrease the intracellular concentration of cyclic AMP. Differential centrifugation of lung homogenates revealed that 78% of A-kinase was present in the 31,000 gmax x 15 min supernatant fraction. Both basal and cyclic AMP-stimulated phosphotransferase activity of this 'soluble' enzyme were abolished by the heat-stable inhibitor of A-kinase. Soluble A-kinase was Mg2(+)-dependent (apparent Km and and Kact 8.6 and 2.6 mM, respectively) and was stimulated nine-fold by saturating concentrations of both cyclic AMP (Kact: 131 nM) and cyclic GMP (Kact: 28.7 microM) at a protein (enzyme) concentration of 1.3 micrograms. Kinetic analysis of the effect of Kemptide and ATP revealed linear, Hanes plots with Michealis constants of ca. 12 and 13 microM, respectively. Chromatography of the soluble enzyme over DEAE-cellulose resolved three peaks of catalytic activity when fractions were assayed in the presence of cyclic AMP (10 microM): (i) free catalytic subunits (5%), (ii) Type I isoenzyme (5%) and (iii) Type II isoenzyme (90%). The A-kinase activity ratio was markedly increased in lung pre-treated with the smooth muscle relaxants isoprenaline and forskolin. This biochemical effect was both time- and concentration-dependent and was temporally associated with the ability of these drugs to reduce lung parenchymal tone. In contrast, the contractile agonists, methacholine (Mch) and leukotriene (LT) D4 exerted opposite effects on A-kinase activity. Thus, Mch significantly reduced cyclic AMP levels and lowered basal A-kinase activity whilst the converse was true for LTD4. For both drugs this biochemical effect accompanied contraction of the lung. Pre-treatment of lung tissue with flurbiprofen, an irreversible

  6. The heptapeptide LSARLAF mediates platelet activation through phospholipase Cgamma2 independently of glycoprotein IIb-IIIa.

    PubMed Central

    Pearce, Andrew C; Wonerow, Peter; Marshall, Stuart J; Frampton, Jon; Gartner, T Kent; Watson, Steve P

    2004-01-01

    The seven-amino-acid peptide LSARLAF has been reported to activate platelets via the integrin GPIIb-IIIa (glycoprotein IIb-IIIa). Activation by LSARLAF is reinforced by release of ADP and thromboxanes, but the initiating event in the signalling cascade is not known. In the present study, we demonstrate that LSARLAF stimulates Src kinase-dependent tyrosine phosphorylation of many of the proteins in the GPIIb-IIIa cascade, including the tyrosine kinase Syk, the adapter SLP-76 (SH2-containing leucocyte phosphoprotein of 76 kDa) and PLCgamma2 (phospholipase Cgamma2). A critical role for PLCgamma2 in signalling by LSARLAF was demonstrated by abolition of aggregation in PLCgamma2-/- murine platelets to low concentrations of the peptide, although a partial recovery was seen with higher concentrations. In sharp contrast with the GPIIb-IIIa-regulated signalling cascade, aggregation was inhibited in murine platelets deficient in the adapter LAT (linker for activation of T-cells) and the Fc receptor gamma-chain. Aggregation was also partially inhibited by the cholesterol-lowering reagent, beta-methyl-cyclodextrin, at concentrations that disrupt membrane rafts, but do not interfere with signalling by GPIIb-IIIa. Furthermore, LSARLAF also stimulated tyrosine phosphorylation in GPIIb-deficient murine platelets, confirming that the integrin is not critical for activation of intracellular signalling pathways. LSARLAF also stimulated Ca2+ elevation in RBL-2H3 cells, which lack the platelet glycoproteins GPIIb, GPVI and GPIb. These results demonstrate that LSARLAF activates platelets through a PLCgamma2-dependent pathway that lies downstream of Src kinases and which is partially dependent on the Fc receptor gamma-chain, LAT and lipid rafts. The mechanism of cell activation by LSARLAF remains to be established, although the present results indicate that more than one surface glycoprotein may mediate this response. PMID:14558887

  7. Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

    SciTech Connect

    Payan, D.G.; Horvath, K.; Graf, L.

    1987-03-23

    The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocyte ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.

  8. The role of side-chain interactions in the early steps of aggregation: Molecular dynamics simulations of an amyloid-forming peptide from the yeast prion Sup35

    NASA Astrophysics Data System (ADS)

    Gsponer, Jörg; Haberthür, Urs; Caflisch, Amedeo

    2003-04-01

    Understanding the early steps of aggregation at atomic detail might be crucial for the rational design of therapeutics preventing diseases associated with amyloid deposits. In this paper, aggregation of the heptapeptide GNNQQNY, from the N-terminal prion-determining domain of the yeast protein Sup35, was studied by 20 molecular dynamics runs for a total simulation time of 20 μs. The simulations generate in-register parallel packing of GNNQQNY -strands that is consistent with x-ray diffraction and Fourier transform infrared data. The statistically preferred aggregation pathway does not correspond to a purely downhill profile of the energy surface because of the presence of enthalpic barriers that originate from out-of-register interactions. The parallel -sheet arrangement is favored over the antiparallel because of side-chain contacts; in particular, stacking interactions of the tyrosine rings and hydrogen bonds between amide groups. No ordered aggregation was found in control simulations with the mutant sequence SQNGNQQRG in accord with experimental data and the strong sequence dependence of aggregation.

  9. A DECam Search for an Optical Counterpart to the LIGO Gravitational-wave Event GW151226

    NASA Astrophysics Data System (ADS)

    Cowperthwaite, P. S.; Berger, E.; Soares-Santos, M.; Annis, J.; Brout, D.; Brown, D. A.; Buckley-Geer, E.; Cenko, S. B.; Chen, H. Y.; Chornock, R.; Diehl, H. T.; Doctor, Z.; Drlica-Wagner, A.; Drout, M. R.; Farr, B.; Finley, D. A.; Foley, R. J.; Fong, W.; Fox, D. B.; Frieman, J.; Garcia-Bellido, J.; Gill, M. S. S.; Gruendl, R. A.; Herner, K.; Holz, D. E.; Kasen, D.; Kessler, R.; Lin, H.; Margutti, R.; Marriner, J.; Matheson, T.; Metzger, B. D.; Neilsen, E. H., Jr.; Quataert, E.; Rest, A.; Sako, M.; Scolnic, D.; Smith, N.; Sobreira, F.; Strampelli, G. M.; Villar, V. A.; Walker, A. R.; Wester, W.; Williams, P. K. G.; Yanny, B.; Abbott, T. M. C.; Abdalla, F. B.; Allam, S.; Armstrong, R.; Bechtol, K.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Burke, D. L.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Castander, F. J.; Cunha, C. E.; D’Andrea, C. B.; da Costa, L. N.; Desai, S.; Dietrich, J. P.; Evrard, A. E.; Fausti Neto, A.; Fosalba, P.; Gerdes, D. W.; Giannantonio, T.; Goldstein, D. A.; Gruen, D.; Gutierrez, G.; Honscheid, K.; James, D. J.; Johnson, M. W. G.; Johnson, M. D.; Krause, E.; Kuehn, K.; Kuropatkin, N.; Lima, M.; Maia, M. A. G.; Marshall, J. L.; Menanteau, F.; Miquel, R.; Mohr, J. J.; Nichol, R. C.; Nord, B.; Ogando, R.; Plazas, A. A.; Reil, K.; Romer, A. K.; Sanchez, E.; Scarpine, V.; Sevilla-Noarbe, I.; Smith, R. C.; Suchyta, E.; Tarle, G.; Thomas, D.; Thomas, R. C.; Tucker, D. L.; Weller, J.; The DES Collaboration

    2016-08-01

    We report the results of a Dark Energy Camera optical follow-up of the gravitational-wave (GW) event GW151226, discovered by the Advanced Laser Interferometer Gravitational-wave Observatory detectors. Our observations cover 28.8 deg2 of the localization region in the i and z bands (containing 3% of the BAYESTAR localization probability), starting 10 hr after the event was announced and spanning four epochs at 2–24 days after the GW detection. We achieve 5σ point-source limiting magnitudes of i≈ 21.7 and z≈ 21.5, with a scatter of 0.4 mag, in our difference images. Given the two-day delay, we search this area for a rapidly declining optical counterpart with ≳ 3σ significance steady decline between the first and final observations. We recover four sources that pass our selection criteria, of which three are cataloged active galactic nuclei. The fourth source is offset by 5.8 arcsec from the center of a galaxy at a distance of 187 Mpc, exhibits a rapid decline by 0.5 mag over 4 days, and has a red color of i-z≈ 0.3 mag. These properties could satisfy a set of cuts designed to identify kilonovae. However, this source was detected several times, starting 94 days prior to GW151226, in the Pan-STARRS Survey for Transients (dubbed as PS15cdi) and is therefore unrelated to the GW event. Given its long-term behavior, PS15cdi is likely a Type IIP supernova that transitioned out of its plateau phase during our observations, mimicking a kilonova-like behavior. We comment on the implications of this detection for contamination in future optical follow-up observations.

  10. A DECam Search for an Optical Counterpart to the LIGO Gravitational-wave Event GW151226

    NASA Astrophysics Data System (ADS)

    Cowperthwaite, P. S.; Berger, E.; Soares-Santos, M.; Annis, J.; Brout, D.; Brown, D. A.; Buckley-Geer, E.; Cenko, S. B.; Chen, H. Y.; Chornock, R.; Diehl, H. T.; Doctor, Z.; Drlica-Wagner, A.; Drout, M. R.; Farr, B.; Finley, D. A.; Foley, R. J.; Fong, W.; Fox, D. B.; Frieman, J.; Garcia-Bellido, J.; Gill, M. S. S.; Gruendl, R. A.; Herner, K.; Holz, D. E.; Kasen, D.; Kessler, R.; Lin, H.; Margutti, R.; Marriner, J.; Matheson, T.; Metzger, B. D.; Neilsen, E. H., Jr.; Quataert, E.; Rest, A.; Sako, M.; Scolnic, D.; Smith, N.; Sobreira, F.; Strampelli, G. M.; Villar, V. A.; Walker, A. R.; Wester, W.; Williams, P. K. G.; Yanny, B.; Abbott, T. M. C.; Abdalla, F. B.; Allam, S.; Armstrong, R.; Bechtol, K.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Burke, D. L.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Castander, F. J.; Cunha, C. E.; D'Andrea, C. B.; da Costa, L. N.; Desai, S.; Dietrich, J. P.; Evrard, A. E.; Fausti Neto, A.; Fosalba, P.; Gerdes, D. W.; Giannantonio, T.; Goldstein, D. A.; Gruen, D.; Gutierrez, G.; Honscheid, K.; James, D. J.; Johnson, M. W. G.; Johnson, M. D.; Krause, E.; Kuehn, K.; Kuropatkin, N.; Lima, M.; Maia, M. A. G.; Marshall, J. L.; Menanteau, F.; Miquel, R.; Mohr, J. J.; Nichol, R. C.; Nord, B.; Ogando, R.; Plazas, A. A.; Reil, K.; Romer, A. K.; Sanchez, E.; Scarpine, V.; Sevilla-Noarbe, I.; Smith, R. C.; Suchyta, E.; Tarle, G.; Thomas, D.; Thomas, R. C.; Tucker, D. L.; Weller, J.; DES Collaboration

    2016-08-01

    We report the results of a Dark Energy Camera optical follow-up of the gravitational-wave (GW) event GW151226, discovered by the Advanced Laser Interferometer Gravitational-wave Observatory detectors. Our observations cover 28.8 deg2 of the localization region in the i and z bands (containing 3% of the BAYESTAR localization probability), starting 10 hr after the event was announced and spanning four epochs at 2-24 days after the GW detection. We achieve 5σ point-source limiting magnitudes of i≈ 21.7 and z≈ 21.5, with a scatter of 0.4 mag, in our difference images. Given the two-day delay, we search this area for a rapidly declining optical counterpart with ≳ 3σ significance steady decline between the first and final observations. We recover four sources that pass our selection criteria, of which three are cataloged active galactic nuclei. The fourth source is offset by 5.8 arcsec from the center of a galaxy at a distance of 187 Mpc, exhibits a rapid decline by 0.5 mag over 4 days, and has a red color of i-z≈ 0.3 mag. These properties could satisfy a set of cuts designed to identify kilonovae. However, this source was detected several times, starting 94 days prior to GW151226, in the Pan-STARRS Survey for Transients (dubbed as PS15cdi) and is therefore unrelated to the GW event. Given its long-term behavior, PS15cdi is likely a Type IIP supernova that transitioned out of its plateau phase during our observations, mimicking a kilonova-like behavior. We comment on the implications of this detection for contamination in future optical follow-up observations.

  11. Cyclic heptapeptides, cordyheptapeptides C-E, from the marine-derived fungus Acremonium persicinum SCSIO 115 and their cytotoxic activities.

    PubMed

    Chen, Ziming; Song, Yongxiang; Chen, Yuchan; Huang, Hongbo; Zhang, Weimin; Ju, Jianhua

    2012-06-22

    Three new cycloheptapeptides, cordyheptapeptides C-E (1-3), were isolated from the fermentation extract of the marine-derived fungus Acremonium persicinum SCSIO 115. Their planar structures were elucidated on the basis of extensive MS, as well as 1D and 2D (COSY, HMQC, and HMBC) NMR spectroscopic data analyses. The absolute configurations of the amino acid residues were determined by single-crystal X-ray diffraction, Marfey's method, and chiral-phase HPLC analysis. Compounds 1 and 3 displayed cytotoxicity against SF-268, MCF-7, and NCI-H460 tumor cell lines with IC(50) values ranging from 2.5 to 12.1 μM.

  12. Role of monomer arrangement in the amyloid self-assembly

    PubMed Central

    Portillo, Alexander; Hashemi, Mohtadin; Zhang, Yuliang; Breydo, Leonid; Uversky, Vladimir N.; Lyubchenko, Yuri L.

    2015-01-01

    Assembly of amyloid proteins into aggregates requires the ordering of the monomers in oligomers and especially in such highly organized structures as fibrils. This ordering is accompanied by structural transitions leading to the formation of ordered β-structural motifs in proteins and peptides lacking secondary structures. To characterize the effect of the monomers arrangements on the aggregation process at various stages, we performed comparative studies of the yeast prion protein Sup35 heptapeptide (GNNQQNY) along with its dimeric form CGNNQQNY-(d-Pro)-G-GNNQQNY. The (d-Pro)-G linker in this construct is capable of adopting a β-turn, facilitating the assembly of the dimer into the dimeric antiparallel hairpin structure (AP-hairpin). We applied Atomic Force Microscopy (AFM) techniques to follow peptide-peptide interactions at the single molecule level, to visualize the morphology of aggregates formed by both constructs, thioflavin T (ThT) fluorescence to follow the aggregation kinetics, and circular dichroism (CD) spectroscopy to characterize the secondary structure of the constructs. The ThT fluorescence data showed that the AP-hairpin aggregation kinetics is insensitive to the external environment such as ionic strength and pH contrary to the monomers the kinetics of which depends dramatically on the ionic strength and pH. The AFM topographic imaging revealed that AP--hairpins primarily assemble into globular aggregates, whereas linear fibrils are primary assemblies of the monomers suggesting that both constructs follow different aggregation pathways during the self-assembly. These morphological differences are in line with the AFM force spectroscopy experiments and CD spectroscopy measurements, suggesting that the AP-hairpin is structurally rigid regardless of changes of environmental factors. PMID:25542374

  13. Crystal Structure of Reduced and of Oxidized Peroxiredoxin IV Enzyme Reveals a Stable Oxidized Decamer and a Non-disulfide-bonded Intermediate in the Catalytic Cycle*

    PubMed Central

    Cao, Zhenbo; Tavender, Timothy J.; Roszak, Aleksander W.; Cogdell, Richard J.; Bulleid, Neil J.

    2011-01-01

    Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue. PMID:21994946

  14. Thermodynamics of Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

    Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

  15. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

    PubMed Central

    Kirillova, Yuliya; Boyarskaya, Nataliya; Dezhenkov, Andrey; Tankevich, Mariya; Prokhorov, Ivan; Varizhuk, Anna; Eremin, Sergei; Esipov, Dmitry; Smirnov, Igor; Pozmogova, Galina

    2015-01-01

    New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA. PMID:26469337

  16. Evaluation of a High-Throughput Peptide Reactivity Format Assay for Assessment of the Skin Sensitization Potential of Chemicals

    PubMed Central

    Wong, Chin Lin; Lam, Ai-Leen; Smith, Maree T.; Ghassabian, Sussan

    2016-01-01

    The direct peptide reactivity assay (DPRA) is a validated method for in vitro assessment of the skin sensitization potential of chemicals. In the present work, we describe a peptide reactivity assay using 96-well plate format and systematically identified the optimal assay conditions for accurate and reproducible classification of chemicals with known sensitizing capacity. The aim of the research is to ensure that the analytical component of the peptide reactivity assay is robust, accurate, and reproducible in accordance with criteria that are used for the validation of bioanalytical methods. Analytical performance was evaluated using quality control samples (QCs; heptapeptides at low, medium, and high concentrations) and incubation of control chemicals (chemicals with known sensitization capacity, weak, moderate, strong, extreme, and non-sensitizers) with each of three synthetic heptapeptides, viz Cor1-C420 (Ac-NKKCDLF), cysteine- (Ac-RFAACAA), and lysine- (Ac-RFAAKAA) containing heptapeptides. The optimal incubation temperature for all three heptapeptides was 25°C. Apparent heptapeptide depletion was affected by vial material composition. Incubation of test chemicals with Cor1-C420, showed that peptide depletion was unchanged in polypropylene vials over 3-days storage in an autosampler but this was not the case for borosilicate glass vials. For cysteine-containing heptapeptide, the concentration was not stable by day 3 post-incubation in borosilicate glass vials. Although the lysine-containing heptapeptide concentration was unchanged in both polypropylene and borosilicate glass vials, the apparent extent of lysine-containing heptapeptide depletion by ethyl acrylate, differed between polypropylene (24.7%) and glass (47.3%) vials. Additionally, the peptide-chemical complexes for Cor1-C420-cinnamaldehyde and cysteine-containing heptapeptide-2, 4-dinitrochlorobenzene were partially reversible during 3-days of autosampler storage. These observations further highlight

  17. A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

    PubMed Central

    Guilfoyle, T J

    1989-01-01

    A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit. PMID:2535525

  18. Structural complexity of a composite amyloid fibril

    PubMed Central

    Lewandowski, Józef R.; van der Wel, Patrick C.A.; Rigney, Mike; Grigorieff, Nikolaus; Griffin, Robert G.

    2011-01-01

    The molecular structure of amyloid fibrils and the mechanism of their formation are of substantial medical and biological importance, but present an ongoing experimental and computational challenge. An early high-resolution view of amyloid-like structure was obtained on amyloid-like crystals of a small fragment of the yeast prion protein Sup35p: the peptide GNNQQNY. As GNNQQNY also forms amyloid-like fibrils under similar conditions, it has been theorized that the crystal's structural features are shared by the fibrils. Here we apply magic-angle-spinning (MAS) NMR to examine the structure and dynamics of these fibrils. Previously multiple NMR signals were observed for such samples, seemingly consistent with the presence of polymorphic fibrils. Here we demonstrate that peptides with these three distinct conformations instead assemble together into composite protofilaments. Electron-microscopy (EM) of the ribbon-like fibrils indicates that these protofilaments combine in differing ways to form striations of variable widths, presenting another level of structural complexity. Structural and dynamical NMR data reveal the presence of highly restricted side chain conformations involved in interfaces between differently structured peptides, likely comprising interdigitated steric zippers. We outline molecular interfaces that are consistent with the observed EM and NMR data. The rigid and uniform structure of the GNNQQNY crystals is found to contrast distinctly with the more complex structural and dynamic nature of these “composite” amyloid fibrils. These results provide insight into the fibril-crystal distinction and also indicate a necessary caution with respect to the extrapolation of crystal structures to the study of fibril structure and formation. PMID:21766841

  19. Fibronectin tetrapeptide is target for syphilis spirochete cytadherence

    SciTech Connect

    Thomas, D.D.; Baseman, J.B.; Alderete, J.F.

    1985-11-01

    The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with SVI-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitism of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.

  20. Pressure-sensitive reaction yield of the TePixD blue-light sensor protein.

    PubMed

    Kuroi, Kunisato; Okajima, Koji; Ikeuchi, Masahiko; Tokutomi, Satoru; Kamiyama, Tadashi; Terazima, Masahide

    2015-02-19

    The effect of pressure on the dissociation reaction of the TePixD decamer was investigated by high-pressure transient grating (TG). The TG signal intensity representing the dissociation reaction of the TePixD decamer significantly decreased by applying a relatively small pressure. On the other hand, the reaction rate increased with increasing pressure. The equilibrium between the pentamer and the decamer was investigated by high-pressure dynamic light scattering. The results indicated that the fraction of the decamer slightly increased in the high-pressure region. From these measurements, it was concluded that the pressure-dependent signal intensity originated from the decrease of the quantum yield of the dissociation reaction of the decamer, indicating that this reaction efficiency is very sensitive to pressure. Using densimetry at high pressures, the compressibility was found to be pressure dependent even in a relatively low pressure range. We attributed the origin of the pressure-sensitive reaction yield to the decrease of compressibility at high pressure. Because the compressibility is related to the volume fluctuation, this observation suggests that the driving force for this reaction is fluctuation of the protein. The relationship between the cavities at the interfaces of the monomer units and the reactivity is also discussed.

  1. System Architecture of the Dark Energy Survey Camera Readout Electronics

    SciTech Connect

    Shaw, Theresa; Ballester, Otger; Cardiel-Sas, Laia; Castilla, Javier; Chappa, Steve; de Vicente, Juan; Holm, Scott; Huffman, Dave; Kozlovsky, Mark; Martinez, Gustavo; Moore, Todd; /Madrid, CIEMAT /Fermilab /Illinois U., Urbana /Fermilab

    2010-05-27

    The Dark Energy Survey makes use of a new camera, the Dark Energy Camera (DECam). DECam will be installed in the Blanco 4M telescope at Cerro Tololo Inter-American Observatory (CTIO). DECam is presently under construction and is expected to be ready for observations in the fall of 2011. The focal plane will make use of 62 2Kx4K and 12 2kx2k fully depleted Charge-Coupled Devices (CCDs) for guiding, alignment and focus. This paper will describe design considerations of the system; including, the entire signal path used to read out the CCDs, the development of a custom crate and backplane, the overall grounding scheme and early results of system tests.

  2. A Scalable and High-Yield Strategy for the Synthesis of Sequence-Defined Macromolecules.

    PubMed

    Solleder, Susanne C; Zengel, Deniz; Wetzel, Katharina S; Meier, Michael A R

    2016-01-18

    The efficient synthesis of a sequence-defined decamer, its characterization, and its straightforward dimerization through self-metathesis are described. For this purpose, a monoprotected AB monomer was designed and used to synthesize a decamer bearing ten different and selectable side chains by iterative Passerini three-component reaction (P-3CR) and subsequent deprotection. The highly efficient procedure provided excellent yields and allows for the multigram-scale synthesis of such perfectly defined macromolecules. An olefin was introduced at the end of the synthesis, allowing the self-metathesis reaction of the resulting decamer to provide a sequence-defined 20-mer with a molecular weight of 7046.40 g mol(-1). The obtained oligomers were carefully characterized by NMR and IR spectroscopy, GPC and GPC coupled to ESI-MS, and mass spectrometry (FAB and orbitrap ESI-MS).

  3. Dark energy camera installation at CTIO: overview

    NASA Astrophysics Data System (ADS)

    Abbott, Timothy M.; Muñoz, Freddy; Walker, Alistair R.; Smith, Chris; Montane, Andrés.; Gregory, Brooke; Tighe, Roberto; Schurter, Patricio; van der Bliek, Nicole S.; Schumacher, German

    2012-09-01

    The Dark Energy Camera (DECam) has been installed on the V. M. Blanco telescope at Cerro Tololo Inter-American Observatory in Chile. This major upgrade to the facility has required numerous modifications to the telescope and improvements in observatory infrastructure. The telescope prime focus assembly has been entirely replaced, and the f/8 secondary change procedure radically changed. The heavier instrument means that telescope balance has been significantly modified. The telescope control system has been upgraded. NOAO has established a data transport system to efficiently move DECam's output to the NCSA for processing. The observatory has integrated the DECam highpressure, two-phase cryogenic cooling system into its operations and converted the Coudé room into an environmentally-controlled instrument handling facility incorporating a high quality cleanroom. New procedures to ensure the safety of personnel and equipment have been introduced.

  4. A Scalable and High-Yield Strategy for the Synthesis of Sequence-Defined Macromolecules.

    PubMed

    Solleder, Susanne C; Zengel, Deniz; Wetzel, Katharina S; Meier, Michael A R

    2016-01-18

    The efficient synthesis of a sequence-defined decamer, its characterization, and its straightforward dimerization through self-metathesis are described. For this purpose, a monoprotected AB monomer was designed and used to synthesize a decamer bearing ten different and selectable side chains by iterative Passerini three-component reaction (P-3CR) and subsequent deprotection. The highly efficient procedure provided excellent yields and allows for the multigram-scale synthesis of such perfectly defined macromolecules. An olefin was introduced at the end of the synthesis, allowing the self-metathesis reaction of the resulting decamer to provide a sequence-defined 20-mer with a molecular weight of 7046.40 g mol(-1). The obtained oligomers were carefully characterized by NMR and IR spectroscopy, GPC and GPC coupled to ESI-MS, and mass spectrometry (FAB and orbitrap ESI-MS). PMID:26663541

  5. Molluscan mega-hemocyanin: an ancient oxygen carrier tuned by a ~550 kDa polypeptide

    PubMed Central

    2010-01-01

    Background The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 × 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia). Results The "typical" KLH2 tridecamer is partially hollow, whereas the cerithioid tridecamer is almost completely filled with material; it was therefore termed "mega-hemocyanin". In both types, the staggering angle between adjoining decamers is 36°. The cerithioid tridecamer comprises two typical decamers based on the canonical 400 kDa subunit, flanking a central "mega-decamer" composed of ten unique ~550 kDa subunits. The additional ~150 kDa per subunit substantially enlarge the internal collar complex. Preliminary oxygen binding measurements indicate a moderate hemocyanin oxygen affinity in Leptoxis (p50 ~9 mmHg), and a very high affinity in Melanoides (~3 mmHg) and Terebralia (~2 mmHg). Species-specific and individual variation in the proportions of the two subunit types was also observed, leading to differences in the oligomeric states found in the hemolymph. Conclusions In cerithioid hemocyanin tridecamers ("mega-hemocyanin") the collar complex of the central decamer is substantially enlarged and modified. The preliminary O2 binding curves indicate that there are species-specific functional differences in the cerithioid mega-hemocyanins which might reflect different physiological

  6. IDENTIFICATION OF MICROCYSTIN TOXINS FROM A STRAIN OF MICROCYSTIS AERUGINOSA BY LIQUID CHROMATOGRAPHY INTRODUCTION INTO A HYBRID LINEAR ION TRAP-FOURIER TRANSFORM ION CYCLOTRON RESONANCE MASS SPECTROMETER

    EPA Science Inventory

    The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment i...

  7. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  8. IDENTIFICATION OF MICROCYSTIN TOXINS FROM A STRAIN OF MICROCYSTIS AERUGINOSA

    EPA Science Inventory

    Microcystin toxins are cyclic heptapeptides produced by several genera and species of cyanobacteria that are responsible for the "green scum" frequently observed on eutrophic surface waters. These toxins, which are a million times more toxic than cyanide ion, have caused deaths o...

  9. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments. PMID:9807828

  10. Association Equilibrium for Cross-Associating Chains in a Good Solvent: Crowding and Other Nonideality Effects.

    PubMed

    Gotlib, Igor Yu; Malov, Ivan K; Victorov, Alexey I; Voznesenskiy, Mikhail A

    2016-07-28

    Association equilibrium has been studied by molecular dynamics (MD) for mixtures of cross-associating molecules (n-decamer+p-dimer and n-decamer+p-decamer) in a good solvent. Each monomer of n-decamers carries an associative site (n-sticker); each molecule of the second component contains two terminal associative sites (p-stickers). To model the univalent association between the n-sticker and the p-sticker, a technique based on introduction of dummy atoms has been used. We report MD data on the effects of temperature, chain flexibility, and location of the sticker within the chain on the association equilibrium. We find that the presence of nonassociating monomer units of p-chain has a substantial effect on the association equilibrium. This effect is similar to "crowding" in reactive mixtures known to be caused by the presence of inert molecules. Widely used mean field theories of associating chains (e.g., SAFT or Semenov-Rubinstein theory) do not account for the effect of crowding caused by the inert fragments of reactive chains. We introduce simple empirical corrections for crowding that describe association equilibrium in the presence of nonassociating fragment in a chain-like molecule.

  11. Confronting JC virus and Homo sapiens biological signatures.

    PubMed

    Lucchese, Guglielmo

    2013-01-01

    The present report describes the peptide commonality between JC virus (JCV) and the human proteome at the heptamer level. In total, 53 viral heptapeptides occur in functionally important human proteins with potential consequences for host functions and JCV pathogenesis. A paradigmatic example of a crucial peptide match is the SGKTTLA sequence, shared by JCV LT antigen and human nicotinamide/nicotinic acid riboside kinase, an enzyme involved in myelination processes. In general, the JCV-versus-host heptapeptide overlap may result in a competition between viral sequences and identical motifs in host enzymic active sites, adhesive domains, regulatory signaling motifs, etc., thus interfering with essential reactions and posing disadvantages to the cell. Overall, this study provides a starting point for investigating the role of peptide commonality in host-pathogen interactions. PMID:23276955

  12. A novel self-assembling peptide with UV-responsive properties.

    PubMed

    Wei, Ran; Jin, Cheng-Cheng; Quan, Jing; Nie, Hua-li; Zhu, Li-Min

    2014-03-01

    A novel heptapeptide comprising Ile-Gln-Ser-Pro-His-Phe-Phe (IQSPHFF) identified and found to undergo self-assembly into microparticles in solution. To understand the effects of ultraviolet (UV) irradiation on the self-assembly process, IQSPHFF solutions were exposed to the UV light of 365 nm at room temperature. This exposure was found to have a profound effect on the morphology of the self-assembled aggregates, converting the microparticles to nanorod shapes. Circular dichroism and FTIR studies indicated distinct structural differences in the arrangements of the peptide moieties before and after UV irradiation. However, Mass spectrum analysis and high performance liquid chromatography of the peptide molecules before and after UV irradiation demonstrated that the chemical structure of IQSPHFF was not changed. UV-visible spectroscopy and fluorescence spectroscopy studies showed that the absorption peak both increased after UV irradiation. Overall, our data show that the heptapeptide with UV-responsive properties.

  13. Identification of a Peptide from Mammal Albumins Responsible for Enhanced Pigment Production by Group B Streptococci

    PubMed Central

    Rosa-Fraile, Manuel; Sampedro, Antonio; Varela, Javier; Garcia-Peña, Marisa; Gimenez-Gallego, Guillermo

    1999-01-01

    The peptide from peptones responsible for enhanced pigment production by Streptococcus agalactiae in culture media has been isolated from a peptic digest of human albumin and has been identified as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe. The related heptapeptide lacking the N-terminal Ile also had pigment-enhancing activity. A sequence similarity search showed that these sequences are present only in mammal albumins. PMID:10225848

  14. Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration.

    PubMed

    Chan, Lai Yue; Craik, David J; Daly, Norelle L

    2015-01-01

    Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics. PMID:26464514

  15. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: A conserved structure with an essential function

    SciTech Connect

    Allison, L.A.; Wong, J.K.C.; Fitzpatrick, V.D.; Moyle, M.; Ingles, C.J.

    1988-01-01

    Using DNA encoding the largest subunit of Drosophila melanogaster RNA polymerase II, the authors isolated the homologous hamster RPO21 gene. Nucleotide sequencing of both the hamster and D. melanogaster RPO21 DNAs confirmed that the RPO21 polypeptides of these two species, like the Saccharomyces cerevisiae RPO21 polypeptide, contain both an N-terminal region homologous to the Escherichia coli RNA polymerase subunit ..beta..' and a unique polymerase II-specific C-terminal domain. This C-terminal domain, encoded by separate exons in the D. melanogaster and hamster genes, consists of a tandemly repeated heptapeptide sequence. By constructing a series of deletions in DNA encoding the 26 heptapeptide repeats normally present in the S. cerevisiae RPO21 polypeptide, the authors have established that a minimum of between 9 and 11 repeats is necessary for RPO21 function in yeast cells. Replacement of the yeast RPO21 heptapeptide repeats by the longer hamster repetitive domain resulted in viable yeast cells with no detectable mutant phenotype, while a similar replacement of the yeast repeats by the more divergent D. melanogaster repeats was a recessive lethal mutation. The authors suggest that this novel repetitive domain is essential for proper initiation of transcription by RNA polymerase II and that it may mediate the functions of TATA boxes, upstream activating sequences, and enhancers.

  16. Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration.

    PubMed

    Chan, Lai Yue; Craik, David J; Daly, Norelle L

    2015-10-13

    Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics.

  17. Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration

    PubMed Central

    Chan, Lai Yue; Craik, David J.; Daly, Norelle L.

    2015-01-01

    Tumour formation is dependent on nutrient and oxygen supply from adjacent blood vessels. Angiogenesis inhibitors can play a vital role in controlling blood vessel formation and consequently tumour progression by inhibiting endothelial cell proliferation, sprouting and migration. The primary aim of the present study was to design cyclic thrombospondin-1 (TSP-1) mimetics using disulfide-rich frameworks for anti-angiogenesis therapies and to determine whether these peptides have better potency than the linear parent peptide. A short anti-angiogenic heptapeptide fragment from TSP-1 (GVITRIR) was incorporated into two cyclic disulfide-rich frameworks, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) and SFTI-1 (sunflower trypsin inhibitor-1). The cyclic peptides were chemically synthesized and folded in oxidation buffers, before being tested in a series of in vitro evaluations. Incorporation of the bioactive heptapeptide fragment into the cyclic frameworks resulted in peptides that inhibited microvascular endothelial cell migration, and had no toxicity against normal primary human endothelial cells or cancer cells. Importantly, all of the designed cyclic TSP-1 mimetics were far more stable than the linear heptapeptide in human serum. The present study has demonstrated a novel approach to stabilize the active region of TSP-1. The anti-angiogenic activity of the native TSP-1 active fragment was maintained in the new TSP-1 mimetics and the results provide a new chemical approach for the design of TSP-1 mimetics. PMID:26464514

  18. Propensities of peptides containing the Asn-Gly segment to form β-turn and β-hairpin structures.

    PubMed

    Kang, Young Kee; Yoo, In Kee

    2016-09-01

    The propensities of peptides that contain the Asn-Gly segment to form β-turn and β-hairpin structures were explored using the density functional methods and the implicit solvation model in CH2 Cl2 and water. The populations of preferred β-turn structures varied depending on the sequence and solvent polarity. In solution, β-hairpin structures with βI' turn motifs were most preferred for the heptapeptides containing the Asn-Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X-ray structures. The sequence, H-bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β-hairpin structure of each heptapeptide, although the β-turn segments played a role in promoting the formation of β-hairpin structures and the β-hairpin propensity varied. In the heptapeptides containing the Asn-Gly segment, the β-hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β-turns and β-hairpins containing the Asn-Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β-hairpin mimics and the design of binding epitopes for protein-protein and protein-nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653-664, 2016.

  19. Short peptides allowing preferential detection of Candida albicans hyphae.

    PubMed

    Kaba, Hani E J; Pölderl, Antonia; Bilitewski, Ursula

    2015-09-01

    Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

  20. Toyz: A framework for scientific analysis of large datasets and astronomical images

    NASA Astrophysics Data System (ADS)

    Moolekamp, F.; Mamajek, E.

    2015-11-01

    As the size of images and data products derived from astronomical data continues to increase, new tools are needed to visualize and interact with that data in a meaningful way. Motivated by our own astronomical images taken with the Dark Energy Camera (DECam) we present Toyz, an open source Python package for viewing and analyzing images and data stored on a remote server or cluster. Users connect to the Toyz web application via a web browser, making it ​a convenient tool for students to visualize and interact with astronomical data without having to install any software on their local machines. In addition it provides researchers with an easy-to-use tool that allows them to browse the files on a server and quickly view very large images (>2 Gb) taken with DECam and other cameras with a large FOV and create their own visualization tools that can be added on as extensions to the default Toyz framework.

  1. Dark Energy Camera for Blanco

    SciTech Connect

    Binder, Gary A.; /Caltech /SLAC

    2010-08-25

    In order to make accurate measurements of dark energy, a system is needed to monitor the focus and alignment of the Dark Energy Camera (DECam) to be located on the Blanco 4m Telescope for the upcoming Dark Energy Survey. One new approach under development is to fit out-of-focus star images to a point spread function from which information about the focus and tilt of the camera can be obtained. As a first test of a new algorithm using this idea, simulated star images produced from a model of DECam in the optics software Zemax were fitted. Then, real images from the Mosaic II imager currently installed on the Blanco telescope were used to investigate the algorithm's capabilities. A number of problems with the algorithm were found, and more work is needed to understand its limitations and improve its capabilities so it can reliably predict camera alignment and focus.

  2. Mass and galaxy distributions of four massive galaxy clusters from Dark Energy Survey Science Verification data

    DOE PAGES

    Melchior, P.; Suchyta, E.; Huff, E.; Hirsch, M.; Kacprzak, T.; Rykoff, E.; Gruen, D.; Armstrong, R.; Bacon, D.; Bechtol, K.; et al

    2015-03-31

    We measure the weak-lensing masses and galaxy distributions of four massive galaxy clusters observed during the Science Verification phase of the Dark Energy Survey. This pathfinder study is meant to 1) validate the DECam imager for the task of measuring weak-lensing shapes, and 2) utilize DECam's large field of view to map out the clusters and their environments over 90 arcmin. We conduct a series of rigorous tests on astrometry, photometry, image quality, PSF modelling, and shear measurement accuracy to single out flaws in the data and also to identify the optimal data processing steps and parameters. We find Sciencemore » Verification data from DECam to be suitable for the lensing analysis described in this paper. The PSF is generally well-behaved, but the modelling is rendered difficult by a flux-dependent PSF width and ellipticity. We employ photometric redshifts to distinguish between foreground and background galaxies, and a red-sequence cluster finder to provide cluster richness estimates and cluster-galaxy distributions. By fitting NFW profiles to the clusters in this study, we determine weak-lensing masses that are in agreement with previous work. For Abell 3261, we provide the first estimates of redshift, weak-lensing mass, and richness. Additionally, the cluster-galaxy distributions indicate the presence of filamentary structures attached to 1E 0657-56 and RXC J2248.7-4431, stretching out as far as 1degree (approximately 20 Mpc), showcasing the potential of DECam and DES for detailed studies of degree-scale features on the sky.« less

  3. Mass and galaxy distributions of four massive galaxy clusters from Dark Energy Survey Science Verification data

    SciTech Connect

    Melchior, P.; et al.

    2015-05-21

    We measure the weak-lensing masses and galaxy distributions of four massive galaxy clusters observed during the Science Verification phase of the Dark Energy Survey. This pathfinder study is meant to 1) validate the DECam imager for the task of measuring weak-lensing shapes, and 2) utilize DECam's large field of view to map out the clusters and their environments over 90 arcmin. We conduct a series of rigorous tests on astrometry, photometry, image quality, PSF modeling, and shear measurement accuracy to single out flaws in the data and also to identify the optimal data processing steps and parameters. We find Science Verification data from DECam to be suitable for the lensing analysis described in this paper. The PSF is generally well-behaved, but the modeling is rendered difficult by a flux-dependent PSF width and ellipticity. We employ photometric redshifts to distinguish between foreground and background galaxies, and a red-sequence cluster finder to provide cluster richness estimates and cluster-galaxy distributions. By fitting NFW profiles to the clusters in this study, we determine weak-lensing masses that are in agreement with previous work. For Abell 3261, we provide the first estimates of redshift, weak-lensing mass, and richness. In addition, the cluster-galaxy distributions indicate the presence of filamentary structures attached to 1E 0657-56 and RXC J2248.7-4431, stretching out as far as 1 degree (approximately 20 Mpc), showcasing the potential of DECam and DES for detailed studies of degree-scale features on the sky.

  4. Mass and galaxy distributions of four massive galaxy clusters from Dark Energy Survey Science Verification data

    SciTech Connect

    Melchior, P.; Suchyta, E.; Huff, E.; Hirsch, M.; Kacprzak, T.; Rykoff, E.; Gruen, D.; Armstrong, R.; Bacon, D.; Bechtol, K.; Bernstein, G. M.; Bridle, S.; Clampitt, J.; Honscheid, K.; Jain, B.; Jouvel, S.; Krause, E.; Lin, H.; MacCrann, N.; Patton, K.; Plazas, A.; Rowe, B.; Vikram, V.; Wilcox, H.; Young, J.; Zuntz, J.; Abbott, T.; Abdalla, F. B.; Allam, S. S.; Banerji, M.; Bernstein, J. P.; Bernstein, R. A.; Bertin, E.; Buckley-Geer, E.; Burke, D. L.; Castander, F. J.; da Costa, L. N.; Cunha, C. E.; Depoy, D. L.; Desai, S.; Diehl, H. T.; Doel, P.; Estrada, J.; Evrard, A. E.; Neto, A. F.; Fernandez, E.; Finley, D. A.; Flaugher, B.; Frieman, J. A.; Gaztanaga, E.; Gerdes, D.; Gruendl, R. A.; Gutierrez, G. R.; Jarvis, M.; Karliner, I.; Kent, S.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Maia, M. A. G.; Makler, M.; Marriner, J.; Marshall, J. L.; Merritt, K. W.; Miller, C. J.; Miquel, R.; Mohr, J.; Neilsen, E.; Nichol, R. C.; Nord, B. D.; Reil, K.; Roe, N. A.; Roodman, A.; Sako, M.; Sanchez, E.; Santiago, B. X.; Schindler, R.; Schubnell, M.; Sevilla-Noarbe, I.; Sheldon, E.; Smith, C.; Soares-Santos, M.; Swanson, M. E. C.; Sypniewski, A. J.; Tarle, G.; Thaler, J.; Thomas, D.; Tucker, D. L.; Walker, A.; Wechsler, R.; Weller, J.; Wester, W.

    2015-03-31

    We measure the weak-lensing masses and galaxy distributions of four massive galaxy clusters observed during the Science Verification phase of the Dark Energy Survey. This pathfinder study is meant to 1) validate the DECam imager for the task of measuring weak-lensing shapes, and 2) utilize DECam's large field of view to map out the clusters and their environments over 90 arcmin. We conduct a series of rigorous tests on astrometry, photometry, image quality, PSF modelling, and shear measurement accuracy to single out flaws in the data and also to identify the optimal data processing steps and parameters. We find Science Verification data from DECam to be suitable for the lensing analysis described in this paper. The PSF is generally well-behaved, but the modelling is rendered difficult by a flux-dependent PSF width and ellipticity. We employ photometric redshifts to distinguish between foreground and background galaxies, and a red-sequence cluster finder to provide cluster richness estimates and cluster-galaxy distributions. By fitting NFW profiles to the clusters in this study, we determine weak-lensing masses that are in agreement with previous work. For Abell 3261, we provide the first estimates of redshift, weak-lensing mass, and richness. Additionally, the cluster-galaxy distributions indicate the presence of filamentary structures attached to 1E 0657-56 and RXC J2248.7-4431, stretching out as far as 1degree (approximately 20 Mpc), showcasing the potential of DECam and DES for detailed studies of degree-scale features on the sky.

  5. Measurements of charge diffusion in deep-depletion CCDs by optical diffraction

    SciTech Connect

    Cease, H.; Diehl, H.T.; Estrada, J.; Flaugher, B.; Scarpine, V.; /Fermilab

    2007-10-01

    The charge diffusion is measured in back illuminated, fully depleted, 250 {micro}m thick CCDs by imaging the diffraction pattern of a double slit. The CCDs studied are the focal plane detectors for the Dark Energy Camera (DECam) instrument currently under construction for the Dark Energy Survey (DES). The results presented here indicate that the dispersion of charge due to diffusion can be kept below the DES specification ({sigma}{sub d} < 7.0 {micro}m).

  6. Physical investigations of the hemocyanin of the chiton, Cryptochiton stelleri (Middendorff).

    PubMed

    Herskovits, T T; Hamilton, M G

    1987-01-01

    The hemocyanin of the giant Pacific chiton, Cryptochiton stelleri has a molecular weight of 4.2 +/- 0.3 X 10(6), determined by light-scattering, and a sedimentation coefficient of 60S. The fully dissociated subunits in nondenaturing solvents, at pH 10.6, 1 X 10(-2)M EDTA and in 8.0 M urea, pH 7.4 have molecular weights of 4.10 X 10(5) and 4.35 X 10(5), close to one-tenth of the molecular mass of the parent hemocyanin decamers. In the pH region from about 3.5 to 11 the molecular weight (Mw), determined at constant protein concentration of 0.10 g1(-1) exhibits a bell-shaped molecular weight profile centering about the physiological pH of the hemolymph of 7.2. The pH-Mw profile is best accounted for in terms of a three state, decamer-dimer-monomer dissociation scheme. Analysis of the Mg2+ and Ca2+ effects on the molecular weight transitions suggest stabilization of the hemocyanin decamers through one bound divalent ion per hemocyanin monomer or dimer. Urea, GdmCl, and the higher members of the chaotropic salt series are effective dissociating agents for Cryptochiton stelleri hemocyanin. The dissociation profile obtained with urea at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+ has been analyzed in terms of both the two- and three-species schemes of subunit-dissociation. Hydrophobic stabilization of the subunit contacts is suggested by the large number of apparent amino acid groups (Napp), of the order of 30 between dimers stabilizing the decamers, and 120 apparent amino acid groups between each monomer forming the constituent dimers.

  7. Direct Observation of a Cytosine Analogue that Forms Five Hydrogen Bonds to Guanosine: Guanyl G-Clamp

    SciTech Connect

    Wilds, C.J.; Maier, M.A.; Tereshko, V.; Manoharan, M.; Egli, M.

    2010-03-08

    A novel heterocyclic base modification, the guanidino G-clamp, is designed to allow two Hoogsteen-type hydrogen bonds to form between the amino and imino nitrogen atoms of a tethered guanidinium group to O6 and N7 of guanosine, which results in a total of five hydrogen bonds (broken lines, see picture). Details of a crystal structure at 1.0-{angstrom} resolution of a modified DNA decamer containing this guanidino G-clamp analogue demonstrate its mechanism of binding.

  8. Structural characterization of d(CAACCCGTTG) and d(CAACGGGTTG) mini-hairpin loops by heteronuclear NMR: the effects of purines versus pyrimidines in DNA hairpins.

    PubMed Central

    Avizonis, D Z; Kearns, D R

    1995-01-01

    The DNA decamers, d(CAACCCGTTG) and d(CAACGGGTTG) were studied in solution by proton and heteronuclear NMR. Under appropriate conditions of pH, temperature, salt concentration and DNA concentration, both decamers form hairpin conformations with similar stabilities [Avizonis and Kearns (1995) Biopolymers, 35, 187-200]. Both decamers adopt mini-hairpin loops, where the first and last four nucleotides are involved in Watson-Crick hydrogen bonding and the central two nucleotides, CC or GG respectively, form the loop. Through the use of proton-proton, proton-phosphorus and natural abundance proton-carbon NMR experiments, backbone torsion angles (beta, gamma and epsilon), sugar puckers and interproton distances were measured. The nucleotides forming the loops of these decamers were found to stack upon one another in an L1 type of loop conformation. Both show gamma tr and unusual beta torsion angles in the loop-closing nucleotide G7, as expected for mini-hairpin loop formation. Our results indicate that the beta and epsilon torsion angles of the fifth and sixth nucleotides that form the loop and the loop-closing nucleotide G7 are not in the standard trans conformation as found in B-DNA. Although the loop structures calculated from NMR-derived constraints are not well defined, the stacking of the bases in the two different hairpins is different. This difference in the base stacking of the loop may provide an explanation as to why the cytosine-containing hairpin is thermodynamically more stable than the guanine-containing hairpin. PMID:7739905

  9. Presentation of Ligands on Hydroxylapatite

    NASA Technical Reports Server (NTRS)

    Chu, Barbara C. F.; Orgel, Leslie E.

    1997-01-01

    Conjugates of biotin with the decamer of glutamic acid (glu(sub 10)) and the trimer of D,L-2-amino-5-phosphonovaleric acid (I) have been synthesized, and it has been shown that they mediate the binding of avidin to hydroxylapatite. In a similar way a conjugate of methotrexate with glu(sub 10) mediates the binding of dihydrofolate reductase to the mineral. The presentation of ligands on the hydroxylapatite component of bone may find applications in clinical medicine.

  10. Surface cleaning of CCD imagers using an electrostatic dissipative formulation of First Contact polymer

    NASA Astrophysics Data System (ADS)

    Derylo, G.; Estrada, J.; Flaugher, B.; Hamilton, J.; Kubik, D.; Kuk, K.; Scarpine, V.

    2008-07-01

    We describe the results obtained cleaning the surface of DECam CCD detectors with a new electrostatic dissipative formulation of First ContactTM polymer from Photonic Cleaning Technologies. We demonstrate that cleaning with this new product is possible without ESD damage to the sensors and without degradation of the antireflective coating used to optimize the optical performance of the detector. We show that First ContactTM is more effective for cleaning a CCD than the commonly used acetone swab.

  11. Evolution of molluscan hemocyanin structures.

    PubMed

    Markl, Jürgen

    2013-09-01

    Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed of many protein subunits that in turn encompass subsets of distinct functional units. The structure and evolution of molluscan hemocyanin have been studied for decades, but it required the recent progress in DNA sequencing, X-ray crystallography and 3D electron microscopy to produce a detailed view of their structure and evolution. The basic quaternary structure is a cylindrical decamer 35nm in diameter, consisting of wall and collar (typically at one end of the cylinder). Depending on the animal species, decamers, didecamers and multidecamers occur in the hemolymph. Whereas the wall architecture of the decamer seems to be invariant, four different types of collar have been identified in different molluscan taxa. Correspondingly, there exist four subunit types that differ in their collar functional units and range from 350 to 550kDa. Thus, molluscan hemocyanin subunits are among the largest polypeptides in nature. In this report, recent 3D reconstructions are used to explain and visualize the different functional units, subunits and quaternary structures of molluscan hemocyanins. Moreover, on the basis of DNA analyses and structural considerations, their possible evolution is traced. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. PMID:23454609

  12. On the quaternary structure of a C-type lectin from Bothrops jararacussu venom--BJ-32 (BjcuL).

    PubMed

    Silva, F P; Alexandre, G M C; Ramos, C H I; De-Simone, S G

    2008-12-15

    BJ-32 (also known as BjcuL) is a C-type lectin from the venom of Bothrops jararacussu with specificity for beta-galactosides and a remarkable ability to agglutinate several species of trypanosomatids. Our objective was to study the oligomerization state of native BJ-32 by using different biophysical and computational methods. Small-angle X-ray light scattering (SAXS) experiments disclosed a compact, globular protein with a radius of gyration of 36.72+/-0.04A and molecular weight calculated as 147.5+/-2.0kDa. From analytical ultracentrifugation analysis, it was determined that the BJ-32 sedimentation profile fits nicely to a decamer model. The analysis of the intrinsic emitted fluorescence spectra for BJ-32 solutions indicated that association of subunits in the decamer is accompanied by changes in the environment of Tryptophan residues. Both ab initio and comparative models of BJ-32 supported the resemblance of the decamer in the crystallographic structure from a close homologue, the rattlesnake venom lectin (RSL) from Crotalus atrox. PMID:18948130

  13. The structure of a fibril-forming sequence, NNQQNY, in the context of a globular fold

    PubMed Central

    Guo, Zhefeng; Eisenberg, David

    2008-01-01

    Numerous human disorders are associated with the formation of protein fibrils. The fibril-forming capacity of a protein has been found in recent studies to be determined by a short segment of residues that forms a dual β-sheet, called a steric zipper, in the spine of the fibril. The question arises as to whether a fibril-forming segment, when inserted within the sequence of a globular protein, will invariably cause the protein to form fibrils. Here we investigate this question by inserting the known fibril-forming segment NNQQNY into the globular enzyme, T7 endonuclease I. From earlier studies, we know that in its fibril form, NNQQNY is in an extended conformation. We first found that the inserted NNQQNY stimulates fibril formation of T7 endonuclease I in solution. Thus NNQQNY within T7 endonuclease I can exist in an extended conformation, capable of forming the steric zipper in the core of a fibril. We also found that T7 endonuclease I folds into a decamer that does not form fibrils. We determined the structure of the decamer by X-ray crystallography, finding an unusual oligomer without point group symmetry, and finding that the NNQQNY segments within the decamer adopt two twisted conformations, neither is apparently able to fibrillize. We conclude that twisting of fibril forming sequences from the fully extended conformation, imposed by the context of their placement in proteins, can interfere with fibril formation. PMID:18552127

  14. A dark energy camera search for an optical counterpart to the first advanced LIGO gravitational wave event GW150914

    DOE PAGES

    Soares-Santos, M.

    2016-05-27

    We report the results of a deep search for an optical counterpart to the gravitational wave (GW) event GW150914, the first trigger from the Advanced LIGO GW detectors. We used the Dark Energy Camera (DECam) to image a 102 deg2 area, corresponding to 38% of the initial trigger high-probability sky region and to 11% of the revised high-probability region. We observed in the i and z bands at 4–5, 7, and 24 days after the trigger. The median 5σ point-source limiting magnitudes of our search images are i = 22.5 and z = 21.8 mag. We processed the images throughmore » a difference-imaging pipeline using templates from pre-existing Dark Energy Survey data and publicly available DECam data. Due to missing template observations and other losses, our effective search area subtends 40 deg2, corresponding to a 12% total probability in the initial map and 3% in the final map. In this area, we search for objects that decline significantly between days 4–5 and day 7, and are undetectable by day 24, finding none to typical magnitude limits of i = 21.5, 21.1, 20.1 for object colors (i – z) = 1, 0, –1, respectively. Lastly, our search demonstrates the feasibility of a dedicated search program with DECam and bodes well for future research in this emerging field.« less

  15. A Dark Energy Camera Search for an Optical Counterpart to the First Advanced LIGO Gravitational Wave Event GW150914

    NASA Astrophysics Data System (ADS)

    Soares-Santos, M.; Kessler, R.; Berger, E.; Annis, J.; Brout, D.; Buckley-Geer, E.; Chen, H.; Cowperthwaite, P. S.; Diehl, H. T.; Doctor, Z.; Drlica-Wagner, A.; Farr, B.; Finley, D. A.; Flaugher, B.; Foley, R. J.; Frieman, J.; Gruendl, R. A.; Herner, K.; Holz, D.; Lin, H.; Marriner, J.; Neilsen, E.; Rest, A.; Sako, M.; Scolnic, D.; Sobreira, F.; Walker, A. R.; Wester, W.; Yanny, B.; Abbott, T. M. C.; Abdalla, F. B.; Allam, S.; Armstrong, R.; Banerji, M.; Benoit-Lévy, A.; Bernstein, R. A.; Bertin, E.; Brown, D. A.; Burke, D. L.; Capozzi, D.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Castander, F. J.; Cenko, S. B.; Chornock, R.; Crocce, M.; D’Andrea, C. B.; da Costa, L. N.; Desai, S.; Dietrich, J. P.; Drout, M. R.; Eifler, T. F.; Estrada, J.; Evrard, A. E.; Fairhurst, S.; Fernandez, E.; Fischer, J.; Fong, W.; Fosalba, P.; Fox, D. B.; Fryer, C. L.; Garcia-Bellido, J.; Gaztanaga, E.; Gerdes, D. W.; Goldstein, D. A.; Gruen, D.; Gutierrez, G.; Honscheid, K.; James, D. J.; Karliner, I.; Kasen, D.; Kent, S.; Kuropatkin, N.; Kuehn, K.; Lahav, O.; Li, T. S.; Lima, M.; Maia, M. A. G.; Margutti, R.; Martini, P.; Matheson, T.; McMahon, R. G.; Metzger, B. D.; Miller, C. J.; Miquel, R.; Mohr, J. J.; Nichol, R. C.; Nord, B.; Ogando, R.; Peoples, J.; Plazas, A. A.; Quataert, E.; Romer, A. K.; Roodman, A.; Rykoff, E. S.; Sanchez, E.; Scarpine, V.; Schindler, R.; Schubnell, M.; Sevilla-Noarbe, I.; Sheldon, E.; Smith, M.; Smith, N.; Smith, R. C.; Stebbins, A.; Sutton, P. J.; Swanson, M. E. C.; Tarle, G.; Thaler, J.; Thomas, R. C.; Tucker, D. L.; Vikram, V.; Wechsler, R. H.; Weller, J.; The DES Collaboration

    2016-06-01

    We report the results of a deep search for an optical counterpart to the gravitational wave (GW) event GW150914, the first trigger from the Advanced LIGO GW detectors. We used the Dark Energy Camera (DECam) to image a 102 deg2 area, corresponding to 38% of the initial trigger high-probability sky region and to 11% of the revised high-probability region. We observed in the i and z bands at 4–5, 7, and 24 days after the trigger. The median 5σ point-source limiting magnitudes of our search images are i = 22.5 and z = 21.8 mag. We processed the images through a difference-imaging pipeline using templates from pre-existing Dark Energy Survey data and publicly available DECam data. Due to missing template observations and other losses, our effective search area subtends 40 deg2, corresponding to a 12% total probability in the initial map and 3% in the final map. In this area, we search for objects that decline significantly between days 4–5 and day 7, and are undetectable by day 24, finding none to typical magnitude limits of i = 21.5, 21.1, 20.1 for object colors (i ‑ z) = 1, 0, ‑1, respectively. Our search demonstrates the feasibility of a dedicated search program with DECam and bodes well for future research in this emerging field.

  16. The Dark Energy Survey instrument design

    SciTech Connect

    Flaugher, B.; /Fermilab

    2006-05-01

    We describe a new project, the Dark Energy Survey (DES), aimed at measuring the dark energy equation of state parameter, w, to a statistical precision of {approx}5%, with four complementary techniques. The survey will use a new 3 sq. deg. mosaic camera (DECam) mounted at the prime focus of the Blanco 4m telescope at the Cerro-Tololo International Observatory (CTIO). DECam includes a large mosaic camera, a five element optical corrector, four filters (g,r,i,z), and the associated infrastructure for operation in the prime focus cage. The focal plane consists of 62 2K x 4K CCD modules (0.27''/pixel) arranged in a hexagon inscribed within the 2.2 deg. diameter field of view. We plan to use the 250 micron thick fully-depleted CCDs that have been developed at the Lawrence Berkeley National Laboratory (LBNL). At Fermilab, we will establish a packaging factory to produce four-side buttable modules for the LBNL devices, as well as to test and grade the CCDs. R&D is underway and delivery of DECam to CTIO is scheduled for 2009.

  17. Formation of intersubunit disulfide bonds and properties of the single histidine and cysteine residues in each subunit relative to the decameric structure of cyanase.

    PubMed

    Anderson, P M; Korte, J J; Holcomb, T A; Cho, Y G; Son, C M; Sung, Y C

    1994-05-27

    Reaction of the single cysteine residue in each subunit of cyanase with certain SH reagents gives an active decameric derivative that dissociates reversibly to an inactive dimer derivative (Anderson, P. M., Johnson, W. V., Korte, J. J., Xiong, X., Sung, Y.-c., and Fuchs, J. A. (1988) J. Biol. Chem. 263, 5674-5680). Reaction of mixed disulfide dimer derivatives of cyanase with dithiothreitol at 0 degree C results in formation of a disulfide bond between the subunits in the dimer. The disulfide dimer was inactive and did not associate to a decamer; the intersubunit disulfide bond could not be formed when the dimers were associated as a decamer. The two SH groups apparently are in close proximity to each other in the dissociated dimer but not when the dimer is associated to a decamer. Substitution of glycine for the cysteine residue or of tyrosine, asparagine, glycine, valine, or leucine for the single histidine residue in each subunit gave mutant enzymes that were active. However, H113N, H113Y, and C83G were unstable at low temperature and/or ionic strength, dissociating reversibly to an inactive dimer. Efficient reassociation required the presence of bicarbonate or cyanate analog. The results are consistent with a proposed single site per subunit model explaining apparent half-site binding of substrates and the requirement of decameric structure for activity.

  18. Evolution of molluscan hemocyanin structures.

    PubMed

    Markl, Jürgen

    2013-09-01

    Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed of many protein subunits that in turn encompass subsets of distinct functional units. The structure and evolution of molluscan hemocyanin have been studied for decades, but it required the recent progress in DNA sequencing, X-ray crystallography and 3D electron microscopy to produce a detailed view of their structure and evolution. The basic quaternary structure is a cylindrical decamer 35nm in diameter, consisting of wall and collar (typically at one end of the cylinder). Depending on the animal species, decamers, didecamers and multidecamers occur in the hemolymph. Whereas the wall architecture of the decamer seems to be invariant, four different types of collar have been identified in different molluscan taxa. Correspondingly, there exist four subunit types that differ in their collar functional units and range from 350 to 550kDa. Thus, molluscan hemocyanin subunits are among the largest polypeptides in nature. In this report, recent 3D reconstructions are used to explain and visualize the different functional units, subunits and quaternary structures of molluscan hemocyanins. Moreover, on the basis of DNA analyses and structural considerations, their possible evolution is traced. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

  19. Embrace the Dark Side: Advancing the Dark Energy Survey

    NASA Astrophysics Data System (ADS)

    Suchyta, Eric

    The Dark Energy Survey (DES) is an ongoing cosmological survey intended to study the properties of the accelerated expansion of the Universe. In this dissertation, I present work of mine that has advanced the progress of DES. First is an introduction, which explores the physics of the cosmos, as well as how DES intends to probe it. Attention is given to developing the theoretical framework cosmologists use to describe the Universe, and to explaining observational evidence which has furnished our current conception of the cosmos. Emphasis is placed on the dark sector - dark matter and dark energy - the content of the Universe not explained by the Standard Model of particle physics. As its name suggests, the Dark Energy Survey has been specially designed to measure the properties of dark energy. DES will use a combination of galaxy cluster, weak gravitational lensing, angular clustering, and supernovae measurements to derive its state of the art constraints, each of which is discussed in the text. The work described in this dissertation includes science measurements directly related to the first three of these probes. The dissertation presents my contributions to the readout and control system of the Dark Energy Camera (DECam); the name of this software is SISPI. SISPI uses client-server and publish-subscribe communication patterns to coordinate and command actions among the many hardware components of DECam - the survey instrument for DES, a 570 megapixel CCD camera, mounted at prime focus of the Blanco 4-m Telescope. The SISPI work I discuss includes coding applications for DECam's filter changer mechanism and hexapod, as well as developing the Scripts Editor, a GUI application for DECam users to edit and export observing sequence SISPI can load and execute. Next, the dissertation describes the processing of early DES data, which I contributed. This furnished the data products used in the first-completed DES science analysis, and contributed to improving the

  20. The upstream activator CTF/NF1 and RNA polymerase II share a common element involved in transcriptional activation.

    PubMed Central

    Xiao, H; Lis, J T; Xiao, H; Greenblatt, J; Friesen, J D

    1994-01-01

    The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of a heptapeptide with the consensus YSPTSPS. It has been shown that the heptapeptide repeat interacts directly with the general transcription factor TFIID. We report here that the CTD activates transcription when fused to the DNA-binding domain of GAL4. More importantly, we find that the proline-rich transcriptional activation domain of the CCAAT-box-binding factor CTF/NF1 contains a sequence with striking similarity to the heptapeptide repeats of the CTD. We show that this CTD-like motif is essential for the transcriptional activator function of the proline-rich domain of CTF/NF1. Deletion of and point mutations in this CTD-like motif abolish the transcriptional activator function of the proline-rich domain, while natural CTD repeats from RNA polymerase II are fully functional in place of the CTD-like motif. We further show that the proline-rich activation domain of CTF/NF1 interacts directly with the TATA-box-binding protein (TBP), and that a mutation in the CTD-like motif that abolishes transcriptional activation reduces the affinity of the proline-rich domain for TBP. These results demonstrate that a class of proline-rich activator proteins and RNA polymerase II possess a common structural and functional component which can interact with the same target in the general transcription machinery. We discuss the implications of these results for the mechanisms of transcriptional activation in eucaryotes. Images PMID:8029001

  1. Angiotensin-(1–7) attenuates angiotensin II-induced cardiac remodeling associated with upregulation of dual-specificity phosphatase 1

    PubMed Central

    McCollum, LaTronya T.; Gallagher, Patricia E.

    2012-01-01

    Chronic hypertension induces cardiac remodeling, including left ventricular hypertrophy and fibrosis, through a combination of both hemodynamic and humoral factors. In previous studies, we showed that the heptapeptide ANG-(1–7) prevented mitogen-stimulated growth of cardiac myocytes in vitro, through a reduction in the activity of the MAPKs ERK1 and ERK2. In this study, saline- or ANG II-infused rats were treated with ANG-(1–7) to determine whether the heptapeptide reduces myocyte hypertrophy in vivo and to identify the signaling pathways involved in the process. ANG II infusion into normotensive rats elevated systolic blood pressure >50 mmHg, in association with increased myocyte cross-sectional area, ventricular atrial natriuretic peptide mRNA, and ventricular brain natriuretric peptide mRNA. Although infusion with ANG-(1–7) had no effect on the ANG II-stimulated elevation in blood pressure, the heptapeptide hormone significantly reduced the ANG II-mediated increase in myocyte cross-sectional area, interstitial fibrosis, and natriuretic peptide mRNAs. ANG II increased phospho-ERK1 and phospho-ERK2, whereas cotreatment with ANG-(1–7) reduced the phosphorylation of both MAPKs. Neither ANG II nor ANG-(1–7) altered the ERK1/2 MAPK kinase MEK1/2. However, ANG-(1–7) infusion, with or without ANG II, increased the MAPK phosphatase dual-specificity phosphatase (DUSP)-1; in contrast, treatment with ANG II had no effect on DUSP-1, suggesting that ANG-(1–7) upregulates DUSP-1 to reduce ANG II-stimulated ERK activation. These results indicate that ANG-(1–7) attenuates cardiac remodeling associated with a chronic elevation in blood pressure and upregulation of a MAPK phosphatase and may be cardioprotective in patients with hypertension. PMID:22140049

  2. Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis

    PubMed Central

    Nocek, Boguslaw; Tikhonov, Anton; Babnigg, Gyorgy; Gu, Minyi; Zhou, Min; Makarova, Kira S.; Vondenhoff, Gaston; Van Aerschot, Arthur; Kwon, Keehwan; Anderson, Wayne F.; Severinov, Konstantin; Joachimiak, Andrzej

    2012-01-01

    Microcin C (McC) is heptapeptide-adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine-peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its AMP-complex reveal specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance. PMID:22516613

  3. A novel Fmoc-based anchorage for the synthesis of protected peptides on solid phase.

    PubMed

    Liu, Y Z; Ding, S H; Chu, J Y; Felix, A M

    1990-02-01

    A novel bifunctional compound, 9-(hydroxymethyl)-2-fluoreneacetic acid, was synthesized, coupled to benzhydrylamine-resin, and evaluated for its application to the solid phase synthesis of protected peptide fragments. Anchor-bond cleavage was achieved with 15% piperidine/DMF. A protected heptapeptide, Boc-Val-Val-Ser(Bzl)-His(Tos)-Phe-Asn-Lys-(Z)-OH, corresponding to the sequence (1-7) of rat-transforming growth factor-alpha, was synthesized using this new support with an overall yield of 46%.

  4. Unguisin F, a new cyclic peptide from the endophytic fungus Mucor irregularis.

    PubMed

    Akone, Sergi H; Daletos, Georgios; Lin, Wenhan; Proksch, Peter

    2016-01-01

    The new cyclic heptapeptide unguisin F (1) and the known congener unguisin E (2), were obtained from the endophytic fungus Mucor irregularis, isolated from the medicinal plant Moringa stenopetala, collected in Cameroon. The structure of the new compound was unambiguously determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configuration of the amino acid residues of 1 and 2 was determined using Marfey's analysis. Compounds 1 and 2 were evaluated for their antibacterial and antifungal potential, but failed to display significant activities. PMID:26812868

  5. Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method

    NASA Astrophysics Data System (ADS)

    Adamcik, Jozef; Lara, Cecile; Usov, Ivan; Jeong, Jae Sun; Ruggeri, Francesco S.; Dietler, Giovanni; Lashuel, Hilal A.; Hamley, Ian W.; Mezzenga, Raffaele

    2012-07-01

    We report the investigation of the mechanical properties of different types of amyloid fibrils by the peak force quantitative nanomechanical (PF-QNM) technique. We demonstrate that this technique correctly measures the Young's modulus independent of the polymorphic state and the cross-sectional structural details of the fibrils, and we show that values for amyloid fibrils assembled from heptapeptides, α-synuclein, Aβ(1-42), insulin, β-lactoglobulin, lysozyme, ovalbumin, Tau protein and bovine serum albumin all fall in the range of 2-4 GPa.

  6. Reniochalistatins A-E, cyclic peptides from the marine sponge Reniochalina stalagmitis.

    PubMed

    Zhan, Kai-Xuan; Jiao, Wei-Hua; Yang, Fan; Li, Jing; Wang, Shu-Ping; Li, Yu-Shan; Han, Bing-Nan; Lin, Hou-Wen

    2014-12-26

    Five new cyclic peptides (including four heptapeptides and one octapeptide), reniochalistatins A-E (1-5), were isolated and characterized from the marine sponge Reniochalina stalagmitis collected off Yongxing Island in the South China Sea. Their structures were assigned on the basis of HRESIMS, 1D and 2D NMR spectroscopic data, and MALDI-TOF/TOF data for sequence analysis. The absolute configurations of all of the amino acid residues were determined using chiral-phase HPLC and Marfey's analysis. The cyclic octapeptide reniochalistatin E showed biological activity in various cytotoxicity assays employing different tumor cell lines (RPMI-8226, MGC-803, HL-60, HepG2, and HeLa).

  7. Indexing amyloid peptide diffraction from serial femtosecond crystallography: New algorithms for sparse patterns

    DOE PAGES

    Brewster, Aaron S.; Sawaya, Michael R.; Rodriguez, Jose; Hattne, Johan; Echols, Nathaniel; McFarlane, Heather T.; Cascio, Duilio; Adams, Paul D.; Eisenberg, David S.; Sauter, Nicholas K.

    2015-01-23

    Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of theComputational Crystallography Toolbox(cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patternsmore » with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.« less

  8. Indexing amyloid peptide diffraction from serial femtosecond crystallography: New algorithms for sparse patterns

    SciTech Connect

    Brewster, Aaron S.; Sawaya, Michael R.; Rodriguez, Jose; Hattne, Johan; Echols, Nathaniel; McFarlane, Heather T.; Cascio, Duilio; Adams, Paul D.; Eisenberg, David S.; Sauter, Nicholas K.

    2015-01-23

    Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of theComputational Crystallography Toolbox(cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.

  9. Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns.

    PubMed

    Brewster, Aaron S; Sawaya, Michael R; Rodriguez, Jose; Hattne, Johan; Echols, Nathaniel; McFarlane, Heather T; Cascio, Duilio; Adams, Paul D; Eisenberg, David S; Sauter, Nicholas K

    2015-02-01

    Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox (cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.

  10. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy.

    PubMed

    Langkilde, Annette E; Morris, Kyle L; Serpell, Louise C; Svergun, Dmitri I; Vestergaard, Bente

    2015-04-01

    Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and α-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods. PMID:25849399

  11. Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns

    SciTech Connect

    Brewster, Aaron S.; Sawaya, Michael R.; Rodriguez, Jose; Hattne, Johan; Echols, Nathaniel; McFarlane, Heather T.; Cascio, Duilio; Adams, Paul D.; Eisenberg, David S.; Sauter, Nicholas K.

    2015-02-01

    Special methods are required to interpret sparse diffraction patterns collected from peptide crystals at X-ray free-electron lasers. Bragg spots can be indexed from composite-image powder rings, with crystal orientations then deduced from a very limited number of spot positions. Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox (cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.

  12. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

    SciTech Connect

    Langkilde, Annette E.; Morris, Kyle L.; Serpell, Louise C.; Svergun, Dmitri I.; Vestergaard, Bente

    2015-04-01

    The aggregation process and the fibril state of an amyloidogenic peptide suggest monomer addition to be the prevailing mechanism of elongation and a model of the peptide packing in the fibrils has been obtained. Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and α-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods.

  13. Lattice model for amyloid peptides: OPEP force field parametrization and applications to the nucleus size of Alzheimer's peptides.

    PubMed

    Tran, Thanh Thuy; Nguyen, Phuong H; Derreumaux, Philippe

    2016-05-28

    Coarse-grained protein lattice models approximate atomistic details and keep the essential interactions. They are, therefore, suitable for capturing generic features of protein folding and amyloid formation at low computational cost. As our aim is to study the critical nucleus sizes of two experimentally well-characterized peptide fragments Aβ16-22 and Aβ37-42 of the full length Aβ1-42 Alzheimer's peptide, it is important that simulations with the lattice model reproduce all-atom simulations. In this study, we present a comprehensive force field parameterization based on the OPEP (Optimized Potential for Efficient protein structure Prediction) force field for an on-lattice protein model, which incorporates explicitly the formation of hydrogen bonds and directions of side-chains. Our bottom-up approach starts with the determination of the best lattice force parameters for the Aβ16-22 dimer by fitting its equilibrium parallel and anti-parallel β-sheet populations to all-atom simulation results. Surprisingly, the calibrated force field is transferable to the trimer of Aβ16-22 and the dimer and trimer of Aβ37-42. Encouraged by this finding, we characterized the free energy landscapes of the two decamers. The dominant structure of the Aβ16-22 decamer matches the microcrystal structure. Pushing the simulations for aggregates between 4-mer and 12-mer suggests a nucleus size for fibril formation of 10 chains. In contrast, the Aβ37-42 decamer is largely disordered with mixed by parallel and antiparallel chains, suggesting that the nucleus size is >10 peptides. Our refined force field coupled to this on-lattice model should provide useful insights into the critical nucleation number associated with neurodegenerative diseases. PMID:27250331

  14. Modulation of TNF-α Secretion in Peripheral Blood Mononuclear Cells by Cocoa Flavanols and Procyanidins

    PubMed Central

    Mao, T. K.; van de Water, J.; Keen, C. L.; Schmitz, H. H.; Gershwin, M. E.

    2002-01-01

    Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-α (TNF-α) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-α released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3–4 fold increase in TNF-α relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-α secretion in the range of 48–128% relative to the PHA control. The monomers and dimers were slightly inhibitory (–1.5 and –15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-α levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-α secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis. PMID:12885154

  15. Lattice model for amyloid peptides: OPEP force field parametrization and applications to the nucleus size of Alzheimer's peptides

    NASA Astrophysics Data System (ADS)

    Tran, Thanh Thuy; Nguyen, Phuong H.; Derreumaux, Philippe

    2016-05-01

    Coarse-grained protein lattice models approximate atomistic details and keep the essential interactions. They are, therefore, suitable for capturing generic features of protein folding and amyloid formation at low computational cost. As our aim is to study the critical nucleus sizes of two experimentally well-characterized peptide fragments Aβ16-22 and Aβ37-42 of the full length Aβ1-42 Alzheimer's peptide, it is important that simulations with the lattice model reproduce all-atom simulations. In this study, we present a comprehensive force field parameterization based on the OPEP (Optimized Potential for Efficient protein structure Prediction) force field for an on-lattice protein model, which incorporates explicitly the formation of hydrogen bonds and directions of side-chains. Our bottom-up approach starts with the determination of the best lattice force parameters for the Aβ16-22 dimer by fitting its equilibrium parallel and anti-parallel β-sheet populations to all-atom simulation results. Surprisingly, the calibrated force field is transferable to the trimer of Aβ16-22 and the dimer and trimer of Aβ37-42. Encouraged by this finding, we characterized the free energy landscapes of the two decamers. The dominant structure of the Aβ16-22 decamer matches the microcrystal structure. Pushing the simulations for aggregates between 4-mer and 12-mer suggests a nucleus size for fibril formation of 10 chains. In contrast, the Aβ37-42 decamer is largely disordered with mixed by parallel and antiparallel chains, suggesting that the nucleus size is >10 peptides. Our refined force field coupled to this on-lattice model should provide useful insights into the critical nucleation number associated with neurodegenerative diseases.

  16. Authentication of the medicinal plant Sennaangustifolia by RAPD profiling

    PubMed Central

    Khan, Salim; Mirza, Khanda Jabeen; Al-Qurainy, Fahad; Abdin, Malik Zainul

    2011-01-01

    In this study “RAPD” molecular marker was employed for the identification of Sennaangustifolia, Sennaacutifolia, Sennatora and Sennasophera. Total 32 decamer primers were screened in amplification with genomic DNA extracted from all species, of which 6 primers yielded species-specific reproducible bands. Out of 42 loci detected, the polymorphic, monomorphic and unique loci were 24, 2 and 16, respectively. Based on dendrogram and similarity matrix, 4 species were differentiated from each other and showed more divergence. Thus, this technique may prove and to contribute the identification of these species of Senna having similar morphology sold in the local markets. PMID:23961137

  17. Conformational and oligomeric effects on the cysteine pK(a) of tryparedoxin peroxidase.

    PubMed

    Yuan, Ye; Knaggs, Michael H; Poole, Leslie B; Fetrow, Jacquelyn S; Salsbury, Freddie R

    2010-08-01

    Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (C(p)) supported by a lowered pK(a) in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that C(p) (C52) pK(a) values are highly affected by oligomeric state; an unshifted C(p) pK(a) (approximately 8.3, comparable to the pK(a) of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the C(p)s evolve dynamically asymmetric pK(a)s during the simulations; one subunit's C(p) pK(a) is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same C(p), showing no preference for a lowered pK(a) in either subunit. Two conserved pathways that communicate the asymmetric pK(a)s between C(p)s of different subunits can be identified. In the decamer, all the C(p) pK(a)s are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent C(p) pK(a) shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the C(p) pK(a). In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the C(p) in dimers relative to the monomers. These side chain and backbone interactions

  18. Cooperativity in beryllium bonds.

    PubMed

    Alkorta, Ibon; Elguero, José; Yáñez, Manuel; Mó, Otilia

    2014-03-01

    A theoretical study of the beryllium bonded clusters of the (iminomethyl)beryllium hydride and (iminomethyl)beryllium fluoride [HC(BeX)=NH, X = H, F] molecules has been carried out at the B3LYP/6-311++G(3df,2p) level of theory. Linear and cyclic clusters have been characterized up to the decamer. The geometric, energetic, electronic and NMR properties of the clusters clearly indicate positive cooperativity. The evolution of the molecular properties, as the size of the cluster increases, is similar to those reported in polymers held together by hydrogen bonds.

  19. VizieR Online Data Catalog: Galaxies in X-ray clusters with DES. I. Stellar mass (Zhang+, 2016)

    NASA Astrophysics Data System (ADS)

    Zhang, Y.; Miller, C.; McKay, T.; Rooney, P.; Evrard, A. E.; Romer, A. K.; Perfecto, R.; Song, J.; Desai, S.; Mohr, J.; Wilcox, H.; Bermeo-Hernandez, A.; Jeltema, T.; Hollowood, D.; Bacon, D.; Capozzi, D.; Collins, C.; Das, R.; Gerdes, D.; Hennig, C.; Hilton, M.; Hoyle, B.; Kay, S.; Liddle, A.; Mann, R. G.; Mehrtens, N.; Nichol, R. C.; Papovich, C.; Sahlen, M.; Soares-Santos, M.; Stott, J.; Viana, P. T.; Abbott, T.; Abdalla, F. B.; Banerji, M.; Bauer, A. H.; Benoit-Levy, A.; Bertin, E.; Brooks, D.; Buckley-Geer, E.; Burke, D. L.; Carnero Rosell, A.; Castander, F. J.; Diehl, H. T.; Doel, P.; Cunha, C. E.; Eifler, T. F.; Fausti Neto, A.; Fernandez, E.; Flaugher, B.; Fosalba, P.; Frieman, J.; Gaztanaga, E.; Gruen, D.; Gruendl, R. A.; Honscheid, K.; James, D.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Maia, M. A. G.; Makler, M.; Marshall, J. L.; Martini, P.; Miquel, R.; Ogando, R.; Plazas, A. A.; Roodman, A.; Rykoff, E. S.; Sako, M.; Sanchez, E.; Scarpine, V.; Schubnell, M.; Sevilla, I.; Smith, R. C.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarle, G.; Thaler, J.; Tucker, D.; Vikram, V.; da Costa, L. N.

    2016-03-01

    The Dark Energy Survey (DES) is a ground-based optical survey that uses the wide-field DECam camera mounted on the 4m Blanco telescope to image 5000deg2 of the southern hemisphere sky (Sanchez et al. 2010JPhCS.259a2080S). The paper is based on 200deg2 DES Science Verification (SV) data. This data set was taken during the 2012B observing season before the main survey (Diehl et al. 2014SPIE.9149E..0VD) began. (1 data file).

  20. Conformational and oligomeric effects on the cysteine pKa of typaredoxin peroxidase

    PubMed Central

    Yuan, Ye; Knaggs, Michael H.; Poole, Leslie B.; Fetrow, Jacquelyn S.; Salsbury, Freddie R.

    2010-01-01

    Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (Cp) supported by a lowered pKa in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that Cp (C52) pKa values are highly affected by oligomeric state; an unshifted Cp pKa (~ 8.3, comparable to the pKa of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the Cps evolve dynamically asymmetric pKas during the simulations; one subunit’s Cp pKa is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same Cp, showing no preference for a lowered pKa in either subunit. Two conserved pathways that communicate the asymmetric pKas between Cps of different subunits can be identified. In the decamer, all the Cp pKas are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent Cp pKa shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the Cp pKa. In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the Cp in dimers relative to the monomers. These side chain and backbone interactions which contribute to pKa modulation indicate the importance

  1. Nuclear magnetic resonance spectroscopy of mussel adhesive protein repeating peptide segment.

    PubMed

    Olivieri, M P; Wollman, R M; Alderfer, J L

    1997-12-01

    Mussel adhesive protein (MAP) is the adhesive agent used by the common blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is generally composed of 75 to 85 repeating decameric units with the reported primary sequence NH2-Ala(1)-Lyst(2)-Pro(3)-Ser(4)-Tyr(5)-Hyp(6)-Hyp(7)-Thr(8)-DOPA( 9)- Lys(10)-COOH. This study examines this peptide's solution-state conformation using proton nuclear magnetic resonance (NMR) spectroscopy. NMR and molecular modeling of the decamer before and after molecular dynamics calculations in water suggests a conformation that retains an overall bent helix.

  2. Hepatotoxic Seafood Poisoning (HSP) Due to Microcystins: A Threat from the Ocean?

    PubMed Central

    Vareli, Katerina; Jaeger, Walter; Touka, Anastasia; Frillingos, Stathis; Briasoulis, Evangelos; Sainis, Ioannis

    2013-01-01

    Cyanobacterial blooms are a major and growing problem for freshwater ecosystems worldwide that increasingly concerns public health, with an average of 60% of blooms known to be toxic. The most studied cyanobacterial toxins belong to a family of cyclic heptapeptide hepatotoxins, called microcystins. The microcystins are stable hydrophilic cyclic heptapeptides with a potential to cause cell damage following cellular uptake via organic anion-transporting proteins (OATP). Their intracellular biologic effects presumably involve inhibition of catalytic subunits of protein phosphatases (PP1 and PP2A) and glutathione depletion. The microcystins produced by cyanobacteria pose a serious problem to human health, if they contaminate drinking water or food. These toxins are collectively responsible for human fatalities, as well as continued and widespread poisoning of wild and domestic animals. Although intoxications of aquatic organisms by microcystins have been widely documented for freshwater ecosystems, such poisonings in marine environments have only occasionally been reported. Moreover, these poisonings have been attributed to freshwater cyanobacterial species invading seas of lower salinity (e.g., the Baltic) or to the discharge of freshwater microcystins into the ocean. However, recent data suggest that microcystins are also being produced in the oceans by a number of cosmopolitan marine species, so that Hepatotoxic Seafood Poisoning (HSP) is increasingly recognized as a major health risk that follows consumption of contaminated seafood. PMID:23921721

  3. Protein interactions between the C-terminus of Aβ-peptide and phospholipase A2--a structure biology based approach to identify novel Alzheimer's therapeutics.

    PubMed

    Mirza, Zeenat; Pillai, Vikram G; Kamal, Mohammad A

    2014-01-01

    Amyloid β (Aβ) polypeptide plays a key role in determining the state of protein aggregation in Alzheimer's disease. The hydrophobic C-terminal part of the Aβ peptide is critical in triggering the transformation from α-helical to β- sheet structure. We hypothesized that phospholipase A2 (PLA2) may inhibit the aggregation of Aβ peptide by interacting with the peptide and keeping the two peptide chains apart. In order to examine the nature of interactions between PLA2 and Aβ peptide, we prepared and crystallized complex of Naja naja sagittifera PLA2 with the C-terminal hepta-peptide Val-Gly-Gly-Val-Val-Ile-Ala. The X-ray intensity data were collected to 2.04 A resolution and the structure was determined by molecular replacement and refined to the crystallographic R factor of 0.186. The structural analysis revealed that the peptide binds to PLA2 at the hydrophobic substrate binding cavity forming at least eight hydrogen bonds and approximately a two dozen Van der Waals interactions. The number and nature of interactions indicate that the affinity between PLA2 and the hepta-peptide is greater than the affinity between two Aβ peptide chains. Therefore, PLA2 is proposed as a probable ligand to prevent the aggregation of Aβ peptides. PMID:25230229

  4. Protein interactions between the C-terminus of Aβ-peptide and phospholipase A2--a structure biology based approach to identify novel Alzheimer's therapeutics.

    PubMed

    Mirza, Zeenat; Pillai, Vikram G; Kamal, Mohammad A

    2014-01-01

    Amyloid β (Aβ) polypeptide plays a key role in determining the state of protein aggregation in Alzheimer's disease. The hydrophobic C-terminal part of the Aβ peptide is critical in triggering the transformation from α-helical to β- sheet structure. We hypothesized that phospholipase A2 (PLA2) may inhibit the aggregation of Aβ peptide by interacting with the peptide and keeping the two peptide chains apart. In order to examine the nature of interactions between PLA2 and Aβ peptide, we prepared and crystallized complex of Naja naja sagittifera PLA2 with the C-terminal hepta-peptide Val-Gly-Gly-Val-Val-Ile-Ala. The X-ray intensity data were collected to 2.04 A resolution and the structure was determined by molecular replacement and refined to the crystallographic R factor of 0.186. The structural analysis revealed that the peptide binds to PLA2 at the hydrophobic substrate binding cavity forming at least eight hydrogen bonds and approximately a two dozen Van der Waals interactions. The number and nature of interactions indicate that the affinity between PLA2 and the hepta-peptide is greater than the affinity between two Aβ peptide chains. Therefore, PLA2 is proposed as a probable ligand to prevent the aggregation of Aβ peptides.

  5. Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin.

    PubMed

    Clark, D P; Durell, S; Maloy, W L; Zasloff, M

    1994-04-01

    Antimicrobial peptides comprise a diverse class of molecules used in host defense by plants, insects, and animals. In this study we have isolated a novel antimicrobial peptide from the skin of the bullfrog, Rana catesbeiana. This 20 amino acid peptide, which we have termed Ranalexin, has the amino acid sequence: NH2-Phe-Leu-Gly-Gly-Leu-Ile-Lys-Ile-Val-Pro-Ala-Met-Ile-Cys-Ala-Val-Thr- Lys-Lys - Cys-COOH, and it contains a single intramolecular disulfide bond which forms a heptapeptide ring within the molecule. Structurally, Ranalexin resembles the bacterial antibiotic, polymyxin, which contains a similar heptapeptide ring. We have also cloned the cDNA for Ranalexin from a metamorphic R. catesbeiana tadpole cDNA library. Based on the cDNA sequence, it appears that Ranalexin is initially synthesized as a propeptide with a putative signal sequence and an acidic amino acid-rich region at its amino-terminal end. Interestingly, the putative signal sequence of the Ranalexin cDNA is strikingly similar to the signal sequence of opioid peptide precursors isolated from the skin of the South American frogs Phyllomedusa sauvagei and Phyllomedusa bicolor. Northern blot analysis and in situ hybridization experiments demonstrated that Ranalexin mRNA is first expressed in R. catesbeiana skin at metamorphosis and continues to be expressed into adulthood.

  6. Mass spectrometry characterization of trypanothione and novel peptides of medical importance isolated from Acanthamoeba polyphaga.

    PubMed

    Ondarza, Raul N; Hernandez, Eva; Hurtado, Gerardo; Woolery, Mathew; Hernandez-Sandoval, Francisco

    2013-04-01

    This paper presents unequivocal results about the presence of trypanothione and its precursor glutathionespermidine from the opportunistic human pathogen Acanthamoeba polyphaga. They were isolated by RP-HPLC as thiolbimane derivatives and characterized using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF). Additionally RP-HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were also present in A. polyphaga. Besides trypanothione, we want to report four new peptides in trophozoites, a tetrapeptide, a hexapeptide, a heptapeptide and a nonapeptide. Trypanothione and two of the thiol peptides, the hexapeptide and heptapeptide, are oxidized since the reduced forms increase in amount when the normal extract is treated by DTT or by electrolytic reduction that convert the oxidized forms to reduced ones. On the other hand, they disappear when the amoeba extract is treated with NEM or when the amoeba culture is treated with various inhibitors of NADPH-dependent disulfidereducing enzymes. Comparison of the thiol peptides, including trypanothione from A. polyphaga with extracts from human lymphocytes showed that they are not present in the latter. Therefore, some of the peptides here reported could be used as antigens for rapid detection of these parasites. In regard to the presence of the enzymes that synthesize and reduce trypanothione in A. polyphaga we suggest that they can be used as drug targets. PMID:23808873

  7. Novel alternatively spliced endoplasmic reticulum retention signal in the cytoplasmic loop of Proteolipid Protein-1.

    PubMed

    Southwood, Cherie; Olson, Kevin; Wu, Chia-Yen; Gow, Alexander

    2007-02-15

    Increased awareness about the importance of protein folding and trafficking to the etiology of gain-of-function diseases has driven extensive efforts to understand the cell and molecular biology underlying the life cycle of normal secretory pathway proteins and the detrimental effects of abnormal proteins. In this regard, the quality-control machinery in the endoplasmic reticulum (ER) has emerged as a major mechanism by which cells ensure that secreted and transmembrane proteins either adopt stable secondary, tertiary, and quaternary structures or are retained in the ER and degraded. Here we examine cellular and molecular aspects of ER retention in transfected fibroblasts expressing missense mutations in the Proteolipid Protein-1 (PLP1) gene that cause mild or severe forms of neurodegenerative disease in humans. Mild mutations cause protein retention in the ER that is partially dependent on the presence of a cytoplasmically exposed heptapeptide, KGRGSRG. In contrast, retention associated with severe mutations occurs independently of this peptide. Accordingly, the function of this novel heptapeptide has a significant impact on pathogenesis and provides new insight into the functions of the two splice isoforms encoded by the PLP1 gene, PLP1 and DM-20. PMID:17171701

  8. MASS SUBSTRUCTURE IN ABELL 3128

    SciTech Connect

    McCleary, J.; Dell’Antonio, I.; Huwe, P.

    2015-05-20

    We perform a detailed two-dimensional weak gravitational lensing analysis of the nearby (z = 0.058) galaxy cluster Abell 3128 using deep ugrz imaging from the Dark Energy Camera (DECam). We have designed a pipeline to remove instrumental artifacts from DECam images and stack multiple dithered observations without inducing a spurious ellipticity signal. We develop a new technique to characterize the spatial variation of the point-spread function that enables us to circularize the field to better than 0.5% and thereby extract the intrinsic galaxy ellipticities. By fitting photometric redshifts to sources in the observation, we are able to select a sample of background galaxies for weak-lensing analysis free from low-redshift contaminants. Photometric redshifts are also used to select a high-redshift galaxy subsample with which we successfully isolate the signal from an interloping z = 0.44 cluster. We estimate the total mass of Abell 3128 by fitting the tangential ellipticity of background galaxies with the weak-lensing shear profile of a Navarro–Frenk–White (NFW) halo and also perform NFW fits to substructures detected in the 2D mass maps of the cluster. This study yields one of the highest resolution mass maps of a low-z cluster to date and is the first step in a larger effort to characterize the redshift evolution of mass substructures in clusters.

  9. Quaternary Structure Heterogeneity of Oligomeric Proteins: A SAXS and SANS Study of the Dissociation Products of Octopus vulgaris Hemocyanin

    PubMed Central

    Spinozzi, Francesco; Mariani, Paolo; Mičetić, Ivan; Ferrero, Claudio; Pontoni, Diego; Beltramini, Mariano

    2012-01-01

    Octopus vulgaris hemocyanin shows a particular self-assembling pattern, characterized by a hierarchical organization of monomers. The highest molecular weight aggregate is a decamer, the stability of which in solution depends on several parameters. Different pH values, buffer compositions, H2O/D2O ratios and Hofmeister’s salts result in modifications of the aggregation state of Octopus vulgaris hemocyanin. The new QUAFIT method, recently applied to derive the structure of the decameric and the monomeric assembly from small-angle scattering data, is used here to model the polydisperse system that results from changing the solution conditions. A dataset of small-angle X-rays and neutron scattering curves is analysed by QUAFIT to derive structure, composition and concentration of different assemblies present in solution. According to the hierarchy of the association/dissociation processes and the possible number of different aggregation products in solution, each sample has been considered as a heterogeneous mixture composed of the entire decamer, the dissociated “loose” monomer and all the intermediate dissociation products. Scattering curves corresponding to given experimental conditions are well fitted by using a linear combination of single particle form factors. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, result to be intrinsically heterogeneous. PMID:23166737

  10. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms

    PubMed Central

    Tairum, Carlos A.; Santos, Melina Cardoso; Breyer, Carlos A.; Geyer, R. Ryan; Nieves, Cecilia J.; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H.; Augusto, Ohara; Netto, Luis E. S.; de Oliveira, Marcos A.

    2016-01-01

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states. PMID:27629822

  11. Protein self-association induced by macromolecular crowding: a quantitative analysis by magnetic relaxation dispersion.

    PubMed

    Snoussi, Karim; Halle, Bertil

    2005-04-01

    In the presence of high concentrations of inert macromolecules, the self-association of proteins is strongly enhanced through an entropic, excluded-volume effect variously called macromolecular crowding or depletion attraction. Despite the predicted large magnitude of this universal effect and its far-reaching biological implications, few experimental studies of macromolecular crowding have been reported. Here, we introduce a powerful new technique, fast field-cycling magnetic relaxation dispersion, for investigating crowding effects on protein self-association equilibria. By recording the solvent proton spin relaxation rate over a wide range of magnetic field strengths, we determine the populations of coexisting monomers and decamers of bovine pancreatic trypsin inhibitor in the presence of dextran up to a macromolecular volume fraction of 27%. Already at a dextran volume fraction of 14%, we find a 30-fold increase of the decamer population and 510(5)-fold increase of the association constant. The analysis of these results, in terms of a statistical-mechanical model that incorporates polymer flexibility as well as the excluded volume of the protein, shows that the dramatic enhancement of bovine pancreatic trypsin inhibitor self-association can be quantitatively rationalized in terms of hard repulsive interactions. PMID:15665132

  12. Investigating interactions of the pentraxins serum amyloid P component and C-reactive protein by mass spectrometry.

    PubMed

    Aquilina, J Andrew; Robinson, Carol V

    2003-10-15

    The oligomeric state of human SAP (serum amyloid P component) in the absence and presence of known ligands has been investigated using nanoelectrospray ionization MS. At pH 8.0, in the absence of Ca2+, SAP has been shown to consist of pentameric and decameric forms. In the presence of physiological levels of Ca2+, SAP was observed to exist primarily as a pentamer, reflecting its in vivo state. dAMP was shown not only to promote decamerization, but also to lead to decamer stacking involving up to 30 monomers. A mechanism for this finding is proposed. CRP (C-reactive protein), a pentraxin closely related to SAP, exists as a pentamer in the presence or absence of Ca2+. Pentamers of CRP and SAP were shown to form mixed decamers in Ca2+-free buffer; however, in the presence of Ca2+, this interaction was not observed. Furthermore, no exchange of monomeric subunits was observed between the SAP and CRP oligomers, suggesting a remarkable stability of the individual pentameric complexes. PMID:12892563

  13. Inhibition of key digestive enzymes by cocoa extracts and procyanidins.

    PubMed

    Gu, Yeyi; Hurst, William J; Stuart, David A; Lambert, Joshua D

    2011-05-25

    This study determined the in vitro inhibitory effects of cocoa extracts and procyanidins against pancreatic α-amylase (PA), pancreatic lipase (PL), and secreted phospholipase A(2) (PLA(2)) and characterized the kinetics of such inhibition. Lavado, regular, and Dutch-processed cocoa extracts as well as cocoa procyanidins (degree of polymerization (DP) = 2-10) were examined. Cocoa extracts and procyanidins dose-dependently inhibited PA, PL, and PLA(2). Lavado cocoa extract was the most potent inhibitor (IC(50) = 8.5-47 μg/mL). An inverse correlation between log IC(50) and DP (R(2) > 0.93) was observed. Kinetic analysis suggested that regular cocoa extract, the pentamer, and decamer inhibited PL activity in a mixed mode. The pentamer and decamer noncompetitively inhibited PLA(2) activity, whereas regular cocoa extract inhibited PLA(2) competitively. This study demonstrates that cocoa polyphenols can inhibit digestive enzymes in vitro and may, in conjunction with a low-calorie diet, play a role in body weight management. PMID:21495725

  14. In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

    PubMed

    Noichri, Y; Palais, G; Ruby, V; D'Autreaux, B; Delaunay-Moisan, A; Nyström, T; Molin, M; Toledano, M B

    2015-12-01

    2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

  15. Is it possible to study the kinetic parameters of interaction between PNA and parallel and antiparallel DNA by stopped-flow fluorescence?

    PubMed

    Barbero, N; Cauteruccio, S; Thakare, P; Licandro, E; Viscardi, G; Visentin, S

    2016-10-01

    Peptide nucleic acids (PNAs) are among the most interesting and versatile artificial structural mimics of nucleic acids and exhibit peculiar and important properties (i.e. high chemical stability, and a high resistance to cellular enzymes and nucleases). Despite their unnatural structure, they are able to recognize and bind DNA and RNA in a very high, specific and selective manner. One of the most popular, easy and reliable method to measure the stability of PNA-DNA hybrid systems is the melting temperature but the thermodynamic data are obtained using a big quantity of materials failing to provide information on the kinetics of the interaction. In the present work, the PNA decamer 6, with the TCACTAGATG sequence of nucleobases, and the corresponding fluorescent PNA-FITU (fluorescein isothiourea) decamer 8 were synthesized with standard manual Boc-based chemistry. The interaction of the PNA-FITU with parallel and antiparallel DNA has been studied by stopped-flow fluorescence, which is proposed as an alternative technique to obtain the kinetic parameters of the binding. The great advantage of using the stopped-flow technique is the possibility of studying the kinetics of the PNA-DNA duplex formation in a physiological environment. In particular, fluorescence stopped-flow technique has been exploited to compare the affinity of two PNA-DNA duplexes since it can discriminate between parallel and antiparallel DNA binding.

  16. DNA polymorphism in crystals: three stable conformations for the decadeoxynucleotide d(GCATGCATGC).

    PubMed

    Thirugnanasambandam, Arunachalam; Karthik, Selvam; Artheswari, Gunanithi; Gautham, Namasivayam

    2016-06-01

    High-resolution structures of DNA fragments determined using X-ray crystallography or NMR have provided descriptions of a veritable alphabet of conformations. They have also shown that DNA is a flexible molecule, with some sequences capable of adopting two different structures. Here, the first example is presented of a DNA fragment that can assume three different and distinct conformations in crystals. The decanucleotide d(GCATGCATGC) was previously reported to assume a single-stranded double-fold structure. In one of the two crystal structures described here the decamer assumes both the double-fold conformation and, simultaneously, the more conventional B-type double-helical structure. In the other crystal the sequence assumes the A-type double-helical conformation. These results, taken together with CD spectra, which were recorded as the decamer was titrated against four metal ions and spermine, indicate that the molecule may exist as a mixed population of structures in solution. Small differences in the environmental conditions, such as the concentration of metal ion, may decide which of these crystallizes out. The results also support the idea that it may be possible for DNA to change its structure to suit the binding requirements of proteins or drugs. PMID:27303798

  17. Amino-Terminal Extended Peptide Single-Chain Trimers are Potent Synthetic Agonists for Memory Human CD8+ T cells

    PubMed Central

    Carreno, Beatriz M.; Becker-Hapak, Michelle; Chan, Megan; Lie, Wen-Rong; Wang, Xiaoli; Hansen, Ted H.; Linette, Gerald P.

    2012-01-01

    Upon antigen exposure, most memory T cells undergo re-stimulation induced cell death. Here we describe a novel synthetic agonist, an amino-terminal extended decamer peptide expressed as a single chain trimer, the AT-SCT, that preferentially promotes the growth of memory human CD8+ T cells with minimal re-stimulation-induced cell death. Using the CMV pp65 and melanoma gp100 antigens, we observe the in vitro numerical expansion of a clonally diverse poly-functional population of antigen-specific CD8+ T cells from normal individuals and vaccinated melanoma patients, respectively. Memory CD8+ T cells stimulated with AT-SCT presented on MHC class I/II null cells show reduced cytokine production, slower kinetics of TCR down-regulation and decreased cell death when compared to native nonamer SCT-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT- and SCT-activated T cells. Probing of SCT and AT-SCT peptide-MHC (p-MHC) complexes using fluorochrome-conjugated TCR multimers suggest that nonamer and decamer-linked peptides may be anchored differently to HLA-A2 peptide binding groove. Our findings demonstrate that modified p-MHC structures such as AT-SCT can be engineered as T cell agonists to promote the growth and expansion of memory human CD8+ T cells. PMID:22573808

  18. Mass Substructure in Abell 3128

    NASA Astrophysics Data System (ADS)

    McCleary, J.; dell'Antonio, I.; Huwe, P.

    2015-05-01

    We perform a detailed two-dimensional weak gravitational lensing analysis of the nearby (z = 0.058) galaxy cluster Abell 3128 using deep ugrz imaging from the Dark Energy Camera (DECam). We have designed a pipeline to remove instrumental artifacts from DECam images and stack multiple dithered observations without inducing a spurious ellipticity signal. We develop a new technique to characterize the spatial variation of the point-spread function that enables us to circularize the field to better than 0.5% and thereby extract the intrinsic galaxy ellipticities. By fitting photometric redshifts to sources in the observation, we are able to select a sample of background galaxies for weak-lensing analysis free from low-redshift contaminants. Photometric redshifts are also used to select a high-redshift galaxy subsample with which we successfully isolate the signal from an interloping z = 0.44 cluster. We estimate the total mass of Abell 3128 by fitting the tangential ellipticity of background galaxies with the weak-lensing shear profile of a Navarro-Frenk-White (NFW) halo and also perform NFW fits to substructures detected in the 2D mass maps of the cluster. This study yields one of the highest resolution mass maps of a low-z cluster to date and is the first step in a larger effort to characterize the redshift evolution of mass substructures in clusters.

  19. Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Alexopoulos, Eftichia; Zhao, Boyu; El Bakkouri, Majida; Thibault, Guillaume; Liu, Kaiyin; Ramachandran, Shaliny; Snider, Jamie; Pai, Emil F; Houry, Walid A

    2011-03-01

    The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses. PMID:21278708

  20. The development of a cryogenic over-pressure pump

    SciTech Connect

    Alvarez, M.; Cease, H.; Flaugher, B.; Flores, R.; Lathrop, A.; Garcia, J.; Ruiz, F.

    2014-01-29

    A cryogenic over-pressure pump (OPP) was tested in the prototype telescope liquid nitrogen (LN2) cooling system for the Dark Energy Survey (DES) Project. This OPP consists of a process cylinder (PC), gas generator, and solenoid operated valves (SOVs). It is a positive displacement pump that provided intermittent liquid nitrogen (LN2) flow to an array of charge couple devices (CCDs) for the prototype Dark Energy Camera (DECam). In theory, a heater submerged in liquid would generate the drive gas in a closed loop cooling system. The drive gas would be injected into the PC to displace that liquid volume. However, due to limitations of the prototype closed loop nitrogen system (CCD cooling system) for DECam, a quasiclosed-loop nitrogen system was created. During the test of the OPP, the CCD array was cooled to its designed set point temperature of 173K. It was maintained at that temperature via electrical heaters. The performance of the OPP was captured in pressure, temperature, and flow rate in the CCD LN2 cooling system at Fermi National Accelerator Laboratory (FNAL)

  1. Peptide nucleic acids tagged with four lysine residues for amperometric genosensors

    PubMed Central

    Zanardi, Chiara; Terzi, Fabio; Seeber, Renato; Baldoli, Clara; Licandro, Emanuela; Maiorana, Stefano

    2012-01-01

    A homothymine PNA decamer bearing four lysine residues has been synthesized as a probe for the development of amperometric sensors. On one hand, the four amino groups introduced make this derivative nine times more soluble than the corresponding homothymine PNA decamer and, on the other hand, allow the stable anchoring of this molecule on Au nanostructured surface through the terminal -NH2 moieties. In particular, XPS and electrochemical investigations performed with hexylamine, as a model molecule, indicate that the stable deposition of primary amine derivatives on such a nanostructured surface is possible and involves the free electron doublet on the nitrogen atom. This finding indicates that this PNA derivative is suitable to act as the probe molecule for the development of amperometric sensors.   Thanks to the molecular probe chosen and to the use of a nanostructured surface as the substrate for the sensor assembly, the device proposed makes possible the selective recognition of the target oligonucleotide sequence with very high sensitivity. PMID:22772036

  2. Evidence That Hydra I is a Tidally Disrupting Milky Way Dwarf Galaxy

    NASA Astrophysics Data System (ADS)

    Hargis, Jonathan R.; Kimmig, Brian; Willman, Beth; Caldwell, Nelson; Walker, Matthew G.; Strader, Jay; Sand, David J.; Grillmair, Carl J.; Yoon, Joo Heon

    2016-02-01

    The Eastern Banded Structure (EBS) and Hydra I halo overdensities are very nearby (d ∼ 10 kpc) objects discovered in Sloan Digital Sky Survey (SDSS) data. Previous studies of the region have shown that EBS and Hydra I are spatially coincident, cold structures at the same distance, suggesting that Hydra I may be the EBS's progenitor. We combine new wide-field Dark Energy Camera (DECam) imaging and MMT/Hectochelle spectroscopic observations of Hydra I with SDSS archival spectroscopic observations to quantify Hydra I's present-day chemodynamical properties, and to infer whether it originated as a star cluster or dwarf galaxy. While previous work using shallow SDSS imaging assumed a standard old, metal-poor stellar population, our deeper DECam imaging reveals that Hydra I has a thin, well-defined main sequence turnoff of intermediate age (∼5–6 Gyr) and metallicity ([Fe/H] = ‑0.9 dex). We measure statistically significant spreads in both the iron and alpha-element abundances of {σ }[{Fe/{{H}}]}=0.13+/- 0.02 dex and {σ }[α /{{Fe}]}=0.09+/- 0.03 dex, respectively, and place upper limits on both the rotation and its proper motion. Hydra I's intermediate age and [Fe/H]—as well as its low [α/Fe], apparent [Fe/H] spread, and present-day low luminosity—suggest that its progenitor was a dwarf galaxy, which has subsequently lost more than 99.99% of its stellar mass.

  3. A Hero's Little Horse: Discovery of a Dissolving Star Cluster in Pegasus

    NASA Astrophysics Data System (ADS)

    Kim, Dongwon; Jerjen, Helmut

    2015-01-01

    We report the discovery of an ultra-faint stellar system in the constellation of Pegasus. This concentration of stars was detected by applying our overdensity detection algorithm to the Sloan Digital Sky Survey Data Release 10 and confirmed with deeper photometry from the Dark Energy Camera (DECam) at the 4 m Blanco telescope. The best-fitting model isochrone indicates that this stellar system, Kim 1, features an old (12 Gyr) and metal-poor ([Fe/H] ~ -1.7) stellar population at a heliocentric distance of 19.8 ± 0.9 kpc. We measure a half-light radius of 6.9 ± 0.6 pc using a Plummer profile. The small physical size and the extremely low luminosity are comparable to the faintest known star clusters Segue 3, Koposov 1 and 2, and Muñoz 1. However, Kim 1 exhibits a lower star concentration and is lacking a well-defined center. It also has an unusually high ellipticity and irregular outer isophotes, which suggests that we are seeing an intermediate mass star cluster being stripped by the Galactic tidal field. An extended search for evidence of an associated stellar stream within the 3 \\deg 2 DECam field remains inconclusive. The finding of Kim 1 is consistent with current overdensity detection limits and supports the hypothesis that there are still a substantial number of extreme low-luminosity star clusters undetected in the wider Milky Way halo.

  4. Is it possible to study the kinetic parameters of interaction between PNA and parallel and antiparallel DNA by stopped-flow fluorescence?

    PubMed

    Barbero, N; Cauteruccio, S; Thakare, P; Licandro, E; Viscardi, G; Visentin, S

    2016-10-01

    Peptide nucleic acids (PNAs) are among the most interesting and versatile artificial structural mimics of nucleic acids and exhibit peculiar and important properties (i.e. high chemical stability, and a high resistance to cellular enzymes and nucleases). Despite their unnatural structure, they are able to recognize and bind DNA and RNA in a very high, specific and selective manner. One of the most popular, easy and reliable method to measure the stability of PNA-DNA hybrid systems is the melting temperature but the thermodynamic data are obtained using a big quantity of materials failing to provide information on the kinetics of the interaction. In the present work, the PNA decamer 6, with the TCACTAGATG sequence of nucleobases, and the corresponding fluorescent PNA-FITU (fluorescein isothiourea) decamer 8 were synthesized with standard manual Boc-based chemistry. The interaction of the PNA-FITU with parallel and antiparallel DNA has been studied by stopped-flow fluorescence, which is proposed as an alternative technique to obtain the kinetic parameters of the binding. The great advantage of using the stopped-flow technique is the possibility of studying the kinetics of the PNA-DNA duplex formation in a physiological environment. In particular, fluorescence stopped-flow technique has been exploited to compare the affinity of two PNA-DNA duplexes since it can discriminate between parallel and antiparallel DNA binding. PMID:27611452

  5. Light Echoes of Galactic Explosions and Eruptions

    NASA Astrophysics Data System (ADS)

    Rest, Armin; Bianco, Federica; Chornock, Ryan; Foley, Ryan; Matheson, Thomas; Narayan, Gautham; Olsen, Knut; Prieto, Jose Luis; Smith, Chris; Smith, Nathan; Suntzeff, Nick; Welch, Doug; Zenteno, Alfredo

    2014-02-01

    We propose to continue our search for the first light echoes (LEs) associated with historical Galactic supernovae and LBV outbursts: SN 1006, Kepler's SN, RCW 86, Crab Nebula, and P Cygni. In previously granted NOAO time, we have discovered LEs of three ancient SNe in the LMC as well as from the historic SN events of Cas A and Tycho [2, 3], which allowed their spectroscopic classification [6, 7, 10] and 3D spectroscopy [8, 9]. Most recently, we discovered light echoes of the mid-19th-century Great Eruption of η Carinae using CTIO 4m Mosaic images [11]. Subsequent spectroscopic follow-up of Eta Carinae revealed that its outburst spectral type was most similar to those of G-type supergiants, rather than reported LBV outburst spectral types of F-type (or earlier) [11]. We propose to continue our search for light echoes of the remaining historical events. With DECam, we have a 10-15 fold improvement in efficiency over the retired CTIO-Mosaic camera, which allows us to cover the bigger search areas of most of the remaining targets. With the KPNO 4-m, we will observe fields too far north for CTIO/DECam. The study of scattered-light echoes from these Galactic supernovae and eruptions will give us the opportunity to directly compare the original outburst and its current remnant, and in favorable cases (like Eta Carinae), it provides a three-dimensional view of the event and/or a spectral time series.

  6. Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA

    PubMed Central

    Kandiah, Eaazhisai; Carriel, Diego; Perard, Julien; Malet, Hélène; Bacia, Maria; Liu, Kaiyin; Chan, Sze W. S.; Houry, Walid A.; Ollagnier de Choudens, Sandrine; Elsen, Sylvie; Gutsche, Irina

    2016-01-01

    The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. PMID:27080013

  7. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms.

    PubMed

    Tairum, Carlos A; Santos, Melina Cardoso; Breyer, Carlos A; Geyer, R Ryan; Nieves, Cecilia J; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H; Augusto, Ohara; Netto, Luis E S; de Oliveira, Marcos A

    2016-09-15

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.

  8. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms.

    PubMed

    Tairum, Carlos A; Santos, Melina Cardoso; Breyer, Carlos A; Geyer, R Ryan; Nieves, Cecilia J; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H; Augusto, Ohara; Netto, Luis E S; de Oliveira, Marcos A

    2016-01-01

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states. PMID:27629822

  9. The identification and structure-activity relations of a cardioactive FMRFamide-related peptide from the blue crab Callinectes sapidus.

    PubMed

    Krajniak, K G

    1991-01-01

    The pericardial organs and thoracic ganglia of the blue crab Callinectes sapidus were resected and extracted. The extracts were fractionated by HPLC and the fractions analyzed by a radioimmunoassay (RIA) to FMRFamide. Multiple peaks of immunoreactivity were present and one of these, upon fast atom bombardment mass spectrometry (FAB-ms) and microsequencing, yielded the sequence GYNRSFLRFamide. The amount of this peptide in each crab is between 7 and 13 pmol. Several incomplete sequences were also characterized, suggesting a precursor with multiple copies of peptides related to GYNRSFLRFamide might occur. The peptide caused a dose-dependent increase in heart rate; threshold was 10 to 30 nM, and the EC50 was 323 +/- 62 nM. A structure-activity study of GYNRSFLRFamide on the crab heart suggests that, for full potency, a peptide should be at least a heptapeptide with the sequence XXZFLRFamide, where X is any amino acid and Z is either asparagine or serine.

  10. Biotransformation of Microcystins in Eukaryotic Cells - Possible Future Research Directions.

    PubMed

    Pflugmacher, Stephan

    2016-01-01

    Due to eutrophication processes in our water bodies, cyanobacterial blooms can develop worldwide. Most of these blooms are toxic. The most prominent cyanobacterial toxins are the group of the microcystins, which are cyclic heptapeptides, currently with more than 100 congeners known. The biotransformation of microcystins starts with the conjugation to the cell internal tripeptide glutathione, catalysed by glutathione S-transferase enzymes. This conjugate is further broken down to a cysteine conjugate, enhancing the cell internal transport and excretion of the conjugated toxin from the organisms. Still many questions remain open, thinking on an obviously good working detoxification system on the one side and the often seen negative effects up to the death of humans on the other sides.

  11. Sub-lethal cadmium exposure increases phytochelatin concentrations in the aquatic snail Lymnaea stagnalis.

    PubMed

    Sf, Gonçalves; Sk, Davies; Bennett, M; Raab, A; Feldmann, J; Kille, P; Loureiro, S; Dj, Spurgeon; Jg, Bundy

    2016-10-15

    Phytochelatins are metal-binding metabolites found in almost all plant species and some animal groups, including nematodes and annelids, where they can play an important role in detoxifying metals such as cadmium. Species from several other taxa contain a phytochelatin synthase (PCS) gene orthologue, including molluscs, indicating they may have the potential to synthesize phytochelatins. However, the presence of a gene alone does not demonstrate that it plays a functional role in metal detoxification. In the present study, we show that the aquatic snail Lymnaea stagnalis produced both penta- and heptapeptide phytochelatins (i.e. phytochelatin-2 and phytochelatin-3), and their levels increased in response to sub-lethal levels of cadmium.

  12. The RNA Polymerase II CTD: The Increasing Complexity of a Low-Complexity Protein Domain.

    PubMed

    Jeronimo, Célia; Collin, Pierre; Robert, François

    2016-06-19

    The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that plays key roles in coordinating transcription with co-transcriptional events. The heptapeptide repeats that form the CTD are dynamically phosphorylated on serine, tyrosine and threonine residues during the various steps of transcription, thereby regulating the recruitment of various proteins involved in gene expression. In this "Perspective," we review the recent literature related to the function of the CTD, to CTD kinases (Kin28, CDK7, CDK9, CDK12, ERK1/2 and DYRK1A) and to CTD phosphatases (Rtr1, RPAP2, Ssu72, Fcp1 and Gcl7). We discuss unresolved and controversial issues and try to provide constructive suggestions. This review also highlights emerging themes in the CTD field, such as crosstalk and feedback mechanisms, as well as gene-specific and tissue-specific functions of the CTD. Finally, promising therapeutic avenues for a recently developed CTD kinase inhibitor are discussed.

  13. Chromopeptides from phycoerythrocyanin. Structure and linkage of the three bilin groups. [Mastigocladus laminosus; Anabaena variabilis

    SciTech Connect

    Bishop, J.E.; Rapoport, H.; Klotz, A.V.; Chan, C.F.; Glazer, A.N.; Fueglistaller, P.; Zuber, H.

    1987-01-01

    Phycoerythrocyanin carries two covalently attached phycocyanobilin (PCB) groups on the ..beta.. subunit and a phycobiliviolinoid (PXB) group on the ..cap alpha.. subunit. Three distinct bilipeptides were obtained by proteolytic digestion of this protein: Asn-Gln-Ala-Ala-Cys(PCB)-Ile-Arg, Gly-Asp-Cys(PCB)-Ser-Gln, and Cys(PXB)-Val-Arg. Correlation 500-MHz /sup 1/H NMR analyses showed that the heptapeptide and pentapeptide were attached by cysteinyl thioether linkage to the A ring of the PCB moiety. /sup 1/H NMR and mass spectrometry determinations led to structural assignment for the hitherto uncharacterized PXB moiety, with peptide-thioether bonding possible to either ring A or D. Amino acid sequence homologies strongly favor A-ring linkage.

  14. Cloning and sequence determination of the Schizosaccharomyces pombe rpb1 gene encoding the largest subunit of RNA polymerase II.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ueshima, R; Ishihama, A

    1991-01-01

    The gene, rpb1, encoding the largest subunit of RNA polymerase II has been cloned from Schizosaccharomyces pombe using the corresponding gene, RPB1, of Saccharomyces cerevisiae as a cross-hybridization probe. We have determined the complete sequence of this gene, and parts of PCR-amplified rpb1 cDNA. The predicted coding sequence, interrupted by six introns, encodes a polypeptide of 1,752 amino acid residues in length with a molecular weight of 194 kilodaltons. This polypeptide contains eight conserved structural domains characteristic of the largest subunit of RNA polymerases from other eukaryotes and, in addition, 29 repetitions of the C-terminal heptapeptide found in all the eukaryotic RNA polymerase II largest subunits so far examined. Images PMID:2011520

  15. [Nootropic and analgesic effects of Semax following different routes of administration].

    PubMed

    Manchenko, D M; Glazova, N Iu; Levitskaia, N G; Andreeva, L A; Kamenskiĭ, A A; Miasoedov, N F

    2010-10-01

    Heptapeptide Semax (MEHFPGP) is the fragment of ACTH(4-10) analogue with prolonged neurotropic activity. The aim of the present work was to study the Semax effects on learning capability and pain sensitivity in white rats following intraperitoneal and intranasal administration in different doses. Semax nootropic effects were studied in the test of acquisition of passive avoidance task. Pain sensitivity was estimated in Randall-Selitto paw-withdrawal test. It was shown that Semax exerts nootropic and analgesic activities following intraperitoneal administration. Analysis of dependence of these effects on dose resulted in different dose-response curves. Following intranasal administration, Semax was more potent in learning improvement compared to intraperitoneal administration. The peptide failed to affect the animal pain sensitivity following intranasal administration as opposed to intraperitoneal administration. The data obtained suggest different mechanisms and brain structures involved in realization of the nootropic and analgesic effects of Semax.

  16. Ranatuerins: antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana.

    PubMed

    Goraya, J; Knoop, F C; Conlon, J M

    1998-09-29

    Nine peptides, termed ranatuerins 1-9, with antimicrobial activity towards Staphylococcus aureus, were isolated from an extract of the skin of the adult American bullfrog, Rana catesbeiana. In common with other cytolytic peptides from Ranid frogs, (e.g. ranalexin, gaegurins, brevinins), ranatuerins 1 and 4 contain an intramolecular disulfide bridge forming a heptapeptide ring whereas in ranatuerins 2 and 3 the disulfide bridge forms a hexapeptide ring. The structurally related ranatuerins 5-9 comprise 12 - 14 amino acids and show sequence similarity towards the hemolytic peptides A1 and B9 previously isolated from the skin of Rana esculenta. Of the peptides purified, ranatuerin 1 (SMLSVLKNLGKVGLG FVACKINKQC) showed the broadest spectrum of antimicrobial action with inhibitory activity against S. aureus, Escherichia coli and Candida albicans.

  17. Sub-lethal cadmium exposure increases phytochelatin concentrations in the aquatic snail Lymnaea stagnalis.

    PubMed

    Sf, Gonçalves; Sk, Davies; Bennett, M; Raab, A; Feldmann, J; Kille, P; Loureiro, S; Dj, Spurgeon; Jg, Bundy

    2016-10-15

    Phytochelatins are metal-binding metabolites found in almost all plant species and some animal groups, including nematodes and annelids, where they can play an important role in detoxifying metals such as cadmium. Species from several other taxa contain a phytochelatin synthase (PCS) gene orthologue, including molluscs, indicating they may have the potential to synthesize phytochelatins. However, the presence of a gene alone does not demonstrate that it plays a functional role in metal detoxification. In the present study, we show that the aquatic snail Lymnaea stagnalis produced both penta- and heptapeptide phytochelatins (i.e. phytochelatin-2 and phytochelatin-3), and their levels increased in response to sub-lethal levels of cadmium. PMID:27358197

  18. Effect of methylene group insertions on the structural rigidity of Aib containing helices.

    PubMed

    Duley, Anju; Gowda, Vasantha; Ganjiwale, Anjali; Raghothama, Srinivasarao; Ramanathan, Gurunath

    2015-11-01

    Nonprotein amino acids are being extensively used in the design of synthetic peptides to create new structure mimics. In this study we report the effect of methylene group insertions in a heptapeptide Boc-Ala1-Leu2-Aib3-Xxx4-Ala5-Leu6-Aib7-OMe which nicely folds into a mixed 310 -/α-helical structure when Xxx= Ala. Analogs of this peptide have been made and studied by replacing central Xxx4 residue with Glycine (α-residue), β-Alanine (β-Αla), γ-aminobutyric acid (Gaba), and ε-aminocaproic acid (ε-Aca). NMR and circular dichroism were used to study the solution structure of these peptides. Crystals of the peptides containing alanine, β-Αla, and Gaba reveal that increasing the number of central methylene (-CH2 -) groups introduces local perturbations even as the helical structure is retained. PMID:26152771

  19. Molecular mechanisms of RIP, an effective inhibitor of chronic infections.

    PubMed

    Lopez-Leban, Florencia; Kiran, Madanahally Divakar; Wolcott, Randall; Balaban, Naomi

    2010-09-01

    Non-healing bacterial infections are often associated with the formation of a biofilm, where bacteria are more resistant to conventional treatment modalities and to host immune responses. We show here that RNAIII inhibiting peptide (RIP), a linear heptapeptide, is very effective in treating severe polymicrobial infections, including drug-resistant staphylococci like MRSA. By functional genomics studies (microarray analysis) on Staphylococcus aureus, we show here that RIP downregulates the expression of genes involved in biofilm formation and toxin production, and upregulates genes involved in stress response. This pattern of gene regulation may explain why RIP has been so effective in treating severe infections and hopefully through the addition of RIP to existing protocols, a new way of tackling chronic persistent infections will be established.

  20. The primary structure of the aridicin aglycon as revealed by long-range J values

    NASA Astrophysics Data System (ADS)

    Mueller, Luciano; Jeffs, Peter W.

    The aglycon of aridicin, which is a member of the vancomycin class of antibiotics, was analyzed by utilizing J spin-spin interactions in two-dimensional NMR experiments.This unusual heptapeptide with the molecular formula C 59H 45N 7O 19Cl 4 (MW 1296.160) has a large number of quateernary carbons in aromatic side chains. For that reason most information was obtained from delayed COSY and COLOC spectra which reveal homo- and heteronuclear connectivities via long-range J couplings. The carbon-13 spectrum was assigned completely. In addition, the primary structure of the aridicin aglycon could be deduced, with the exception of the ether linkages between the side chains A, B, and C, by solely relying on J-connectivity maps.

  1. Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.).

    PubMed

    Chang, K S; Lee, S H; Hwang, S B; Park, K Y

    2000-10-01

    Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

  2. A Functional Interaction between the Carboxy-Terminal Domain of RNA Polymerase II and Pre-mRNA Splicing

    PubMed Central

    Du, Lei; Warren, Stephen L.

    1997-01-01

    In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human β-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing. PMID:9008699

  3. A biocompatible cobaltporphyrin-based complex micelle constructed via supramolecular assembly for oxygen transfer.

    PubMed

    Shen, Liangliang; Qu, Rui; Shi, Hejin; Huang, Fan; An, Yingli; Shi, Linqi

    2016-05-26

    Herein, a complex micelle as an oxygen nano-carrier is constructed through the hierarchical assembly of the diblock copolymer poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLys), tetrakis(4-sulfonatophenyl)porphinato cobalt(ii) (Co(ii)TPPS), a heptapeptide (Cys-His-His-His-His-His-His) and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD). Co(ii)TPPS was encapsulated into the cavities of TM-β-CDs driven by the host-guest interaction so that the irreversible formation of a μ-oxo-dimer of Co(ii)TPPS can be effectively prevented. The imidazole groups of the heptapeptide were selected as good axial ligands coordinating to the centric cobalt of Co(ii)TPPS, which subtly constituted the five-coordinated precursor serving as an active functional centre for oxygen binding. The sixth position of Co(ii)TPPS can bind oxygen. Furthermore, the host-guest inclusion (TM-β-CD/Co(ii)TPPS) was loaded into the hydrophobic core of the complex micelle and tightly fixed with PLys chains. The hydrophilic PEG blocks stretched in the aqueous solution constitute the shells which stabilize the structure of the complex micelle as well as impart the complex micelle sufficient blood circulation time. Moreover, the complex micelle exhibited excellent biocompatibility and cellular uptake. Therefore, the rationally designed amphiphilic structure can work as promising artificial O2 carriers in vivo. Potentially, the complex micelle can be expected to change the anaerobic microenvironment and find applications in the repair of the cells damaged by cellular hypoxia. PMID:27009911

  4. Observation of two new L4 Neptune Trojans in the Dark Energy Survey supernova fields

    DOE PAGES

    Gerdes, D. W.

    2016-01-28

    We report the discovery of the eighth and ninth known Trojans in stable orbits around Neptune's leading Lagrange point, L4. The objects 2014 QO441 and 2014 QP441 were detected in data obtained during the 2013-14 and 2014-15 observing seasons by the Dark Energy Survey, using the Dark Energy Camera (DECam) on the 4-meter Blanco telescope at Cerro Tololo Inter- American Observatory. Both are in high-inclination orbits (18.8° and 19.4° respectively). Furthermore, with an eccentricity of 0.104, 2014 QO441 has the most eccentric orbit of the eleven known stable Neptune Trojans. We describe the search procedure and investigate the objects' long-termmore » dynamical stability and physical properties.« less

  5. PreCam: A Step Towards the Photometric Calibration of the Dark Energy Survey

    NASA Astrophysics Data System (ADS)

    Allam, S. S.; Tucker, D. L.; PreCam Team; DES Collaboration

    2016-05-01

    The Dark Energy Survey (DES) will be taking the next step in probing the properties of Dark Energy and in understanding the physics of cosmic acceleration. A step towards the photometric calibration of DES is to have a quick, bright survey in the DES footprint (PreCam), using a pre-production set of the Dark Energy Camera (DECam) CCDs and a set of 100 mm×100 mm DES filters. The objective of the PreCam Survey is to create a network of calibrated DES grizY standard stars that will be used for DES nightly calibrations and to improve the DES global relative calibrations. Here, we describe the first year of PreCam observation, results, and photometric calibrations.

  6. Efficacy of random primer-pair arrays in plant genome analysis: a case study of Cucumis (Cucurbitaceae) for identification of wild and cultivated species.

    PubMed

    Gatphoh, E M; Sharma, S K; Rajkumari, K; Rama Rao, S

    2011-01-01

    The efficacy of random primer-pair arrays compared to conventional RAPD method with a single decamer primer was evaluated using DNA from two species of Cucumis. The banding patterns of amplicons revealed enhanced utility of primer-pair arrays over conventional RAPDs, producing more bands and a higher degree of polymorphism, both at intra- and inter-specific levels. Amplification produced by both methods clearly distinguished a wild from a cultivated species of the genus Cucumis. The main advantage of the primer-pair RAPD over single-primer-based RAPD is the increase in the number of reactions and amplification products in the form of novel/unique bands with a limited number of primers. It also enables the generation of reliable amplicons with a large number of polymorphic bands, which can be linked to gene-governing traits, allowing sequence-characterized partial genome analysis. PMID:21823091

  7. Identical linkage and cooperativity of oxygen and carbon monoxide binding to Octopus dofleini hemocyanin.

    PubMed

    Connelly, P R; Gill, S J; Miller, K I; Zhou, G; van Holde, K E

    1989-02-21

    Employment of high-precision thin-layer methods has enabled detailed functional characterization of oxygen and carbon monoxide binding for (1) the fully assembled form with 70 binding sites and (2) the isolated chains with 7 binding sites of Octopus dofleini hemocyanin. The striking difference in the cooperativities of the two ligands for the assembled decamer is revealed through an examination of the binding capacities and the partition coefficient, determined as functions of the activities of both ligands. A global analysis of the data sets supported a two-state allosteric model assuming an allosteric unit of 7. Higher level allosteric interactions were not indicated. This contrasts to results obtained for arthropod hemocyanins. Oxygen and carbon monoxide experiments performed on the isolated subunit chain confirmed the presence of functional heterogeneity reported previously [Miller, K. (1985) Biochemistry 24, 4582-4586]. The analysis shows two types of binding sites in the ratio of 4:3. PMID:2719937

  8. Assembly of Octopus dofleini hemocyanin. A study of the kinetics by sedimentation, light scattering and electron microscopy.

    PubMed

    van Holde, K E; Miller, K; Schabtach, E; Libertini, L

    1991-01-20

    The kinetics of association of Octopus dofleini hemocyanin subunits to form the native decameric molecule have been studied with a combination of sedimentation, light scattering and electron microscopy. The reaction, initiated by addition of magnesium, is relatively slow, requiring hours to reach completion, with monomer and decamer as predominant molecular species throughout. Analysis of the light-scattering data, including stopped-flow studies, reveals an initial lag period in the reaction, followed by a second-order process that is rate limiting. The lag period depends on both protein and magnesium ion concentration. Electron microscope studies reveal intermediates in the process, and support a model of assembly in which nucleation begins at the dimer level. Theoretical models for the process are compared. PMID:1992165

  9. Automated three-dimensional reconstruction of keyhole limpet hemocyanin type 1.

    PubMed

    Mouche, Fabrice; Zhu, Yuanxin; Pulokas, James; Potter, Clinton S; Carragher, Bridget

    2003-12-01

    We have reconstructed a three-dimensional map of keyhole limpet hemocyanin isoform 1 (KLH1), using our automated data collection software, Leginon, integrated with particle selection algorithms, and the SPIDER reconstruction package. KLH1, a 7.9 MDa macromolecule, is an extracellular respiratory pigment composed of two asymmetric decamers, and presents an overall D(5) point-group symmetry. The reconstruction is in agreement with previous data published on molluscan hemocyanins. The reconstructed map (11.3A resolution, 3sigma criterion) was used to fit an available X-ray crystallography structure of Octopus dofleini Odg, solved at 2.3A [J. Mol. Biol. 278 (4) (1998) 855], with satisfactory results. The results validate the approach of automating the cryoEM process and demonstrate that the quality of the images acquired and the particles selected is comparable to those obtained using manual methods. Several problems remain to be solved however before these results can be generalized. PMID:14643198

  10. Variable Stars in the Field of the Hydra II Ultra-Faint Dwarf Galaxy

    NASA Astrophysics Data System (ADS)

    Vivas, Anna Katherina; Olsen, Knut A.; Blum, Robert D.; Nidever, David L.; Walker, Alistair R.; Martin, Nicolas; Besla, Gurtina; Gallart, Carme; Van Der Marel, Roeland P.; Majewski, Steven R.; Munoz, Ricardo; Kaleida, Catherine C.; Saha, Abhijit; Conn, Blair; Jin, Shoko

    2016-06-01

    We searched for variable stars in Hydra II, one of the recently discovered ultra-faint dwarf satellites of the Milky Way, using gri time-series obtained with the Dark Energy Camera (DECam) at Cerro Tololo Inter-American Observatory, Chile. We discovered one RR Lyrae star in the galaxy which was used to derive a distance of 154±8 kpc to this system and to re-calculate its absolute magnitude and half-light radius.A comparison with other RR Lyrae stars in ultra-faint systems indicates similar pulsational properties among them, which are different to those found among halo field stars and those in the largest of the Milky Way satellites. We also report the discovery of 31 additional short period variables in the field of view (RR Lyrae, SX Phe, eclipsing binaries, and a likely anomalous cepheid) which are likely not related with Hydra II.

  11. Prediction of binding to MHC class I molecules.

    PubMed

    Adams, H P; Koziol, J A

    1995-09-25

    The binding of antigenic peptide sequences to major histocompatibility complex (MHC) molecules is a prerequisite for stimulation of cytotoxic T cell responses. Neural networks are here used to predict the binding capacity of polypeptides to MHC class I molecules encoded by the gene HLA-A*0201. Given a large database of 552 nonamers and 486 decamers and their known binding capacities, the neural networks achieve a predictive hit rate of 0.78 for classifying peptides which might induce an immune response (good or intermediate binders) vs. those which cannot (weak or non-binders). The neural nets also depict specific motifs for different binding capacities. This approach is in principle applicable to all MHC class I and II molecules, given a suitable set of known binding capacities. The trained networks can then be used to perform a systematic search through all pathogen or tumor antigen protein sequences for potential cytotoxic T lymphocyte epitopes.

  12. Efficacy of random primer-pair arrays in plant genome analysis: a case study of Cucumis (Cucurbitaceae) for identification of wild and cultivated species.

    PubMed

    Gatphoh, E M; Sharma, S K; Rajkumari, K; Rama Rao, S

    2011-01-01

    The efficacy of random primer-pair arrays compared to conventional RAPD method with a single decamer primer was evaluated using DNA from two species of Cucumis. The banding patterns of amplicons revealed enhanced utility of primer-pair arrays over conventional RAPDs, producing more bands and a higher degree of polymorphism, both at intra- and inter-specific levels. Amplification produced by both methods clearly distinguished a wild from a cultivated species of the genus Cucumis. The main advantage of the primer-pair RAPD over single-primer-based RAPD is the increase in the number of reactions and amplification products in the form of novel/unique bands with a limited number of primers. It also enables the generation of reliable amplicons with a large number of polymorphic bands, which can be linked to gene-governing traits, allowing sequence-characterized partial genome analysis.

  13. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F.W.

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient. 2 figs.

  14. Glutamine synthetase isoforms in nitrogen-fixing soybean nodules: distinct oligomeric structures and thiol-based regulation.

    PubMed

    Masalkar, Pintu D; Roberts, Daniel M

    2015-01-16

    Legume root nodule glutamine synthetase (GS) catalyzes the assimilation of ammonia produced by nitrogen fixation. Two GS isoform subtypes (GS1β and GS1γ) are present in soybean nodules. GS1γ isoforms differ from GS1β isoforms in terms of their susceptibility to reversible inhibition by intersubunit disulfide bond formation between C159 and C92 at the shared active site at subunit interfaces. Although nodule GS enzymes share 86% amino acid sequence identity, analytical ultracentrifugation experiments showed that GS1γ is a dodecamer, whereas the GS1β is a decamer. It is proposed that this difference contributes to the differential thiol sensitivity of each isoform, and that GS1γ1 may be a target of thiol-based regulation.

  15. Disentangling the Contribution of Multiple Isomers to the Infrared Spectrum of the Protonated Water Heptamer.

    PubMed

    Heine, Nadja; Fagiani, Matias R; Asmis, Knut R

    2015-06-18

    We use infrared/infrared double-resonance population labeling (IR(2)MS(2)) spectroscopy in the spectral region of the free and hydrogen-bonded OH stretching fundamentals (2880-3850 cm(-1)) to identify the number and to isolate the vibrational signatures of individual isomers contributing to the gas-phase IR spectra of the cryogenically cooled protonated water clusters H(+)(H2O)n·H2/D2 with n = 7-10. For n = 7, four isomers are identified and assigned. Surprisingly, the IR(2)MS(2) spectra of the protonated water octa-, nona-, and decamer show no evidence for multiple isomers. The present spectra support the prediction that the quasi-2D to 3D structural transition occurs in between n = 8 and 9 in the cold cluster regime. However, the same models have difficulty explaining the remarkable size dependence of the isomer population reported here.

  16. High-Molecular-Weight Proanthocyanidins in Foods: Overcoming Analytical Challenges in Pursuit of Novel Dietary Bioactive Components.

    PubMed

    Neilson, Andrew P; O'Keefe, Sean F; Bolling, Bradley W

    2016-01-01

    Proanthocyanidins (PACs) are an abundant but complex class of polyphenols found in foods and botanicals. PACs are polymeric flavanols with a variety of linkages and subunits. Connectivity and degree of polymerization (DP) determine PAC bioavailability and bioactivity. Current quantitative and qualitative methods may ignore a large percentage of dietary PACs. Subsequent correlations between intake and activity are hindered by a lack of understanding of the true PAC complexity in many foods. Additionally, estimates of dietary intakes are likely inaccurate, as nutrient databank values are largely based on standards from cocoa (monomers to decamers) and blueberries (mean DP of 36). Improved analytical methodologies are needed to increase our understanding of the biological roles of these complex compounds. PMID:26735794

  17. Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 Å Resolution.

    PubMed

    Gai, Zuoqi; Matsuno, Asuka; Kato, Koji; Kato, Sanae; Khan, Md Rafiqul Islam; Shimizu, Takeshi; Yoshioka, Takeya; Kato, Yuki; Kishimura, Hideki; Kanno, Gaku; Miyabe, Yoshikatsu; Terada, Tohru; Tanaka, Yoshikazu; Yao, Min

    2015-12-01

    Molluscan hemocyanin, a copper-containing oxygen transporter, is one of the largest known proteins. Although molluscan hemocyanins are currently applied as immunotherapeutic agents, their precise structure has not been determined because of their enormous size. Here, we have determined the first X-ray crystal structure of intact molluscan hemocyanin. The structure unveiled the architecture of the 3.8-MDa supermolecule composed of homologous functional units (FUs), wherein the dimers of FUs hierarchically associated to form the entire cylindrical decamer. Most of the specific inter-FU interactions were localized at narrow regions in the FU dimers, suggesting that rigid FU dimers formed by specific interactions assemble with flexibility. Furthermore, the roles of carbohydrates in assembly and allosteric effect, and conserved sulfur-containing residues in copper incorporation, were revealed. The precise structural information obtained in this study will accelerate our understanding of the molecular basis of hemocyanin and its future applications.

  18. PreCam

    SciTech Connect

    Allam, Sahar S.; Tucker, Douglas L.

    2015-01-01

    The Dark Energy Survey (DES) will be taking the next step in probing the properties of Dark Energy and in understanding the physics of cosmic acceleration. A step towards the photometric calibration of DES is to have a quick, bright survey in the DES footprint (PreCam), using a pre-production set of the Dark Energy Camera (DECam) CCDs and a set of 100 mm×100 mm DES filters. The objective of the PreCam Survey is to create a network of calibrated DES grizY standard stars that will be used for DES nightly calibrations and to improve the DES global relative calibrations. Here, we describe the first year of PreCam observation, results, and photometric calibrations.

  19. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    PubMed Central

    Kim, Young Eun; Kim, Yu-na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-01-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein–protein interactions and tools to manipulate receptor clustering on live cell surfaces. PMID:25972078

  20. Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

    NASA Astrophysics Data System (ADS)

    Kim, Young Eun; Kim, Yu-Na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

    2015-05-01

    Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein-protein interactions and tools to manipulate receptor clustering on live cell surfaces.

  1. RAPD-inferred genetic variability of some indigenous Rhizobium leguminosarum isolates from red clover (Trifolium pratense L.) nodules.

    PubMed

    Stefan, Andrei; Rosu, Craita M; Stedel, Catalina; Gorgan, Lucian D; Efrose, Rodica C

    2015-09-01

    The application of commercial rhizobial inoculants to legume crops is proving to be an alternative to synthetic fertilizer use. The challenge for sustainable agriculture resides in the compatibility between crop, inoculants and environmental conditions. The evaluation of symbiotic efficiency and genetic diversity of indigenous rhizobial strains could lead to the development of better inoculants and increased crop production. The genetic variability of 32 wild indigenous rhizobial isolates was assessed by RAPD (Random Amplified Polymorphic DNA). The strains were isolated from red clover (Trifolium pratense L.) nodules from two distinct geographical regions of Northern and Eastern Romania. Three decamer primers were used to resolve the phylogenetic relationships between the investigated isolates. Cluster analysis revealed a high diversity; most strains clustered together based on their geographical location. PMID:26344027

  2. Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

    DOEpatents

    Studier, F. William

    1995-04-18

    Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

  3. The DES Science Verification weak lensing shear catalogues

    NASA Astrophysics Data System (ADS)

    Jarvis, M.; Sheldon, E.; Zuntz, J.; Kacprzak, T.; Bridle, S. L.; Amara, A.; Armstrong, R.; Becker, M. R.; Bernstein, G. M.; Bonnett, C.; Chang, C.; Das, R.; Dietrich, J. P.; Drlica-Wagner, A.; Eifler, T. F.; Gangkofner, C.; Gruen, D.; Hirsch, M.; Huff, E. M.; Jain, B.; Kent, S.; Kirk, D.; MacCrann, N.; Melchior, P.; Plazas, A. A.; Refregier, A.; Rowe, B.; Rykoff, E. S.; Samuroff, S.; Sánchez, C.; Suchyta, E.; Troxel, M. A.; Vikram, V.; Abbott, T.; Abdalla, F. B.; Allam, S.; Annis, J.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Buckley-Geer, E.; Burke, D. L.; Capozzi, D.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Castander, F. J.; Clampitt, J.; Crocce, M.; Cunha, C. E.; D'Andrea, C. B.; da Costa, L. N.; DePoy, D. L.; Desai, S.; Diehl, H. T.; Doel, P.; Fausti Neto, A.; Flaugher, B.; Fosalba, P.; Frieman, J.; Gaztanaga, E.; Gerdes, D. W.; Gruendl, R. A.; Gutierrez, G.; Honscheid, K.; James, D. J.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Li, T. S.; Lima, M.; March, M.; Martini, P.; Miquel, R.; Mohr, J. J.; Neilsen, E.; Nord, B.; Ogando, R.; Reil, K.; Romer, A. K.; Roodman, A.; Sako, M.; Sanchez, E.; Scarpine, V.; Schubnell, M.; Sevilla-Noarbe, I.; Smith, R. C.; Soares-Santos, M.; Sobreira, F.; Swanson, M. E. C.; Tarle, G.; Thaler, J.; Thomas, D.; Walker, A. R.; Wechsler, R. H.

    2016-08-01

    We present weak lensing shear catalogues for 139 square degrees of data taken during the Science Verification (SV) time for the new Dark Energy Camera (DECam) being used for the Dark Energy Survey (DES). We describe our object selection, point spread function estimation and shear measurement procedures using two independent shear pipelines, IM3SHAPE and NGMIX, which produce catalogues of 2.12 million and 3.44 million galaxies, respectively. We detail a set of null tests for the shear measurements and find that they pass the requirements for systematic errors at the level necessary for weak lensing science applications using the SV data. We also discuss some of the planned algorithmic improvements that will be necessary to produce sufficiently accurate shear catalogues for the full 5-yr DES, which is expected to cover 5000 square degrees.

  4. Estimation of genetic diversity Among Turkish kale populations (Brassica oleracea var. acephala L.) using RAPD markers.

    PubMed

    Okumus, A; Balkaya, A

    2007-04-01

    20 populations of kale (B. oleracea var. acephala L.) selected from 127 populations for fresh consumption terms of yield and leaf quality characteristics as superior types using weight-based ranking method from the Black Sea Region of Turkey were evaluated at the DNA level using randomly amplified polymorphic DNA (RAPD) markers compared to some morphological characters. The 7 primers selected from 100 decamers used generated 110 bands, of which 60 (54.5%) were polymorphic. Jaccard's genetic distances were calculated and dendogram was generated using the UPGMA algorithm. The dendogram obtained were classified into three main groups and four subgroups. The accessions showed a limited clustering in compare to morphological characters such as the number of leaf, leaf intentation of the margin, leaf and midrib color and thickness of midrib than geographical characteristics. Leaf color and midrib thickness characters clustered in the same group as OR49 and G18 accessions; S20, G6 and OR37 accessions, respectively.

  5. Synthesis and properties of oligonucleotides containing aminodeoxythymidine units.

    PubMed Central

    Gryaznov, S M; Letsinger, R L

    1992-01-01

    Procedures are described for synthesis via solid support methodology of oligonucleotide analogues derived in part from 3'-amino-3'-deoxythymidine or 5'-amino-5'-deoxythymidine. Oligothymidylate decamers terminated with a 3'-amino group or containing a 3'-NHP(O)(O-)O-5' internucleoside link are found to form unusually stable complexes with poly(dA), poly(A), and oligo(dA). For related derivatives of 5'-amino-5'-deoxythymidine enhancement is less or absent, and in the case of multiple substitution destabilization of the heteroduplex may be observed. That the effect of the 3'-amino group is general for oligonucleotide derivatives is indicated by enhanced Tm values for heteroduplex complexes of the mixed-base oligomer, d(TATTCAGTCAT(NH2)), and the methyl phosphonate derivatives, TmTmTmTmTmTmTmTmTmT(NH2) and d(TmAmTmTmCmAmGmTmCmAmT(NH2)). PMID:1630911

  6. Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets.

    PubMed

    Han, Linjie; Ruotolo, Brandon T

    2015-07-01

    Electrospray ionization coupled to mass spectrometry is a key technology for determining the stoichiometries of multiprotein complexes. Despite highly accurate results for many assemblies, challenging samples can generate signals for artifact protein-protein binding born of the crowding forces present within drying electrospray droplets. Here, for the first time, we study the formation of preferred protein quaternary structures within such rapidly evaporating nanodroplets. We use ion mobility and tandem mass spectrometry to investigate glutamate dehydrogenase dodecamers and serum amyloid P decamers as a function of protein concentration, along with control experiments using carefully chosen protein analogues, to both establish the formation of operative mechanisms and assign the bimodal conformer populations observed. Further, we identify an unprecedented symmetric collision-induced dissociation pathway that we link directly to the quaternary structures of the precursor ions selected.

  7. Genetic characterization and authentication of Embelia ribes using RAPD-PCR and SCAR marker.

    PubMed

    Devaiah, K M; Venkatasubramanian, Padma

    2008-02-01

    Embelia ribes Burm. f. (Myrsinaceae) is one of the important plants used in Indian traditional medicine. RAPD-PCR analysis was performed to obtain species-specific DNA fragments. A band of 906 bp that was specific to Embelia ribes irrespective of the geographical source was obtained using the random decamer primer OPF 05. SCAR primers ER 1 (27 mer) and ER 2 (26 mer) were designed from the sequence of the RAPD marker which yielded an expected amplicon of 594 bp with the Embelia ribes DNA only. This methodology can be used for species identification of genuine Embelia ribes and to distinguish it from common substitutes and adulterants. BLAST: basic local alignment search tool ER 1: Embelia ribes forward primer ER 2: Embelia ribes reverse primer RAPD-PCR: random amplification of polymorphic DNA polymerase chain reaction SCAR: sequence characterized amplified region.

  8. 2-O-[2-(Methylthio)ethyl]-Modified Oligonucleotide: An Analog of 2-O-[2-(Methoxy)ethyl]-Modified Oligonucleotide with Improved Protein Binding Properties and High Binding Affinity to Target RNA

    SciTech Connect

    Prakash, T.P.; Manoharan, M.; Fraser, A.S.; Kawasaki, A.M.; Lesnik, E.; Sioufi, N.; Leeds, J.M.; Teplova, M.; Egli, M.

    2010-03-08

    A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.

  9. The Canarias Einstein ring: a newly discovered optical Einstein ring

    NASA Astrophysics Data System (ADS)

    Bettinelli, M.; Simioni, M.; Aparicio, A.; Hidalgo, S. L.; Cassisi, S.; Walker, A. R.; Piotto, G.; Valdes, F.

    2016-09-01

    We report the discovery of an optical Einstein ring in the Sculptor constellation, IAC J010127-334319, in the vicinity of the Sculptor dwarf spheroidal galaxy. It is an almost complete ring (˜300°) with a diameter of ˜4.5 arcsec. The discovery was made serendipitously from inspecting Dark Energy Camera (DECam) archive imaging data. Confirmation of the object nature has been obtained by deriving spectroscopic redshifts for both components, lens and source, from observations at the 10.4 m Gran Telescopio CANARIAS (GTC) with the spectrograph OSIRIS. The lens, a massive early-type galaxy, has a redshift of z = 0.581, while the source is a starburst galaxy with redshift of z = 1.165. The total enclosed mass that produces the lensing effect has been estimated to be Mtot = (1.86 ± 0.23) × 1012 M⊙.

  10. Sampling saddle points on the free energy surface

    NASA Astrophysics Data System (ADS)

    Samanta, Amit

    2014-03-01

    We develop an algorithm for finding the saddle points on the free energy surface ``on-the-fly'' without having to find the free energy function itself. This is done by using the general strategy of the heterogeneous multi-scale method, applying a macro-scale solver, here the gentlest ascent dynamics algorithm, with the needed force and Hessian values computed on-the-fly using a micro-scale model such as molecular dynamics. The algorithm is capable of dealing with problems involving many coarse-grained variables. The utility of the algorithm is illustrated by studying the saddle points associated with (a) the isomerization transition of the alanine dipeptide using two coarse-grained variables, specifically the Ramachandran dihedral angles, and (b) the beta-hairpin structure of the alanine decamer using twenty coarse-grained variables, specifically the full set of Ramachandran angle pairs associated with each residue.

  11. Observation of Two New L4 Neptune Trojans in the Dark Energy Survey Supernova Fields

    NASA Astrophysics Data System (ADS)

    Gerdes, D. W.; Jennings, R. J.; Bernstein, G. M.; Sako, M.; Adams, F.; Goldstein, D.; Kessler, R.; Hamilton, S.; Abbott, T.; Abdalla, F. B.; Allam, S.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Buckley-Geer, E.; Burke, D. L.; Capozzi, D.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Cunha, C. E.; D'Andrea, C. B.; da Costa, L. N.; DePoy, D. L.; Desai, S.; Dietrich, J. P.; Doel, P.; Eifler, T. F.; Fausti Neto, A.; Flaugher, B.; Frieman, J.; Gaztanaga, E.; Gruen, D.; Gruendl, R. A.; Gutierrez, G.; Honscheid, K.; James, D. J.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Li, T. S.; Maia, M. A. G.; March, M.; Martini, P.; Miller, C. J.; Miquel, R.; Nichol, R. C.; Nord, B.; Ogando, R.; Plazas, A. A.; Romer, A. K.; Roodman, A.; Sanchez, E.; Santiago, B.; Schubnell, M.; Sevilla-Noarbe, I.; Smith, R. C.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarlé, G.; Thaler, J.; Walker, A. R.; Wester, W.; Zhang, Y.; DES Collaboration

    2016-02-01

    We report the discovery of the eighth and ninth known Trojans in stable orbits around Neptune’s leading Lagrange point, L4. The objects 2014 QO441 and 2014 QP441 were detected in data obtained during the 2013-14 and 2014-15 observing seasons by the Dark Energy Survey, using the Dark Energy Camera (DECam) on the 4-m Blanco telescope at Cerro Tololo Inter-American Observatory. Both are in high-inclination orbits (18.°8 and 19.°4, respectively). With an eccentricity of 0.104, 2014 QO441 has the most eccentric orbit of the 11 known stable Neptune Trojans. Here we describe the search procedure and investigate the objects’ long-term dynamical stability and physical properties.

  12. A Radiometric All-Sky Infrared Camera (RASICAM) for DES/CTIO

    SciTech Connect

    Lewis, Peter M.; Rogers, Howard; Schindler, Rafe H.; /SLAC

    2010-08-25

    A novel radiometric all-sky infrared camera [RASICAM] has been constructed to allow automated real-time quantitative assessment of night sky conditions for the Dark Energy Camera [DECam] located on the Blanco Telescope at the Cerro Tololo Inter-American Observatory in Chile. The camera is optimized to detect the position, motion and optical depth of thin, high (8-10km) cirrus clouds and contrails by measuring their apparent temperature above the night sky background. The camera system utilizes a novel wide-field equiresolution catadioptic mirror system that provides sky coverage of 2{pi} azimuth and 14-90{sup o} from zenith. Several new technological and design innovations allow the RASICAM system to provide unprecedented cloud detection and IR-based photometricity quantification. The design of the RASICAM system is presented.

  13. Identical linkage and cooperativity of oxygen and carbon monoxide binding to Octopus dofleini hemocyanin

    SciTech Connect

    Connelly, P.R.; Gill, S.J.; Miller, K.I.; Zhou, G.; van Holde, K.E. )

    1989-02-21

    Employment of high-precision thin-layer methods has enabled detailed functional characterization of oxygen and carbon monoxide binding for (1) the fully assembled form with 70 binding sites and (2) the isolated chains with 7 binding sites of octopus dofleini hemocyanin. The striking difference in the cooperativities of the two ligands for the assembled decamer is revealed through an examination of the binding capacities and the partition coefficient, determined as functions of the activities of both ligands. A global analysis of the data sets supported by a two-state allosteric model assuming an allosteric unit of 7. Higher level allosteric interactions were not indicated. This contrasts to results obtained for arthropod hemocyanins. Oxygen and carbon monoxide experiments performed on the isolated subunit chain confirmed the presence of functional heterogeneity reported previously. The analysis shows two types of binding sites in the ratio of 4:3.

  14. The DES Science Verification Weak Lensing Shear Catalogs

    DOE PAGES

    Jarvis, M.

    2016-05-01

    We present weak lensing shear catalogs for 139 square degrees of data taken during the Science Verification (SV) time for the new Dark Energy Camera (DECam) being used for the Dark Energy Survey (DES). We describe our object selection, point spread function estimation and shear measurement procedures using two independent shear pipelines, IM3SHAPE and NGMIX, which produce catalogs of 2.12 million and 3.44 million galaxies respectively. We also detail a set of null tests for the shear measurements and find that they pass the requirements for systematic errors at the level necessary for weak lensing science applications using the SVmore » data. Furthermore, we discuss some of the planned algorithmic improvements that will be necessary to produce sufficiently accurate shear catalogs for the full 5-year DES, which is expected to cover 5000 square degrees.« less

  15. The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

    SciTech Connect

    Diaz, Z.; Xavier, K; Miller, S

    2009-01-01

    Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2). Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5-phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (??)8-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology with aldolases that process phosphorylated sugars. Ligand binding sites and key catalytic residues are structurally conserved, strongly implicating LsrF as a class I aldolase.

  16. The Mayall z-band Legacy Survey

    NASA Astrophysics Data System (ADS)

    Silva, David R.; Blum, Robert D.; Allen, Lori; Dey, Arjun; Schlegel, David J.; Lang, Dustin; Moustakas, John; Meisner, Aaron M.; Valdes, Francisco; Patej, Anna; Myers, Adam D.; Sprayberry, David; Saha, Abi; Olsen, Knut A.; Safonova, Sasha; Yang, Qian; Burleigh, Kaylan J.; MzLS Team

    2016-06-01

    The Mayall z-band Legacy Survey (MzLS) is conducting a deep z-band imaging survey covering 5000 square degrees in the north Galactic cap as part of the Legacy Survey, which is associated with the Dark Energy Spectroscopic Instrument (DESI) redshift survey. The Legacy Survey covers 14000 square degrees in the g, r, and z bands and is being executed on the Blanco 4-m, Mayall 4-m, and Bok 2.3-m telescopes. The MzLS footprint will be observed in the g and r bands using the Bok 2.3-m telescope also on Kitt Peak. The Beijing Arizona Sky Survey (BASS) is being conducted by a parallel team from Beijing and the University of Arizona. MzLS will cover the sky north of declination 30 degrees and reach a depth of z=23.0. The survey began in January 2016 and will run through June 2017 comprising approximately 230 nights on the Mayall telescope. The data are being obtained with an upgraded Mosaic camera that deploys with newred-sensitive CCDs from Lawrence Berkeley Lab (LBL) whose throughput is in excess of 80% at 8000 to approximately 9800 Angstrom. The upgrade project was a collaboration of Yale, LBL, and NOAO. MzLS images are public as soon as they are taken and delivered to the NOAO archive. Catalogs based on Tractor photometry for all available Legacy Survey images are released soon after they are constructed and MzLS sources will be included in next release planned for summer 2016. The Dark Energy Spectroscopic Instrument (DESI) will observe 30+ million galaxies and quasars in a 14,000 square degree extragalactic footprint. The targeting in that footprint will be provided by a combination of these MzLS data, DECam data from the DECam Legacy Survey, and data from the BASS survey.

  17. Oxygen equilibria of Octopus dofleini hemocyanin.

    PubMed

    Miller, K I

    1985-08-13

    Oxygen binding by Octopus dofleini hemocyanin was examined under very nearly physiological conditions. The effects of pH, ionic composition, temperature, and aggregation were controlled so that the role each plays in modulating oxygen binding can be isolated. There is a very large effect of pH on affinity, the Bohr effect (delta log P50/delta pH = -1.7), which is the same at 10 and 20 degrees C. However, cooperativity is substantially altered over the same range of pHs at the two temperatures. The allosteric properties were examined by comparing the experimental data points to curves generated by use of the Monod-Wyman-Changeux model. A computer-fitting process was developed which allowed the individual allosteric parameters to be varied independently until the best fit could be determined. The relationship between kR and kT is responsible for the effect of pH on cooperativity. A change in the allosteric properties of the T form is primarily responsible for the differences due to temperature. Changing cation concentrations when the molecule is in the fully aggregated 51S form alters affinity without influencing cooperativity. The effect of Mg2+ is much greater than that of Na+. If the 51S decamer is dissociated to 11S monomers by removing divalent cations, oxygen binding is noncooperative. There is evidence for negative cooperativity, indicating heterogeneity of function within the subunit which contains seven oxygen binding domains. Association into decamers generates conformational change which results in a much wider range of allosteric function. PMID:4063340

  18. Kriging atomic properties with a variable number of inputs.

    PubMed

    Davie, Stuart J; Di Pasquale, Nicodemo; Popelier, Paul L A

    2016-09-14

    A new force field called FFLUX uses the machine learning technique kriging to capture the link between the properties (energies and multipole moments) of topological atoms (i.e., output) and the coordinates of the surrounding atoms (i.e., input). Here we present a novel, general method of applying kriging to chemical systems that do not possess a fixed number of (geometrical) inputs. Unlike traditional kriging methods, which require an input system to be of fixed dimensionality, the method presented here can be readily applied to molecular simulation, where an interaction cutoff radius is commonly used and the number of atoms or molecules within the cutoff radius is not constant. The method described here is general and can be applied to any machine learning technique that normally operates under a fixed number of inputs. In particular, the method described here is also useful for interpolating methods other than kriging, which may suffer from difficulties stemming from identical sets of inputs corresponding to different outputs or input biasing. As a demonstration, the new method is used to predict 54 energetic and electrostatic properties of the central water molecule of a set of 5000, 4 Å radius water clusters, with a variable number of water molecules. The results are validated against equivalent models from a set of clusters composed of a fixed number of water molecules (set to ten, i.e., decamers) and against models created by using a naïve method of treating the variable number of inputs problem presented. Results show that the 4 Å water cluster models, utilising the method presented here, return similar or better kriging models than the decamer clusters for all properties considered and perform much better than the truncated models. PMID:27634248

  19. Sampling saddle points on a free energy surface.

    PubMed

    Samanta, Amit; Chen, Ming; Yu, Tang-Qing; Tuckerman, Mark; E, Weinan

    2014-04-28

    Many problems in biology, chemistry, and materials science require knowledge of saddle points on free energy surfaces. These saddle points act as transition states and are the bottlenecks for transitions of the system between different metastable states. For simple systems in which the free energy depends on a few variables, the free energy surface can be precomputed, and saddle points can then be found using existing techniques. For complex systems, where the free energy depends on many degrees of freedom, this is not feasible. In this paper, we develop an algorithm for finding the saddle points on a high-dimensional free energy surface "on-the-fly" without requiring a priori knowledge the free energy function itself. This is done by using the general strategy of the heterogeneous multi-scale method by applying a macro-scale solver, here the gentlest ascent dynamics algorithm, with the needed force and Hessian values computed on-the-fly using a micro-scale model such as molecular dynamics. The algorithm is capable of dealing with problems involving many coarse-grained variables. The utility of the algorithm is illustrated by studying the saddle points associated with (a) the isomerization transition of the alanine dipeptide using two coarse-grained variables, specifically the Ramachandran dihedral angles, and (b) the beta-hairpin structure of the alanine decamer using 20 coarse-grained variables, specifically the full set of Ramachandran angle pairs associated with each residue. For the alanine decamer, we obtain a detailed network showing the connectivity of the minima obtained and the saddle-point structures that connect them, which provides a way to visualize the gross features of the high-dimensional surface.

  20. Early Science Results from the Dark Energy Survey

    NASA Astrophysics Data System (ADS)

    Ross, Ashley

    2015-04-01

    The Dark Energy Survey (DES) is a next-generation large galaxy survey designed to unravel the mystery of the nature of the dark energy that powers the current accelerated expansion of the Universe. The DES collaboration built and participated in the installation and commissioning of DECam, a 570 mega-pixel optical and near-infrared camera with a large 3 deg2 field of view, set at the prime focus of the Víctor M. Blanco 4-meter telescope in at the Cerro Tololo Inter-American Observatory in Chile. Using DECam, DES will map 5000 deg2 to a depth IAB ~ 24 and observe designated supernova survey fields at high cadence. These data will allow DES to measure positions, approximate redshifts, and shapes for 300 million galaxies, the light-curves of several thousand supernovae, and the masses of tens of thousands of galaxy clusters. Using these data, DES will use four main probes to study the properties of dark energy: galaxy clustering on large scales, weak gravitational lensing, galaxy-cluster abundance, and supernova distances. I describe the early progress of the survey and provide highlights of the science analyses that have been completed so far. These include: large-scale galaxy clustering measurements; significant detection of a cross-correlation with SPT CMB lensing maps; galaxy-shear and shear-shear correlation function measurements; discoveries of super-luminous supernovae, dozens of strong lenses, and redshift > 6 quasars; and characterization of DES galaxy clusters and SNe1a light-curves.

  1. Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

    PubMed

    Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

    2015-12-28

    The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.

  2. Hydrophobic stabilization of chiton hemocyanins: effects of ureas, Hofmeister salts and pH on their dissociation.

    PubMed

    Herskovits, T T; Hamilton, M G

    1987-09-24

    The subunit dissociation of the hemocyanins from five members of the Polyplacophora families, Acanthochitonidae, Callistoplacidae, Chitonidae, Ischnochitonidae and Mopalidae, represented by the chitons Cryptochiton stelleri, Nutallina fluxa, Acanthopleura granulata, Stenoplax conspicua and Mopalia mucosa, respectively, have been investigated by light-scattering molecular-weight and ultracentrifugation methods, using the hydrophobic reagents of the urea series and the Hofmeister salt series as probes of the contact areas of the hemocyanin subunits. The polyplacophoran hemocyanins are decamers with molecular weights of (4.2-4.6) X 10(6). The effectiveness of dissociation by the ureas follows the order of increasing hydrophobicity of the reagent, i.e., urea, methyl-, ethyl-, propyl- and butylurea, as expected of hydrophobically stabilized subunit systems. The urea dissociation is found to be a two-step reaction: the dissociation of parent decamers to dimers followed by dissociation of the dimers to monomers. Analysis of the observed decrease in molecular weight requires the interaction with urea of about 27 to 35 apparent amino-acid groups (Napp) at the contact areas of the dimers, and a much larger number of apparent binding groups ranging from about 100 to 120 per monomer at the contact areas of the monomers. Fitting of the pH dissociation profiles of A. granulata, C. stelleri, M. muscosa and S. conspicua requires the participation of a much smaller number of amino-acid residues in the interaction with the probe-solvent components. The ionization or protonation of one acidic and one basic group per dimer, and five to eight acidic and basic groups per monomer is found to be adequate for the description of the two-step pH dissociation reaction.

  3. Application of the Dark Energy Survey Data Management System to the Blanco Cosmology Survey Data

    NASA Astrophysics Data System (ADS)

    Ngeow, Chow Choong; Mohr, J. J.; Barkhouse, W.; Alam, T.; Beldica, C.; Cai, D.; Daues, G.; Duda, P.; Annis, J.; Lin, H.; Tucker, D.; Rest, A.; Smith, C.; Lin, Y.; High, W.; Hansen, S.; Brodwin, M.; Allam, S.; BCS Collaboration

    2006-12-01

    The Dark Energy Survey (DES; operations 2010-2016) will image 5000 deg2 of the southern sky using a new 3 deg2 imager (DECam) for the CTIO Blanco 4-m telescope. The total data volume after the end of the survey will exceed 1 peta-byte, which requires our data management system (DMS) to offer a high degree of automated processing. Our DMS leverages the existing high performance computing infrastructure to meet the project's goals. The DESDMS consists of (1) processing pipelines with built in quality assurance testing, (2) a distributed archive to support automated data processing and calibration, (3) a catalog archive database to support scientific analysis, (4) web portals for control, monitoring, user data access and scientific analysis, and (5) hardware platforms required for operations. We have tested our early version of DMS using both of the simulated DECam data from Fermilab and the observed data from the Blanco Cosmology Survey (BCS), which is a 45-night NOAO survey program. The aim of BCS is to study the cosmic acceleration using the galaxy cluster survey (in coordination with APEX, ACT and SPT) and the galaxy power spectrum. The BCS employs the MOSAIC-II imager currently installed on the Blanco telescope to carry out the deep, griz photometric survey of two 50 deg2 patches of the southern sky. The flexibility and scalabity of our DMS allows the automatic reduction of the BCS data to be done on local workstations, which is convenient because of the two orders of magnitude lower data volume compared to DES. We report our preliminary results from reducing the BCS data for the first two observing semesters with our DESDMS. We present survey completeness limits, astrometric and photometric accuracy, photometric redshift estimates and a preliminary summary of optical cluster finding and the galaxy angular power spectrum.

  4. Kriging atomic properties with a variable number of inputs

    NASA Astrophysics Data System (ADS)

    Davie, Stuart J.; Di Pasquale, Nicodemo; Popelier, Paul L. A.

    2016-09-01

    A new force field called FFLUX uses the machine learning technique kriging to capture the link between the properties (energies and multipole moments) of topological atoms (i.e., output) and the coordinates of the surrounding atoms (i.e., input). Here we present a novel, general method of applying kriging to chemical systems that do not possess a fixed number of (geometrical) inputs. Unlike traditional kriging methods, which require an input system to be of fixed dimensionality, the method presented here can be readily applied to molecular simulation, where an interaction cutoff radius is commonly used and the number of atoms or molecules within the cutoff radius is not constant. The method described here is general and can be applied to any machine learning technique that normally operates under a fixed number of inputs. In particular, the method described here is also useful for interpolating methods other than kriging, which may suffer from difficulties stemming from identical sets of inputs corresponding to different outputs or input biasing. As a demonstration, the new method is used to predict 54 energetic and electrostatic properties of the central water molecule of a set of 5000, 4 Å radius water clusters, with a variable number of water molecules. The results are validated against equivalent models from a set of clusters composed of a fixed number of water molecules (set to ten, i.e., decamers) and against models created by using a naïve method of treating the variable number of inputs problem presented. Results show that the 4 Å water cluster models, utilising the method presented here, return similar or better kriging models than the decamer clusters for all properties considered and perform much better than the truncated models.

  5. Oligomeric-state-dependent conformational change of the BLUF protein TePixD (Tll0078).

    PubMed

    Tanaka, Keisuke; Nakasone, Yusuke; Okajima, Koji; Ikeuchi, Masahiko; Tokutomi, Satoru; Terazima, Masahide

    2009-03-13

    The photochemical reaction dynamics of a BLUF (sensors of blue light using FAD) protein, PixD, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) were studied by pulsed laser-induced transient grating method. After the formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as expansion of partial molar volume with a time constant of 40 micros, whereas the other reaction phase represented a change in the diffusion coefficient D [i.e., the diffusion-sensitive conformational change (DSCC)]. D decreased from 4.9 x 10(-11) to 4.4 x 10(-11) m2 s(-1) upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 x 10(-11) m2 s(-1) upon formation of the second intermediate. From a global analysis of signals at various grating wavenumbers, the time constant of D-change was determined to be 4 ms. Although the magnitude and rate constant of the faster volume change were independent of protein concentration, the amplitude of the signal that reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution: a reactive species exhibiting the DSCC, and a second species that is nonreactive. The fraction of these species was found to be dependent on the concentration. The difference in reactivity was attributed to the different oligomeric states of TePixD (i.e., pentamer and decamer). The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in the signal transduction of this photosensory protein.

  6. Static and statistical bending of DNA evaluated by Monte Carlo simulations.

    PubMed Central

    Zhurkin, V B; Ulyanov, N B; Gorin, A A; Jernigan, R L

    1991-01-01

    To investigate the influence of thermal fluctuations on DNA curvature the Metropolis procedure at 300 K was applied to B-DNA decamers containing A5.T5 and A4.T4 blocks. Monte Carlo simulations have confirmed the DNA bending anisotropy: B-DNA bends most easily in a groove direction (roll). The A5.T5 block is more rigid than the other sequences; the pyrimidine-purine dimers are found to be the most flexible. For A5TCTCT, A5CTCTC, and A5GAGAG, the average bend angle per decamer is 20-25 degrees in a direction toward the minor groove in the center of the A5.T5 tract, which is consistent with both the "junction" and "wedge AA" models. However, in A5T5, A4T4CG, and T4A4GC, bending is directed into the grooves at the 5' and 3' ends of purine tracts. Thus, directionality of bending caused by An.Tn blocks strongly depends on their neighboring sequences. These calculations demonstrate that the sequence-dependent variation of the minor-groove width mimics the observed hydroxyl radical cleavage pattern. To estimate the effect of fluctuations on the overall shape of curved DNA fragments, longer pieces of DNA (up to 200 base pairs) were generated. For sequences with strong curvature (A5X5 and A4T4CG), the static model and Monte Carlo ensemble give similar results but, for moderately and slightly curved sequences (A5T5 or T4A4GC), the static model predicts a much smaller degree of bending than does the statistical representation. Considering fluctuations is important for quantitative interpretation of the gel electrophoresis measurements of DNA curvature, where both the static and statistical bends are operative. PMID:1871119

  7. A HERO'S LITTLE HORSE: DISCOVERY OF A DISSOLVING STAR CLUSTER IN PEGASUS

    SciTech Connect

    Kim, Dongwon; Jerjen, Helmut E-mail: helmut.jerjen@anu.edu.au

    2015-01-20

    We report the discovery of an ultra-faint stellar system in the constellation of Pegasus. This concentration of stars was detected by applying our overdensity detection algorithm to the Sloan Digital Sky Survey Data Release 10 and confirmed with deeper photometry from the Dark Energy Camera (DECam) at the 4 m Blanco telescope. The best-fitting model isochrone indicates that this stellar system, Kim 1, features an old (12 Gyr) and metal-poor ([Fe/H] ∼ -1.7) stellar population at a heliocentric distance of 19.8 ± 0.9 kpc. We measure a half-light radius of 6.9 ± 0.6 pc using a Plummer profile. The small physical size and the extremely low luminosity are comparable to the faintest known star clusters Segue 3, Koposov 1 and 2, and Muñoz 1. However, Kim 1 exhibits a lower star concentration and is lacking a well-defined center. It also has an unusually high ellipticity and irregular outer isophotes, which suggests that we are seeing an intermediate mass star cluster being stripped by the Galactic tidal field. An extended search for evidence of an associated stellar stream within the 3 deg{sup 2} DECam field remains inconclusive. The finding of Kim 1 is consistent with current overdensity detection limits and supports the hypothesis that there are still a substantial number of extreme low-luminosity star clusters undetected in the wider Milky Way halo.

  8. Sampling saddle points on a free energy surface

    NASA Astrophysics Data System (ADS)

    Samanta, Amit; Chen, Ming; Yu, Tang-Qing; Tuckerman, Mark; E, Weinan

    2014-04-01

    Many problems in biology, chemistry, and materials science require knowledge of saddle points on free energy surfaces. These saddle points act as transition states and are the bottlenecks for transitions of the system between different metastable states. For simple systems in which the free energy depends on a few variables, the free energy surface can be precomputed, and saddle points can then be found using existing techniques. For complex systems, where the free energy depends on many degrees of freedom, this is not feasible. In this paper, we develop an algorithm for finding the saddle points on a high-dimensional free energy surface "on-the-fly" without requiring a priori knowledge the free energy function itself. This is done by using the general strategy of the heterogeneous multi-scale method by applying a macro-scale solver, here the gentlest ascent dynamics algorithm, with the needed force and Hessian values computed on-the-fly using a micro-scale model such as molecular dynamics. The algorithm is capable of dealing with problems involving many coarse-grained variables. The utility of the algorithm is illustrated by studying the saddle points associated with (a) the isomerization transition of the alanine dipeptide using two coarse-grained variables, specifically the Ramachandran dihedral angles, and (b) the beta-hairpin structure of the alanine decamer using 20 coarse-grained variables, specifically the full set of Ramachandran angle pairs associated with each residue. For the alanine decamer, we obtain a detailed network showing the connectivity of the minima obtained and the saddle-point structures that connect them, which provides a way to visualize the gross features of the high-dimensional surface.

  9. Deep Surveys for Inner Oort Cloud Objects

    NASA Astrophysics Data System (ADS)

    Trujillo, Chadwick A.; Tholen, David J.; Sheppard, Scott S.

    2015-11-01

    We are undertaking two deep wide-field surveys to discover extremely distant solar system objects. While our target solar system population is the Inner Oort Cloud objects such as 2012 VP113 and Sedna, we are also sensitive to other populations with high perihelia such as the Scattered Kuiper Belt Objects and the highest perihelion Kuiper Belt Objects which have similar arguments of perihelion to the Inner Oort Cloud Objects. These unusual populations are thought to consist primarily of highly eccentric objects which spend most of their orbits hundreds or thousands of AU from the sun. Large aperture telescopes are needed to reach the faintness limits, red magnitudes of 23.5 to 25, required for detection of even the large members of the population. In addition, wide fields of view are also needed since the sky density of the detectable members of the populations approach 1 in 100 square degrees even with large telescopes.Our primary discovery instruments are the Dark Energy Camera (DECam) on the 4 meter Blanco Telescope at the Cerro Tololo Inter-American Observatory and Hyper Suprime-Cam (HSC) on Subaru Telescope at Maunakea. Each of these instruments has a tremendously wide field of view considering the size of the telescope they are mounted on. DECam has a field of view of about 3 square degrees and HSC has a field of view of about 1.75 square degrees. We will present our survey progress in terms of sky area covered and new objects discovered and highlight some of our more interesting findings.

  10. Capture of a Transition State Using Molecular Dynamics: Creation of an Intercalation Site in dsDNA with Ethidium Cation.

    PubMed

    Monaco, Regina R

    2010-03-31

    The mechanism of intercalation and the ability of double stranded DNA (dsDNA) to accommodate a variety of ligands in this manner has been well studied. Proposed mechanistic steps along this pathway for the classical intercalator ethidium have been discussed in the literature. Some previous studies indicate that the creation of an intercalation site may occur spontaneously, with the energy for this interaction arising either from solvent collisions or soliton propagation along the helical axis. A subsequent 1D diffusional search by the ligand along the helical axis of the DNA will allow the ligand entry to this intercalation site from its external, electrostatically stabilized position. Other mechanistic studies show that ethidium cation participates in the creation of the site, as a ligand interacting closely with the external surface of the DNA can cause unfavorable steric interactions depending on the ligands' orientation, which are relaxed during the creation of an intercalation site. Briefly, such a site is created by the lengthening of the DNA molecule via bond rotation between the sugars and phosphates along the DNA backbone, causing an unwinding of the dsDNA itself and separation between the adjacent base pairs local to the position of the ligand, which becomes the intercalation site. Previous experimental measurements of this interaction measure the enthalpic cost of this part of the mechanism to be about -8 kcal/mol. This paper reports the observation, during a computational study, of the spontaneous opening of an intercalation site in response to the presence of a single ethidium cation molecule in an externally bound configuration. The concerted motions between this ligand and the host, a dsDNA decamer, are clear. The dsDNA decamer AGGATGCCTG was studied; the central ...GATG... site was the intercalation site.

  11. Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site

    PubMed Central

    Walsh, Martin A; Otwinowski, Zbyszek; Perrakis, Anatassis; Anderson, Paul M; Joachimiak, Andrzej

    2011-01-01

    Background Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. Results We have determined the crystal structure of cyanase at 1.65 Å resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. Conclusions The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding α-helix bundle motif. The C-terminal domain has an ‘open fold’ with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed. PMID:10801492

  12. Reaction of the N-terminal methionine residues in cyanase with diethylpyrocarbonate.

    PubMed

    Anderson, P M; Korte, J J; Holcomb, T A

    1994-11-29

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The enzyme is a decamer of identical subunits (M(r) = 17,000). Previous studies have shown that modification of either the single cysteine residue or the single histidine residue in each subunit gives an active decameric derivative that dissociates reversibly to inactive dimer derivative, indicating that decameric structure is required for activity and that the SH and imidazole groups are not required for catalytic activity [Anderson, P. M., Korte, J. J., Holcomb, T. A., Cho, Y.-G., Son, C.-M., & Sung, Y.-C. (1994) J. Biol. Chem. 269, 15036-15045]. Here the effects of reaction of the reagent diethylpyrocarbonate (DEPC) with cyanase or mutant cyanases are reported. DEPC reacts stoichiometrically with the histidine residue and at one additional site in each subunit when the enzyme is in the inactive dimer form, preventing reactivation. DEPC reacts stoichiometrically (with the same result on reactivation) at only one site per subunit with the inactive dimer form of cyanase mutants in which the single histidine residue has been replaced by one of several different amino acids by site-directed mutagenesis; the site of the reaction was identified as the amino group of the N-terminal methionine. DEPC does not react with the histidine residue of the active decameric form of wild-type cyanase and does not affect activity of the active decameric form of wild-type or mutant cyanases. Reaction with the N-terminal amino group of methionine apparently prevents reactivation of the mutant enzymes by blocking association to decamer.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Kriging atomic properties with a variable number of inputs.

    PubMed

    Davie, Stuart J; Di Pasquale, Nicodemo; Popelier, Paul L A

    2016-09-14

    A new force field called FFLUX uses the machine learning technique kriging to capture the link between the properties (energies and multipole moments) of topological atoms (i.e., output) and the coordinates of the surrounding atoms (i.e., input). Here we present a novel, general method of applying kriging to chemical systems that do not possess a fixed number of (geometrical) inputs. Unlike traditional kriging methods, which require an input system to be of fixed dimensionality, the method presented here can be readily applied to molecular simulation, where an interaction cutoff radius is commonly used and the number of atoms or molecules within the cutoff radius is not constant. The method described here is general and can be applied to any machine learning technique that normally operates under a fixed number of inputs. In particular, the method described here is also useful for interpolating methods other than kriging, which may suffer from difficulties stemming from identical sets of inputs corresponding to different outputs or input biasing. As a demonstration, the new method is used to predict 54 energetic and electrostatic properties of the central water molecule of a set of 5000, 4 Å radius water clusters, with a variable number of water molecules. The results are validated against equivalent models from a set of clusters composed of a fixed number of water molecules (set to ten, i.e., decamers) and against models created by using a naïve method of treating the variable number of inputs problem presented. Results show that the 4 Å water cluster models, utilising the method presented here, return similar or better kriging models than the decamer clusters for all properties considered and perform much better than the truncated models.

  14. Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

    PubMed

    Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

    2015-01-01

    The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level. PMID:26782491

  15. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

    PubMed Central

    Winkler, F K; Banner, D W; Oefner, C; Tsernoglou, D; Brown, R S; Heathman, S P; Bryan, R K; Martin, P D; Petratos, K; Wilson, K S

    1993-01-01

    The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B-form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base-specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co-operative as they are all made through one short surface loop comprising residues 182-186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme. Images PMID:8491171

  16. Effect of Electrostatics on Aggregation of Prion Protein Sup35 Peptide

    PubMed Central

    Portillo, Alexander M.; Krasnoslobodtsev, Alexey V.; Lyubchenko, Yuri L.

    2012-01-01

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic Force Microscopy (AFM) and Thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI=5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0) suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by the experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 hours, whereas the lag time decreases ~5 times when ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 hours under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. Ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics on dissociation of transient peptide dimers. PMID:22466073

  17. Effect of electrostatics on aggregation of prion protein Sup35 peptide

    NASA Astrophysics Data System (ADS)

    Portillo, Alexander M.; Krasnoslobodtsev, Alexey V.; Lyubchenko, Yuri L.

    2012-04-01

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. The GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI = 5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0), suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 h, whereas the lag time decreases ˜5 times when the ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 h under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. The ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics to dissociation of transient peptide dimers.

  18. Exploring the role of hydration and confinement in the aggregation of amyloidogenic peptides Aβ16-22 and Sup357-13 in AOT reverse micelles

    NASA Astrophysics Data System (ADS)

    Martinez, Anna Victoria; Małolepsza, Edyta; Rivera, Eva; Lu, Qing; Straub, John E.

    2014-12-01

    Knowledge of how intermolecular interactions of amyloid-forming proteins cause protein aggregation and how those interactions are affected by sequence and solution conditions is essential to our understanding of the onset of many degenerative diseases. Of particular interest is the aggregation of the amyloid-β (Aβ) peptide, linked to Alzheimer's disease, and the aggregation of the Sup35 yeast prion peptide, which resembles the mammalian prion protein linked to spongiform encephalopathies. To facilitate the study of these important peptides, experimentalists have identified small peptide congeners of the full-length proteins that exhibit amyloidogenic behavior, including the KLVFFAE sub-sequence, Aβ16-22, and the GNNQQNY subsequence, Sup357-13. In this study, molecular dynamics simulations were used to examine these peptide fragments encapsulated in reverse micelles (RMs) in order to identify the fundamental principles that govern how sequence and solution environment influence peptide aggregation. Aβ16-22 and Sup357-13 are observed to organize into anti-parallel and parallel β-sheet arrangements. Confinement in the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles is shown to stabilize extended peptide conformations and enhance peptide aggregation. Substantial fluctuations in the reverse micelle shape are observed, in agreement with earlier studies. Shape fluctuations are found to facilitate peptide solvation through interactions between the peptide and AOT surfactant, including direct interaction between non-polar peptide residues and the aliphatic surfactant tails. Computed amide I IR spectra are compared with experimental spectra and found to reflect changes in the peptide structures induced by confinement in the RM environment. Furthermore, examination of the rotational anisotropy decay of water in the RM demonstrates that the water dynamics are sensitive to the presence of peptide as well as the peptide sequence. Overall, our results

  19. Ir-Uv Double Resonance Spectroscopy of a Cold Protonated Fibril-Forming Peptide: NNQQNY\\cdotH+

    NASA Astrophysics Data System (ADS)

    DeBlase, Andrew F.; Harrilal, Christopher P.; Walsh, Patrick S.; McLuckey, Scott A.; Zwier, Timothy S.

    2016-06-01

    Protein aggregation to form amyloid-like fibrils is a purported molecular manifestation that leads to Alzheimer's, Huntington's, and other neurodegenerative diseases. The propensity for a protein to aggregate is often driven by the presence of glutamine (Q) and asparagine (N) rich tracts within the primary sequence. For example, Eisenberg and coworkers [Nature 2006, 435, 773] have shown by X-ray crystallography that the peptides NNQQNY and GNNQQNY aggregate into a parallel β-sheet configuration with side chains that intercalate into a "steric zipper". These sequences are commonly found at the N-terminus of the prion-determining domain in the yeast protein Sup35, a typical fibril-forming protein. Herein, we invoke recent advances in cold ion spectroscopy to explore the nascent conformational preferences of the protonated peptides that are generated by electrospray ionization. Towards this aim, we have used UV and IR spectroscopy to record conformation-specific photofragment action spectra of the NNQQNY monomer cryogenically cooled in an octopole ion trap. This short peptide contains 20 hydride stretch oscillators, leading to a rich infrared spectrum with at least 18 resolved transitions in the 2800-3800 cm-1 region. The infrared spectrum suggests the presence of both a free acid OH moiety and an H-bonded tyrosine OH group. We compare our results with resonant ion dip infrared spectra (RIDIRS) of the acyl/NH-benzyl capped neutral glutamine amino acid and its corresponding dipeptide: Ac-Q-NHBn and Ac-QQ-NHBn, respectively. These comparisons bring empirical insight to the NH stretching region of the spectrum, which contains contributions from free and singly H-bonded NH2 side-chain groups, and from peptide backbone amide NH groups. We further compare our spectrum to harmonic calculations at the M05-2X/6-31+G* level of theory, which were performed on low energy structures obtained from Monte Carlo conformational searches using the Amber* and OPLS force fields to assess

  20. Exploring the role of hydration and confinement in the aggregation of amyloidogenic peptides Aβ(16-22) and Sup35(7-13) in AOT reverse micelles.

    PubMed

    Martinez, Anna Victoria; Małolepsza, Edyta; Rivera, Eva; Lu, Qing; Straub, John E

    2014-12-14

    Knowledge of how intermolecular interactions of amyloid-forming proteins cause protein aggregation and how those interactions are affected by sequence and solution conditions is essential to our understanding of the onset of many degenerative diseases. Of particular interest is the aggregation of the amyloid-β (Aβ) peptide, linked to Alzheimer's disease, and the aggregation of the Sup35 yeast prion peptide, which resembles the mammalian prion protein linked to spongiform encephalopathies. To facilitate the study of these important peptides, experimentalists have identified small peptide congeners of the full-length proteins that exhibit amyloidogenic behavior, including the KLVFFAE sub-sequence, Aβ16-22, and the GNNQQNY subsequence, Sup357-13. In this study, molecular dynamics simulations were used to examine these peptide fragments encapsulated in reverse micelles (RMs) in order to identify the fundamental principles that govern how sequence and solution environment influence peptide aggregation. Aβ16-22 and Sup357-13 are observed to organize into anti-parallel and parallel β-sheet arrangements. Confinement in the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles is shown to stabilize extended peptide conformations and enhance peptide aggregation. Substantial fluctuations in the reverse micelle shape are observed, in agreement with earlier studies. Shape fluctuations are found to facilitate peptide solvation through interactions between the peptide and AOT surfactant, including direct interaction between non-polar peptide residues and the aliphatic surfactant tails. Computed amide I IR spectra are compared with experimental spectra and found to reflect changes in the peptide structures induced by confinement in the RM environment. Furthermore, examination of the rotational anisotropy decay of water in the RM demonstrates that the water dynamics are sensitive to the presence of peptide as well as the peptide sequence. Overall, our results

  1. Exploring the role of hydration and confinement in the aggregation of amyloidogenic peptides Aβ16−22 and Sup357−13 in AOT reverse micelles

    PubMed Central

    Martinez, Anna Victoria; Małolepsza, Edyta; Rivera, Eva; Lu, Qing; Straub, John E.

    2014-01-01

    Knowledge of how intermolecular interactions of amyloid-forming proteins cause protein aggregation and how those interactions are affected by sequence and solution conditions is essential to our understanding of the onset of many degenerative diseases. Of particular interest is the aggregation of the amyloid-β (Aβ) peptide, linked to Alzheimer's disease, and the aggregation of the Sup35 yeast prion peptide, which resembles the mammalian prion protein linked to spongiform encephalopathies. To facilitate the study of these important peptides, experimentalists have identified small peptide congeners of the full-length proteins that exhibit amyloidogenic behavior, including the KLVFFAE sub-sequence, Aβ16−22, and the GNNQQNY subsequence, Sup357−13. In this study, molecular dynamics simulations were used to examine these peptide fragments encapsulated in reverse micelles (RMs) in order to identify the fundamental principles that govern how sequence and solution environment influence peptide aggregation. Aβ16−22 and Sup357−13 are observed to organize into anti-parallel and parallel β-sheet arrangements. Confinement in the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles is shown to stabilize extended peptide conformations and enhance peptide aggregation. Substantial fluctuations in the reverse micelle shape are observed, in agreement with earlier studies. Shape fluctuations are found to facilitate peptide solvation through interactions between the peptide and AOT surfactant, including direct interaction between non-polar peptide residues and the aliphatic surfactant tails. Computed amide I IR spectra are compared with experimental spectra and found to reflect changes in the peptide structures induced by confinement in the RM environment. Furthermore, examination of the rotational anisotropy decay of water in the RM demonstrates that the water dynamics are sensitive to the presence of peptide as well as the peptide sequence. Overall, our results

  2. Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

    SciTech Connect

    Wang, Wenhui; Chen, Xilei; Li, Tao; Li, Yanmei; Wang, Ruixue; He, Dan; Luo, Wu; Li, Xiaokun; Wu, Xiaoping

    2013-05-01

    Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.

  3. Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus

    PubMed Central

    Ojeda, Nicolás; Cárdenas, Constanza; Guzmán, Fanny

    2016-01-01

    ABSTRACT Infectious salmon anemia virus (ISAV) is the etiological agent of the disease by the same name and causes major losses in the salmon industry worldwide. Epizootic ISAV outbreaks have occurred in Norway and, to a lesser degree, in Canada. In 2007, an ISAV outbreak in Chile destroyed most of the seasonal production and endangered the entire Chilean salmon industry. None of the existing prophylactic approaches have demonstrated efficacy in providing absolute protection from or even a palliative effect on ISAV proliferation. Sanitary control measures for ISAV, based on molecular epidemiology data, have proven insufficient, mainly due to high salmon culture densities and a constant presence of a nonpathogenic strain of the virus. This report describes an alternative treatment approach based on interfering peptides selected from a phage display library. The screening of a phage display heptapeptide library resulted in the selection of a novel peptide with significant in vitro antiviral activity against ISAV. This peptide specifically interacted with the viral hemagglutinin-esterase protein, thereby impairing virus binding, with plaque reduction assays showing a significant reduction in viral yields. The identified peptide acts at micromolar concentrations against at least two different pathogenic strains of the virus, without detectable cytotoxic effects on the tested fish cells. Therefore, antiviral peptides represent a novel alternative for controlling ISAV and, potentially, other fish pathogens. IMPORTANCE Identifying novel methods for the efficient control of infectious diseases is imperative for the future of global aquaculture. The present study used a phage display heptapeptide library to identify a peptide with interfering activity against a key protein of the infectious salmon anemia virus (ISAV). A piscine orthomyxovirus, ISAV is a continuous threat to the commercial sustainability of cultured salmon production worldwide. The complex epidemiological

  4. Measuring the Flatness of Focal Plane for Very Large Mosaic CCD Camera

    SciTech Connect

    Hao, Jiangang; Estrada, Juan; Cease, Herman; Diehl, H.Thomas; Flaugher, Brenna L.; Kubik, Donna; Kuk, Keivin; Kuropatkine, Nickolai; Lin, Huan; Montes, Jorge; Scarpine, Vic; /Fermilab

    2010-06-08

    Large mosaic multiCCD camera is the key instrument for modern digital sky survey. DECam is an extremely red sensitive 520 Megapixel camera designed for the incoming Dark Energy Survey (DES). It is consist of sixty two 4k x 2k and twelve 2k x 2k 250-micron thick fully-depleted CCDs, with a focal plane of 44 cm in diameter and a field of view of 2.2 square degree. It will be attached to the Blanco 4-meter telescope at CTIO. The DES will cover 5000 square-degrees of the southern galactic cap in 5 color bands (g, r, i, z, Y) in 5 years starting from 2011. To achieve the science goal of constraining the Dark Energy evolution, stringent requirements are laid down for the design of DECam. Among them, the flatness of the focal plane needs to be controlled within a 60-micron envelope in order to achieve the specified PSF variation limit. It is very challenging to measure the flatness of the focal plane to such precision when it is placed in a high vacuum dewar at 173 K. We developed two image based techniques to measure the flatness of the focal plane. By imaging a regular grid of dots on the focal plane, the CCD offset along the optical axis is converted to the variation the grid spacings at different positions on the focal plane. After extracting the patterns and comparing the change in spacings, we can measure the flatness to high precision. In method 1, the regular dots are kept in high sub micron precision and cover the whole focal plane. In method 2, no high precision for the grid is required. Instead, we use a precise XY stage moves the pattern across the whole focal plane and comparing the variations of the spacing when it is imaged by different CCDs. Simulation and real measurements show that the two methods work very well for our purpose, and are in good agreement with the direct optical measurements.

  5. New dwarf galaxy candidates in the Centaurus group

    NASA Astrophysics Data System (ADS)

    Müller, Oliver; Jerjen, Helmut; Binggeli, Bruno

    2015-11-01

    Context. Recent studies of the distribution and kinematics of the Milky Way and Andromeda satellite galaxy systems have confirmed the existence of coplanar, corotating structures of galaxies. In addition to the "missing satellite problem", these structures pose a major challenge to the standard ΛCDM scenario of structure formation. Aims: We complement the efforts made by the dwarf galaxy community to extend these studies to other nearby galaxy groups by systematically searching for faint unresolved dwarf members with a low surface brightness in the Southern Centaurus group of galaxies. The aim is to determine whether these coplanar, corotating structures are a universal phenomenon. Methods: We imaged an area of 60 sq. deg (0.3 Mpc2) around the M 83 subgroup with the wide-field Dark Energy Camera (DECam) at the CTIO 4 m Blanco telescope in g and r down to a limiting surface brightness of μr ≈ 30 mag arcsec-2. Various image-filtering techniques were applied to the DECam data to enhance the visibility of extremely low-surface brightness objects. Results: We report the discovery of 16 new dwarf galaxy candidates in the direction of the M 83 subgroup, roughly doubling the number of known dwarfs in that region. The photometric properties of the candidates, when compared to those of the Local Group, suggest membership in the M 83 subgroup. The faintest objects have a central star density of ≈1.3 L⊙ pc-2 and a total magnitude of g = 20.25, corresponding to Mg = -9.55 at the nominal distance of 4.9 Mpc. The sky distribution of the new objects is significantly prolonged toward Cen A, suggesting that many of them belong to the Cen A subgroup or a common halo. We also provide updated surface photometry for the brighter, known dwarf members in the surveyed area. Conclusions: Modern survey CCD cameras and sophisticated detection algorithms can be used to systematically probe the faint end of the galaxy luminosity function around the M 83 subgroup of galaxies. We aim at

  6. Discovery of Rare and Highly Toxic Microcystins from Lichen-Associated Cyanobacterium Nostoc sp. Strain IO-102-I

    PubMed Central

    Oksanen, Ilona; Jokela, Jouni; Fewer, David P.; Wahlsten, Matti; Rikkinen, Jouko; Sivonen, Kaarina

    2004-01-01

    The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda5]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda5]microcystin-LR and [d-Asp3,ADMAdda5]microcystin-LR and a partial structure of three new [ADMAdda5]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis. PMID:15466511

  7. 1,4-disubstituted-[1,2,3]triazolyl-containing analogues of MT-II: design, synthesis, conformational analysis, and biological activity.

    PubMed

    Testa, Chiara; Scrima, Mario; Grimaldi, Manuela; D'Ursi, Anna M; Dirain, Marvin L; Lubin-Germain, Nadège; Singh, Anamika; Haskell-Luevano, Carrie; Chorev, Michael; Rovero, Paolo; Papini, Anna M

    2014-11-26

    Side chain-to-side chain cyclizations represent a strategy to select a family of bioactive conformations by reducing the entropy and enhancing the stabilization of functional ligand-induced receptor conformations. This structural manipulation contributes to increased target specificity, enhanced biological potency, improved pharmacokinetic properties, increased functional potency, and lowered metabolic susceptibility. The Cu(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen's cycloaddition, the prototypic click reaction, presents a promising opportunity to develop a new paradigm for an orthogonal bioorganic and intramolecular side chain-to-side chain cyclization. In fact, the proteolytic stable 1,4- or 4,1-disubstituted [1,2,3]triazolyl moiety is isosteric with the peptide bond and can function as a surrogate of the classical side chain-to-side chain lactam forming bridge. Herein we report the design, synthesis, conformational analysis, and functional biological activity of a series of i-to-i+5 1,4- and 4,1-disubstituted [1,2,3]triazole-bridged cyclopeptides derived from MT-II, the homodetic Asp(5) to Lys(10) side chain-to-side chain bridged heptapeptide, an extensively studied agonist of melanocortin receptors. PMID:25347033

  8. The Structural Basis for Peptidomimetic Inhibition of Eukaryotic Ribonucleotide Reductase: A Conformationally Flexible Pharmacophore

    SciTech Connect

    Xu, Hai; Fairman, James W.; Wijerathna, Sanath R.; Kreischer, Nathan R.; LaMacchia, John; Helmbrecht, Elizabeth; Cooperman, Barry S.; Dealwis, Chris

    2008-08-19

    Eukaryotic ribonucleotide reductase (RR) catalyzes nucleoside diphosphate conversion to deoxynucleoside diphosphate. Crucial for rapidly dividing cells, RR is a target for cancer therapy. RR activity requires formation of a complex between subunits R1 and R2 in which the R2 C-terminal peptide binds to R1. Here we report crystal structures of heterocomplexes containing mammalian R2 C-terminal heptapeptide, P7 (Ac-{sup 1}FTLDADF{sup 7}) and its peptidomimetic P6 ({sup 1}Fmoc(Me)PhgLDChaDF{sup 7}) bound to Saccharomyces cerevisiae R1 (ScR1). P7 and P6, both of which inhibit ScRR, each bind at two contiguous sites containing residues that are highly conserved among eukaryotes. Such binding is quite distinct from that reported for prokaryotes. The Fmoc group in P6 peptide makes several hydrophobic interactions that contribute to its enhanced potency in binding to ScR1. Combining all of our results, we observe three distinct conformations for peptide binding to ScR1. These structures provide pharmacophores for designing highly potent nonpeptide class I RR inhibitors.

  9. Functional characterization of the C-terminal domain of mouse capping enzyme.

    PubMed

    Liu, Li

    2006-01-01

    Mouse capping enzyme (Mce1) consists of two functional domains: the amino-terminal triphosphatase domain and the carboxyl-terminal guanylyltransferase (GTase) domain. The bifunctional Mce1 gene encodes 597 a.a. with a molecular weight approximately 68 kDa. Mce1 cDNA is located on chromosome 4A4 approximately 4A5 and is composed of 17 exons. To functionally characterize the C-terminus of Mce1, we generated four truncated proteins with 12, 30, 37, or 60 a.a. deletions from the C-terminus of either the wild type (Mce1) or the isolated GTase domain (211-597), respectively. Plasmid shuffling experiment with Saccharomyces cerevisiae GTase subunit gene CEG1 null mutant demonstrated that deletion mutants 211-567 and 211-585 were able to support cell viability in the presence of 5-fluoroorotic acid, whereas 211-537 and 211-560 were not. Consistent with the yeast genetic study, both 211-567 and 211-585 had significant GTase activity in vitro, while 211-537 and 211-560 that were only detected in the insoluble fraction in the bacterial expression system, were completely inactive. Overall, both in vivo and in vitro studies indicate that the functional domain of Mce1 is between a.a. 211 and 567, and the heptapeptide sequence between 561 and 567 may play an important role in the enzyme activity. PMID:16342145

  10. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Dewdney, Tamaria G.; Reiter, Samuel J.; Brunzelle, Joseph S.; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  11. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

  12. Mapping of the gene encoding the melanocortin-1 ([alpha]-melanocyte stimulating hormone) receptor (MC1R) to human chromosome 16q24. 3 by fluorescence in situ hybridization

    SciTech Connect

    Gantz, I.; Yamada, Tadataka; Tashiro, Takao; Konda, Yoshitaka; Shimoto, Yoshimasa; Miwa, Hiroto; Trent, J.M. )

    1994-01-15

    [alpha]-Melanocyte stimulating hormone ([alpha]-MSH), a hormone originally named for its ability to regulate pigmentation of melanocytes, is a 13-amino-acid post-translational product of the pro-opiomelanocortin (POMC) gene. [alpha]-MSH and the other products of POMC processing, which share the core heptapeptide amino acid sequence Met-Glu (Gly)-His-Phe-Arg-Trp-Gly (Asp), the adrenocorticotropic hormone (ACTH), [beta]-MSH, and [gamma]-MSH, are collectively referred to as melanocortins. While best known for their effects on the melanocyte (pigmentation) and adrenal cortical cells (steroidogenesis), melanocortins have been postulated to function in diverse activities, including enhancement of learning and memory, control of the cardiovascular system, analgesia, thermoregulation, immunomodulation, parturition, and neurotrophism. To identify the chromosomal band encoding the human melanocortin-1 receptor gene, 1 [mu]g of an EMBL clone coding region of the human MC1R and approximately 15 kb of surrounding DNA was labeled with biotin and hybridized to human metaphase chromosomes as previously described. The results indicate that the human MC1R gene is localized to 16q24.3. 15 refs., 1 fig.

  13. The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver.

    PubMed Central

    Campos-Cavieres, M; Milstein, C P

    1975-01-01

    The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase. PMID:1180894

  14. Are fish fed with cyanobacteria safe, nutritious and delicious? A laboratory study.

    PubMed

    Liang, Hualei; Zhou, Wenshan; Zhang, Yulei; Qiao, Qin; Zhang, Xuezhen

    2015-10-16

    Toxic cyanobacterial blooms, which produce cyclic heptapeptide toxins known as microcystins, are worldwide environmental problems. On the other hand, the cyanobacteria protein (30-50%) has been recommended as substitute protein for aquaculture. The present laboratory study verified the feasibility of cyanobacteria protein substitution and risk assessment. Goldfish were fed diets supplemented lyophilised cyanobacteria powder for 16 weeks with the various doses: 0% (control), 10%, 20%, 30% and 40%. Low doses (10% and 20%) promoted growth whereas high doses (30% and 40%) inhibited growth. In cyanobacteria treated fish, the proximate composition of ash, crude fat content and crude protein content decreased in 16 weeks; the saturated fatty acid (SFA) content significantly increased; the n-3 polyunsaturated fatty acid content, collagen content and muscle pH significantly decreased; cooking loss percents increased significantly. Muscle fiber diameter and myofibril length were negatively correlation. Additionally, flavour compounds (e.g., amino acids, nucleotides, organic acids and carnosine) changed significantly in the treated fish, and odour compounds geosmin and 2-methylisoborneol increased significantly. The estimated daily intake (EDI) of microcystins in muscle was close to or exceeded the World Health Organization (WHO) tolerable daily intake (TDI), representing a great health risk. Cyanobacterie is not feasible for protein sources use in aquaculture.

  15. UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space.

    PubMed

    Yang, Zhen; Kong, Fanxiang

    2015-10-01

    Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m² not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine), MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space. PMID:26492272

  16. [The role of opioid system in peculiarities of anti-anxiety effect of peptide anxiolytic selank].

    PubMed

    Kozlovskiĭ, I I; Andreeva, L A; Kozlovskaia, M M; Nadorova, A V; Kolik, L G

    2012-01-01

    Peculiarities of the anxiolytic effects of selank (heptapeptide analog of taftsin) under reduced activity of opioid system upon acute administration of naloxone have been studied in BALB/C and C57BL/6 inbred mice with high and low levels of anxiety, with passive and active emotional stress reaction phenotypes in the open field (OF) test. Selank (0.25 mg/kg, i.p.) per se exhibited anxiolytic effect in BALB/C mice by increasing the general locomotor activity, with no effects on the behavior of C57BL/6 mice in the OF test. Naloxone (1.0 mg/kg, i.p.) per se evoked swift runaway in OF peripheral areas in BALB/C mice while "freezing" the reaction in C57BL/6 mice with active response to stress under the same conditions. Pretreatment with naloxone attenuated the sensitivity to selank in BALB/C mice whereas the response to anxiolytic effects of peptide was increased in C57BL/6 mice. The data obtained reveal a new target for selank in CNS and indicate significance of the activity of enkephalin-opioid system in individual sensitivity to selank.

  17. The NLP toxin family in Phytophthora sojae includes rapidly evolving groups that lack necrosis-inducing activity.

    PubMed

    Dong, Suomeng; Kong, Guanghui; Qutob, Dinah; Yu, Xiaoli; Tang, Junli; Kang, Jixiong; Dai, Tingting; Wang, Hai; Gijzen, Mark; Wang, Yuanchao

    2012-07-01

    Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

  18. Structural and functional analysis of Aplysia attractins, a family of water-borne protein pheromones with interspecific attractiveness

    PubMed Central

    Painter, Sherry D.; Cummins, Scott F.; Nichols, Amy E.; Akalal, David-B. G.; Schein, Catherine H.; Braun, Werner; Smith, John S.; Susswein, Abraham J.; Levy, Miriam; de Boer, Pamela A. C. M.; ter Maat, Andries; Miller, Mark W.; Scanlan, Cory; Milberg, Richard M.; Sweedler, Jonathan V.; Nagle, Gregg T.

    2004-01-01

    Mate attraction in Aplysia involves a long-distance water-borne signal (the protein pheromone attractin), which is released during egg laying. Aplysia californica attractin attracts species that produce closely related attractins, such as Aplysia brasiliana, whose geographic distribution does not overlap that of A. californica. This finding suggests that other mollusks release attractin-related pheromones to form and maintain breeding aggregations. We describe four additional members of the attractin family: A. brasiliana, Aplysia fasciata, Aplysia depilans (which aggregates with A. fasciata aggregations), and Aplysia vaccaria (which aggregates with A. californica aggregations). On the basis of their sequence similarity with A. californica attractin, the attractin proteins fall into two groups: A. californica, A. brasiliana, and A. fasciata (91–95% identity), and A. depilans and A. vaccaria (41–43% identity). The sequence similarity within the attractin family, the conserved six cysteines, and the compact fold of the NMR solution structure of A. californica attractin suggest a common fold for this pheromone family containing two antiparallel helices. The second helix contains the IEECKTS sequence conserved in Aplysia attractins. Mutating surface-exposed charged residues within this heptapeptide sequence abolishes attractin activity, suggesting that the second helix is an essential part of the receptor-binding interface. PMID:15118100

  19. Highly sensitive electrochemical lead ion sensor harnessing peptide probe molecules on porous gold electrodes.

    PubMed

    Su, Wenqiong; Cho, Misuk; Nam, Jae-Do; Choe, Woo-Seok; Lee, Youngkwan

    2013-10-15

    Lead ion is one of the most hazardous and ubiquitous heavy metal pollutants and poses an increasing threat to the environment and human health. This necessitates rapid and selective detection and/or removal of lead ions from various soil and water resources. Recently, we identified several Pb²⁺ binding peptides via phage display technique coupled with chromatographic biopanning (Nian et al., 2010) where a heptapeptide (TNTLSNN) capable of recognizing Pb²⁺ with high affinity and specificity evolved. In the present study, an electrochemical sensor harnessing this Pb²⁺ affinity peptide as a probe on a porous gold electrode was developed. The three dimensional porous gold electrode was obtained from electrochemical deposition using the dynamic hydrogen bubble template method. A thin layer of poly(thiophene acetic acid) (PTAA) was coated on the porous gold surface. The Pb²⁺ recognizing peptide was immobilized via amide linkage on the PTAA. The developed biosensor was demonstrated to be fast, selective and reproducible in Pb²⁺ etection, exhibiting Pb²⁺-specific peak current values around -0.15 V in a broad concentration range (1-1×10⁷ nM) in 10 min despite the repeated use after regeneration.

  20. Comparison of the level of residual coagulant activity in different cheese varieties.

    PubMed

    Bansal, Nidhi; Fox, Patrick F; McSweeney, Paul L H

    2009-08-01

    The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify residual coagulant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, followed in order by Feta=Port du Salut=Cheddar>Gouda>Emmental=Parmigiano Reggiano=low-moisture part-skim Mozzarella=Mozzarella di Bufala Campana. The high cooking temperature (50-54 degrees C) used during the manufacture of Emmental and Parmigiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity was higher in Feta cheese made from milk concentrated by ultrafiltration than in conventional Feta.

  1. UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space.

    PubMed

    Yang, Zhen; Kong, Fanxiang

    2015-10-20

    Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m² not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine), MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space.

  2. Novel fluorescently labeled peptide compounds for detection of oxidized low-density lipoprotein at high specificity.

    PubMed

    Sato, Akira; Yamanaka, Hikaru; Oe, Keitaro; Yamazaki, Yoji; Ebina, Keiichi

    2014-10-01

    The probes for specific detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. In this study, to develop a fluorescent peptide probe for specific detection of ox-LDL, we investigated the interaction of fluorescein isothiocyanate (FITC)-labeled peptides with ox-LDL using polyacrylamide gel electrophoresis. Two heptapeptides (KWYKDGD and KP6) coupled through the ε-amino group of K at the N-terminus to FITC in the presence/absence of 6-amino-n-caproic acid (AC) linker to FITC--(FITC-AC)KP6 and (FITC)KP6--both bound with high specificity to ox-LDL in a dose-dependent manner. In contrast, a tetrapeptide (YKDG) labeled with FITC at the N-terminus and a pentapeptide (YKDGK) coupled through the ε-amino group of K at the C-terminus to FITC did not bind selectively to ox-LDL. Furthermore, (FITC)KP6 and (FITC-AC)KP6 bound with high specificity to the protein in mouse plasma (probably ox-LDL fraction). These findings strongly suggest that (FITC)KP6 and (FITC-AC)KP6 may be effective novel fluorescent probes for specific detection of ox-LDL.

  3. Structural Basis for Microcin C7 Inactivation by the MccE Acetyltransferase

    SciTech Connect

    Agarwal, Vinayak; Metlitskaya, Anastasiya; Severinov, Konstantin; Nair, Satish K.

    2015-10-15

    The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE{sup AcTase}). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through p-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE{sup AcTase} can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.

  4. 3D-printed biosensor with poly(dimethylsiloxane) reservoir for magnetic separation and quantum dots-based immunolabeling of metallothionein.

    PubMed

    Heger, Zbynek; Zitka, Jan; Cernei, Natalia; Krizkova, Sona; Sztalmachova, Marketa; Kopel, Pavel; Masarik, Michal; Hodek, Petr; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

    2015-06-01

    Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous versus healthy tissue of various types of malignant tumors, including head and neck cancer. Thus, we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of MT, based on affinity of its sulfhydryl groups toward gold. Biosensor was crafted from PDMS combined with technology of 3D printing and contained reservoir with volume of 50 μL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunolabeling of immobilized MT anti-MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabeling (120 min, 20°C, and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of MT in cell lines derived from spinocellular carcinoma (cell line 122P-N) and fibroblasts (122P-F) and levels of the biomarker were found to be about 90 nM in tumor cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 μL), with low acquisition costs and high portability. PMID:25735231

  5. In the african lungfish Met-enkephalin and leu-enkephalin are derived from separate genes: cloning of a proenkephalin cDNA.

    PubMed

    Dores, R M; Lee, J; Sollars, C; Danielson, P; Lihrmann, I; Vallarino, M; Vaudry, H

    2000-10-01

    A full-length proenkephalin cDNA (accession number: AF232670) was cloned from an African lungfish (Protopterus annectens) brain cDNA library. The 1,351-bp African lungfish proenkephalin contains an open reading frame that codes 266 amino acids and a stop codon. Within the sequence of lungfish proenkephalin there are 5 pentapeptide opioid sequences (all YGGFM), 1 octapeptide opioid sequence (YGGFMRSL) and 1 heptapeptide opioid sequence (YGGFMGY). A Leu-enkephalin sequence was conspicuously absent in lungfish proenkephalin. These results, coupled with observations on the organization of amphibian proenkephalin and mammalian proenkephalin, indicate that among the Sarcopterygii (lobed finned fish and tetrapods), the appearance of a Leu-enkephalin sequence in proenkephalin may have evolved in either the ancestral amniotes or the ancestral mammals, but not earlier in sarcopterygian evolution. Furthermore, the detection of neurons in the lungfish CNS that are only immunopositive for Met-enkephalin, coupled with earlier anatomical studies on the presence of neurons in the lungfish CNS that are only immunopositive for Leu-enkephalin, indicates that a Leu-enkephalin-coding opioid gene must be present in the CNS of the lungfish. This gene may be the lungfish form of prodynorphin. Given the phylogenetic position of the lungfish in vertebrate evolution, the putative Leu-enkephalin-coding gene must have evolved in the ancestral sarcopterygian vertebrates, or in the ancestral gnathostomes. The apparent slow rate of lungfish evolution makes these organisms interesting models for investigating the evolution of the opioid/orphanin gene family.

  6. Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

    PubMed Central

    Rocha, Antônio Augusto; Moretti, Nilmar Silvio

    2014-01-01

    The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing. PMID:24813189

  7. Novel Pharmacophores of Connexin43 Based on the “RXP” Series of Cx43-binding Peptides

    PubMed Central

    Verma, Vandana; Larsen, Bjarne Due; Coombs, Wanda; Lin, Xianming; Spagnol, Gaelle; Sorgen, Paul L; Taffet, Steven M; Delmar, Mario

    2009-01-01

    Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-amino acid peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71), and a linear octapeptide of sequence RRNYRRNY. These two small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function. PMID:19556520

  8. Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae.

    PubMed

    Iwakoshi-Ukena, Eiko; Ukena, Kazuyoshi; Okimoto, Aiko; Soga, Miyuki; Okada, Genya; Sano, Naomi; Fujii, Tamotsu; Sugawara, Yoshiaki; Sumida, Masayuki

    2011-04-01

    The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus. PMID:21193000

  9. Are fish fed with cyanobacteria safe, nutritious and delicious? A laboratory study

    PubMed Central

    Liang, Hualei; Zhou, Wenshan; Zhang, Yulei; Qiao, Qin; Zhang, Xuezhen

    2015-01-01

    Toxic cyanobacterial blooms, which produce cyclic heptapeptide toxins known as microcystins, are worldwide environmental problems. On the other hand, the cyanobacteria protein (30–50%) has been recommended as substitute protein for aquaculture. The present laboratory study verified the feasibility of cyanobacteria protein substitution and risk assessment. Goldfish were fed diets supplemented lyophilised cyanobacteria powder for 16 weeks with the various doses: 0% (control), 10%, 20%, 30% and 40%. Low doses (10% and 20%) promoted growth whereas high doses (30% and 40%) inhibited growth. In cyanobacteria treated fish, the proximate composition of ash, crude fat content and crude protein content decreased in 16 weeks; the saturated fatty acid (SFA) content significantly increased; the n-3 polyunsaturated fatty acid content, collagen content and muscle pH significantly decreased; cooking loss percents increased significantly. Muscle fiber diameter and myofibril length were negatively correlation. Additionally, flavour compounds (e.g., amino acids, nucleotides, organic acids and carnosine) changed significantly in the treated fish, and odour compounds geosmin and 2-methylisoborneol increased significantly. The estimated daily intake (EDI) of microcystins in muscle was close to or exceeded the World Health Organization (WHO) tolerable daily intake (TDI), representing a great health risk. Cyanobacterie is not feasible for protein sources use in aquaculture. PMID:26470644

  10. Structural diversity of the microbial surfactin derivatives from selective esterification approach.

    PubMed

    Shao, Chuanshi; Liu, Lin; Gang, Hongze; Yang, Shizhong; Mu, Bozhong

    2015-01-15

    Surfactin originated from genus Bacillus is composed of a heptapeptide moiety bonded to the carboxyl and hydroxyl groups of a β-hydroxy fatty acid and it can be chemically modified to prepare the derivatives with different structures, owing to the existence of two free carboxyl groups in its peptide loop. This article presents the chemical modification of surfactin esterified with three different alcohols, and nine novel surfactin derivatives have been separated from products by the high performance liquid chromatography (HPLC). The novel derivatives, identified with Fourier transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectrometry (ESI-MS), are the mono-hexyl-surfactin C14 ester, mono-hexyl-surfactin C15 ester, mono-2-methoxy-ethyl-surfactin C14 ester, di-hexyl-surfactin C14 ester, di-hexyl-surfactin ester C15, di-2-methoxy-ethyl-surfactin ester C14, di-2-methoxy-ethyl-surfactin ester C15, di-6-hydoxyl-hexyl-surfactin C14 ester and, di-6-hydoxyl-hexyl-surfactin C15 ester. The reaction conditions for esterification were optimized and the dependence of yields on different alcohols and catalysts were discussed. This study shows that esterification is one of the most efficient ways of chemical modification for surfactin and it can be used to prepare more derivatives to meet the needs of study in biological and interfacial activities.

  11. Development and in vitro/in vivo evaluation of immediate release perindopril tablets.

    PubMed

    Ölçer, Muharrem; Ölçer, Aysel; İnce, İskender; Karasulu, Ercument

    2015-01-01

    Perindopril erbumine (PE) is a BCS (Biopharmaceutics Classification System) class 3 drug with high solubility and low permeability. It is an inhibitor of the enzyme that converts angiotensin I (Angiotensin Converting Enzyme, ACE) into angiotensin II as well as causing the degradation of the vasodilator bradykinin into an inactive heptapeptide. The aim of this study was to develop an alternative drug product by using a different salt of perindopril and to evaluate the bioequivalence between PE, not still licensed, and perindopril arginine (PA), licensed in many countries, and to prepare PE tablets by using direct compression method. Many different formulations were prepared, among which F3-coded formulation was only selected due to releasing of 98.03% active substance at 45th minute. Bioequivalence study was planned as a cross-designed, randomized, open-labeled, single-dose, single-center study and conducted in 24 male healthy volunteers via peroral route. The results of bioequivalence study were evaluated for Perindopril and Perindoprilat according to Cmax, tmax and AUC criteria. The geometric mean ratios (90% CI) of perindopril and perindoprilat followed test and reference drug were calculated for AUC0-t and Cmax, 105.946% (100.218-112.002%) and 110.437% (102.534-118.948%); 109.542% (98.364-121.992%) and 115.729% (101.031-132.565%), respectively. The 90% confidence intervals of them were found within the standard bioequivalence range (80-125%).

  12. Loss of Internal Backbone Carbonyls: Additional Evidence for Sequence-Scrambling in Collision-Induced Dissociation of y-Type Ions

    NASA Astrophysics Data System (ADS)

    Harper, Brett; Miladi, Mahsan; Solouki, Touradj

    2014-10-01

    It is shown that y-type ions, after losing C-terminal H2O or NH3, can lose an internal backbone carbonyl (CO) from different peptide positions and yield structurally different product fragment ions upon collision-induced dissociation (CID). Such CO losses from internal peptide backbones of y-fragment ions are not unique to a single peptide and were observed in four of five model peptides studied herein. Experimental details on examples of CO losses from y-type fragment ions for an isotopically labeled AAAAH AA-NH2 heptapeptide and des-acetylated-α-melanocyte-stimulating hormone (dα-MSH) (SYSMEHFRWGKPV-NH2) are reported. Results from isotope labeling, tandem mass spectrometry (MSn), and ion mobility-mass spectrometry (IM-MS) confirm that CO losses from different amino acids of m/ z-isolated y-type ions yield structurally different ions. It is shown that losses of internal backbone carbonyls (as CID products of m/ z-isolated y-type ions) are among intermediate steps towards formation of rearranged or permutated product fragment ions. Possible mechanisms for generation of the observed sequence-scrambled a-"like" ions, as intermediates in sequence-scrambling pathways of y-type ions, are proposed and discussed.

  13. Activated Scavenger Receptor A Promotes Glial Internalization of Aβ

    PubMed Central

    Zhou, Wei-wei; Wang, Shao-wei; Xu, Peng-xin; Yu, Xiao-lin; Liu, Rui-tian

    2014-01-01

    Beta-amyloid (Aβ) aggregates have a pivotal role in pathological processing of Alzheimer’s disease (AD). The clearance of Aβ monomer or aggregates is a causal strategy for AD treatment. Microglia and astrocytes are the main macrophages that exert critical neuroprotective roles in the brain. They may effectively clear the toxic accumulation of Aβ at the initial stage of AD, however, their functions are attenuated because of glial overactivation. In this study, we first showed that heptapeptide XD4 activates the class A scavenger receptor (SR-A) on the glia by increasing the binding of Aβ to SR-A, thereby promoting glial phagocytosis of Aβ oligomer in microglia and astrocytes and triggering intracellular mitogen-activated protein kinase (MAPK) signaling cascades. Moreover, XD4 enhances the internalization of Aβ monomers to microglia and astrocytes through macropinocytosis or SR-A-mediated phagocytosis. Furthermore, XD4 significantly inhibits Aβ oligomer-induced cytotoxicity to glial cells and decreases the production of proinflammatory cytokines, such as TNF-α and IL-1β, in vitro and in vivo. Our findings may provide a novel strategy for AD treatment by activating SR-A. PMID:24718459

  14. Molluscan attractins, a family of water-borne protein pheromones with interspecific attractiveness.

    PubMed

    Cummins, Scott F; Schein, Catherine H; Xu, Yuan; Braun, Werner; Nagle, Gregg T

    2005-01-01

    The marine mollusk Aplysia releases the water-borne pheromone attractin during egg laying. This small protein stimulates the formation and maintenance of mating and egg-laying aggregations. Attractin has been characterized from five Aplysia species: A. californica, A. brasiliana, A. fasciata, A. vaccaria, and A. depilans. We describe here the isolation of attractin from Bursatella leachii, and show that it belongs to the same protein family. The pattern of residue conservation, especially the six invariant cysteines, suggests that all of these attractins have a common fold. The nuclear magnetic resonance solution structure of A. californica attractin contains two antiparallel alpha-helices, the second of which contains the heptapeptide sequence IEECKTS that has been implicated in attractin function. Synthetic peptides containing this IEECKTS region are attractive, and mutating surface exposed charged residues within this region of attractin abolishes attractin activity. This suggests that the second helix is an essential part of the receptor-binding interface. In contrast to the peptide pheromonal attractants in amphibians, which are species specific, the attractins are, to our knowledge, the first water-borne peptide or protein pheromone family in invertebrates and vertebrates that are not species specific.

  15. The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats.

    PubMed

    Bowman, Andrew; Lercher, Lukas; Singh, Hari R; Zinne, Daria; Timinszky, Gyula; Carlomagno, Teresa; Ladurner, Andreas G

    2016-04-20

    Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from(1)H-(15)N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.

  16. Evidence for a "wattle and daub" model of the cyst wall of entamoeba.

    PubMed

    Chatterjee, Anirban; Ghosh, Sudip K; Jang, Ken; Bullitt, Esther; Moore, Landon; Robbins, Phillips W; Samuelson, John

    2009-07-01

    The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a "wattle and daub" model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).

  17. Interactions between lipid-free apolipoprotein-AI and a lipopeptide incorporating the RGDS cell adhesion motif

    NASA Astrophysics Data System (ADS)

    Castelletto, V.; Hamley, I. W.; Reza, M.; Ruokolainen, J.

    2014-11-01

    The interaction of a designed bioactive lipopeptide C16-GGGRGDS, comprising a hexadecyl lipid chain attached to a functional heptapeptide, with the lipid-free apoliprotein, Apo-AI, is examined. This apolipoprotein is a major component of high density lipoprotein and it is involved in lipid metabolism and may serve as a biomarker for cardiovascular disease and Alzheimers' disease. We find via isothermal titration calorimetry that binding between the lipopeptide and Apo-AI occurs up to a saturation condition, just above equimolar for a 10.7 μM concentration of Apo-AI. A similar value is obtained from circular dichroism spectroscopy, which probes the reduction in α-helical secondary structure of Apo-AI upon addition of C16-GGGRGDS. Electron microscopy images show a persistence of fibrillar structures due to self-assembly of C16-GGGRGDS in mixtures with Apo-AI above the saturation binding condition. A small fraction of spheroidal or possibly ``nanodisc'' structures was observed. Small-angle X-ray scattering (SAXS) data for Apo-AI can be fitted using a published crystal structure of the Apo-AI dimer. The SAXS data for the lipopeptide/Apo-AI mixtures above the saturation binding conditions can be fitted to the contribution from fibrillar structures coexisting with flat discs corresponding to Apo-AI/lipopeptide aggregates.

  18. Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

    SciTech Connect

    Li, Zhi; Rupasinghe, Sanjeewa G.; Schuler, Mary A.; Nair, Satish K.

    2012-02-08

    The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

  19. Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive oxygen species formation in primary cultured rat hepatocytes.

    PubMed Central

    Ding, W X; Shen, H M; Shen, Y; Zhu, H G; Ong, C N

    1998-01-01

    Cyanobacteria contamination of water has become a growing public health problem worldwide. Microcystis aeruginosa is one of the most common toxic cyanobacteria. It is capable of producing microcystins, a group of cyclic heptapeptide compounds with potent hepatotoxicity and tumor promotion activity. The present study investigated the effect of microcystic cyanobacteria on primary cultured rat hepatocytes by examining mitochondrial membrane potential (MMP) changes and intracellular reactive oxygen species (ROS) formation in cells treated with lyophilized freshwater microcystic cyanobacteria extract (MCE). Rhodamine 123 (Rh-123) was used as a fluorescent probe for changes in mitochondrial fluorescence intensity. The mitochondrial Rh-123 fluorescence intensity in MCE-treated hepatocytes, examined using a laser confocal microscope, responded in a dose- and time-dependent manner. The results thus indicate that the alteration of MMP might be an important event in the hepatotoxicity caused by cyanobacteria. Moreover, the parallel increase of ROS formation detected using another fluorescent probe, 2',7'-dichlorofluorescin diacetate also suggests the involvement of oxidative stress in the hepatotoxicity caused by cyanobacteria. The fact that MMP changes precede other cytotoxic parameters such as nuclear staining by propidium iodide and cell morphological changes suggests that mitochondrial damage is closely associated with MCE-induced cell injury in cultured rat hepatocytes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9637798

  20. Primary structures of skin antimicrobial peptides indicate a close, but not conspecific, phylogenetic relationship between the leopard frogs Lithobates onca and Lithobates yavapaiensis (Ranidae).

    PubMed

    Conlon, J Michael; Coquet, Laurent; Leprince, Jérôme; Jouenne, Thierry; Vaudry, Hubert; King, Jay D

    2010-04-01

    The phylogenetic relationship between the relict leopard frog Lithobates (Rana) onca (Cope, 1875) and the lowland leopard frog Lithobates (Rana) yavapaiensis (Platz and Frost, 1984) is unclear. Chromatographic analysis of norepinephrine-stimulated skin secretions from L. onca led to the identification of six peptides with antimicrobial activity. Determination of their primary structures indicated that four of the peptides were identical to brevinin-1Ya, brevinin-1Yb, brevinin-1Yc and ranatuerin-2Ya previously isolated from skin secretions of L. yavapaiensis. However, a peptide belonging to the temporin family (temporin-ONa: FLPTFGKILSGLF.NH(2)) and an atypical member of the ranatuerin-2 family containing a C-terminal cyclic heptapeptide domain (ranatuerin-2ONa: GLMDTVKNAAKNLAGQMLDKLKCKITGSC) were isolated from the L. onca secretions but were not present in the L. yavapaiensis secretions. Ranatuerin-2ONa inhibited the growth of Escherichia coli (MIC=50muM) and Candida albicans (MIC=100muM ) and showed hemolytic activity (LC(50)=90muM) but was inactive against Staphylococcus aureus. The data indicate a close phylogenetic relationship between L. onca and L. yavapaiensis but suggest that they are not conspecific species.

  1. Effects of microcystins on the growth and the activity of superoxide dismutase and peroxidase of rape (Brassica napus L.) and rice (Oryza sativa L.).

    PubMed

    Chen, Jianzhong; Song, Lirong; Dai, Jian; Gan, Nanqin; Liu, Zhili

    2004-03-15

    Microcystins are naturally occurring hepatotoxic cyclic heptapeptides produced by some toxic freshwater cyanobacterial species. In this study, crude extract of toxic cyanobacterial blooms from Dianchi Lake in southwestern China was used to determine the effects of microcystins on rape (Brassica napus L.) and rice (Oryza sativa L.). Experiments were carried out on a range of doses of the extract (equivalent to 0, 0.024, 0.12, 0.6 and 3 microg MC-LR/ml). Investigations showed that exposure to microcystins inhibited the growth and development of both rice and rape seedlings, however, microcystins had more powerful inhibition effect on rape than rice in germination percentage of seeds and seedling height. Microcystins significantly inhibited the elongation of primary roots of rape and rice seedlings. Determination of the activities of peroxidase and superoxide dismutase demonstrated that microcystin stress was manifested as an oxidative stress. Using ELISA, microcystins were examined from the extract of exposed rape and rice seedlings, indicating that consumption of edible plants exposed to microcystins via irrigation route may have health risks. Significantly different levels of recovered microcystins between exposed rice and rape seedlings suggested that there might be different tolerant mechanisms toward microcystins. PMID:15051402

  2. Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains

    PubMed Central

    Gilmore, Michael S.; Rauch, Marcus; Ramsey, Matthew M.; Himes, Paul R.; Varahan, Sriram; Manson, Janet M.; Lebreton, Francois; Hancock, Lynn Ernest

    2015-01-01

    Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains. PMID:26039987

  3. Identification of peptides that bind hepatitis C virus envelope protein E2 and inhibit viral cellular entry from a phage-display peptide library.

    PubMed

    Lü, Xin; Yao, Min; Zhang, Jian-Min; Yang, Jing; Lei, Ying-Feng; Huang, Xiao-Jun; Jia, Zhan-Sheng; Ma, Li; Lan, Hai-Yun; Xu, Zhi-Kai; Yin, Wen

    2014-05-01

    Hepatitis C virus (HCV) envelope protein E2 is required for the entry of HCV into cells. Viral envelope proteins interact with cell receptors in a multistep process, which may be a promising target for the development of novel antiviral agents. In this study, a heptapeptide M13 phage-display library was screened for peptides that bind specifically to prokaryotically expressed, purified truncated HCV envelope protein E2. ELISA assay was used to quantify the binding of the peptides to HCV E2 protein. Flow cytometry, quantitative reverse-transcription PCR and western blotting were used to investigate the inhibition effect of one peptide on HCV infection in hepatoma cells (Huh7.5) in vitro. Four peptides capable of binding specifically to HCV E2 protein were obtained after three rounds of biopanning. Peptide C18 (WPWHNHR), with the highest affinity for binding HCV E2 protein, was synthesized. The results showed that peptide C18 inhibited the viral infectivity of both HCV pseudotype particles (HCVpp) harboring HCV envelope glycoproteins and cell-culture produced HCV (HCVcc). Thus, this study demonstrated that peptide C18 is a potential candidate for anti-HCV therapy as a novel viral entry inhibitor.

  4. A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.

    PubMed Central

    Lee, J M; Greenleaf, A L

    1989-01-01

    The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts. Images PMID:2657724

  5. A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver.

    PubMed Central

    Serizawa, H; Conaway, R C; Conaway, J W

    1992-01-01

    We previously purified RNA polymerase II transcription factor delta from rat liver and found that it has an associated DNA-dependent ATPase (dATPase) activity. In this report, we show that delta is also closely associated with a protein kinase activity that catalyzes phosphorylation of the largest subunit of RNA polymerase II. Kinase activity copurifies with transcription and DNA-dependent ATPase (dATPase) activities when delta is analyzed by anion- and cation-exchange HPLC as well as by sucrose gradient sedimentation, arguing that delta possesses all three activities. Phosphorylation of the largest subunits of both rat and yeast RNA polymerase II is stimulated by DNA, whereas phosphorylation of a synthetic peptide containing multiple copies of the carboxyl-terminal heptapeptide repeat is not. Although both ATP and GTP appear to function as phosphate donors, GTP is utilized less than 10% as well as ATP. These findings suggest that delta may exert its action in transcription at least in part through a mechanism involving phosphorylation of the largest subunit of RNA polymerase II. Images PMID:1386928

  6. Expression of prtA from Photorhabdus luminescens in Bacillus thuringiensis enhances mortality in lepidopteran larvae by sub-cutaneous but not oral infection.

    PubMed

    Bishop, A H

    2014-09-01

    The prtA gene from Photorhabdus luminescens encodes the virulence factor Protease A. When P. luminescens is injected into the hemocoel of insects by entomopathogenic nematodes, PrtA is a key component of pathogenicity thought to help degrade the immune system. The prtA gene was cloned and introduced on a plasmid into Bacillus thuringiensis. PrtA was shown to be actively expressed in vitro by cleavage of a specific Dabcyl-Edans heptapeptide substrate. There was no difference in the speed or level of mortality when spores and δ-endotoxins crystals of the transformed strain were fed to larvae of Pieris brassicae, as compared to the wild-type strain. When vegetative cells were injected into the hemocoel of larvae of Galleriamellonella, however, there was a significant increase in the rate and level of mortality over the wild type. The yield of B. thuringiensis per cadaver was a hundred-fold greater in the PrtA-secreting strain. The increased pathogenicity from intrahemocoelic infection may have been due to a greater ability to overcome the immune response of G. mellonella while other factors such as resident gut bacteria may have negated this advantage after oral dosage. PMID:25036004

  7. Suppression Analysis Reveals a Functional Difference between the Serines in Positions Two and Five in the Consensus Sequence of the C-Terminal Domain of Yeast RNA Polymerase II

    PubMed Central

    Yuryev, A.; Corden, J. L.

    1996-01-01

    The largest subunit of RNA polymerase II contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4) Ser(5)Pro(6) Ser(7). Substitution of nonphosphorylatable amino acids at positions two or five of the Saccharomyces cerevisiae CTD is lethal. We developed a selection ssytem for isolating suppressors of this lethal phenotype and cloned a gene, SCA1 (suppressor of CTD alanine), which complements recessive suppressors of lethal multiple-substitution mutations. A partial deletion of SCA1 (sca1Δ::hisG) suppresses alanine or glutamate substitutions at position two of the consensus CTD sequence, and a lethal CTD truncation mutation, but SCA1 deletion does not suppress alanine or glutamate substitutions at position five. SCA1 is identical to SRB9, a suppressor of a cold-sensitive CTD truncation mutation. Strains carrying dominant SRB mutations have the same suppression properties as a sca1Δ::hisG strain. These results reveal a functional difference between positions two and five of the consensus CTD heptapeptide repeat. The ability of SCA1 and SRB mutant alleles to suppress CTD truncation mutations suggest that substitutions at position two, but not at position five, cause a defect in RNA polymerase II function similar to that introduced by CTD truncation. PMID:8725217

  8. RPRD1A and RPRD1B Are Human RNA Polymerase II C-Terminal Domain Scaffolds for Ser5 Dephosphorylation

    PubMed Central

    Guo, Xinghua; Hunter, Gerald O.; Kuznetsova, Olga V.; Tempel, Wolfram; Marcon, Edyta; Zhong, Guoqing; Guo, Hongbo; Kuo, Wei-Hung William; Li, Joyce; Young, Peter; Olsen, Jonathan B.; Wan, Cuihong; Loppnau, Peter; El Bakkouri, Majida; Senisterra, Guillermo A.; He, Hao; Huang, Haiming; Sidhu, Sachdev S.; Emili, Andrew; Murphy, Shona; Mosley, Amber L.; Arrowsmith, Cheryl H.; Min, Jinrong; Greenblatt, Jack F.

    2014-01-01

    SUMMARY The RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) heptapeptide repeats (Y1-S2-P3-T4-S5-P6-S7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD interaction domains (CIDs) with CTD repeats phosphorylated at S2 and S7. Our high resolution crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an interesting example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2. PMID:24997600

  9. Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity

    SciTech Connect

    Zhou, Yuan; Wang, Hui; Wang, Cong; Qiu, Xuefeng; Benson, Mikael; Yin, Xiaoqin; Xiang, Zou; Li, Dongmei; and others

    2015-08-15

    Microcystin (MC)-LR, a cyclic heptapeptide, is a potent reproductive system toxin. To understand the molecular mechanisms of MC-induced reproductive system cytotoxicity, we evaluated global changes of miRNA and mRNA expression in mouse Sertoli cells following MC-LR treatment. Our results revealed that the exposure to MC-LR resulted in an altered miRNA expression profile that might be responsible for the modulation of mRNA expression. Bio-functional analysis indicated that the altered genes were involved in specific cellular processes, including cell death and proliferation. Target gene analysis suggested that junction injury in Sertoli cells exposed to MC-LR might be mediated by miRNAs through the regulation of the Sertoli cell-Sertoli cell pathway. Collectively, these findings may enhance our understanding on the modes of action of MC-LR on mouse Sertoli cells as well as the molecular mechanisms underlying the toxicity of MC-LR on the male reproductive system. - Highlights: • miRNAs were altered in Sertoli cells exposed to MC-LR. • Alerted genes were involved in different cell functions including the cell morphology. • MC-LR adversely affected Sertoli cell junction formation through the regulating miRNAs.

  10. The Periplasmic Bacterial Molecular Chaperone SurA Adapts Its Structure to Bind Peptides in Different Conformations to Assert a Sequence Preference for Aromatic Residues

    SciTech Connect

    Xu, X.; Wang, S.; Hu, Y.-X.; McKay, D.B.

    2009-06-04

    The periplasmic molecular chaperone protein SurA facilitates correct folding and maturation of outer membrane proteins in Gram-negative bacteria. It preferentially binds peptides that have a high fraction of aromatic amino acids. Phage display selections, isothermal titration calorimetry and crystallographic structure determination have been used to elucidate the basis of the binding specificity. The peptide recognition is imparted by the first peptidyl-prolyl isomerase (PPIase) domain of SurA. Crystal structures of complexes between peptides of sequence WEYIPNV and NFTLKFWDIFRK with the first PPIase domain of the Escherichia coli SurA protein at 1.3 A resolution, and of a complex between the dodecapeptide and a SurA fragment lacking the second PPIase domain at 3.4 A resolution, have been solved. SurA binds as a monomer to the heptapeptide in an extended conformation. It binds as a dimer to the dodecapeptide in an alpha-helical conformation, predicated on a substantial structural rearrangement of the SurA protein. In both cases, side-chains of aromatic residues of the peptides contribute a large fraction of the binding interactions. SurA therefore asserts a recognition preference for aromatic amino acids in a variety of sequence configurations by adopting alternative tertiary and quaternary structures to bind peptides in different conformations.

  11. The RNA Polymerase II CTD: The Increasing Complexity of a Low-Complexity Protein Domain.

    PubMed

    Jeronimo, Célia; Collin, Pierre; Robert, François

    2016-06-19

    The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that plays key roles in coordinating transcription with co-transcriptional events. The heptapeptide repeats that form the CTD are dynamically phosphorylated on serine, tyrosine and threonine residues during the various steps of transcription, thereby regulating the recruitment of various proteins involved in gene expression. In this "Perspective," we review the recent literature related to the function of the CTD, to CTD kinases (Kin28, CDK7, CDK9, CDK12, ERK1/2 and DYRK1A) and to CTD phosphatases (Rtr1, RPAP2, Ssu72, Fcp1 and Gcl7). We discuss unresolved and controversial issues and try to provide constructive suggestions. This review also highlights emerging themes in the CTD field, such as crosstalk and feedback mechanisms, as well as gene-specific and tissue-specific functions of the CTD. Finally, promising therapeutic avenues for a recently developed CTD kinase inhibitor are discussed. PMID:26876604

  12. 1,4-Disubstituted-[1,2,3]triazolyl-Containing Analogues of MT-II: Design, Synthesis, Conformational Analysis, and Biological Activity

    PubMed Central

    2015-01-01

    Side chain-to-side chain cyclizations represent a strategy to select a family of bioactive conformations by reducing the entropy and enhancing the stabilization of functional ligand-induced receptor conformations. This structural manipulation contributes to increased target specificity, enhanced biological potency, improved pharmacokinetic properties, increased functional potency, and lowered metabolic susceptibility. The CuI-catalyzed azide–alkyne 1,3-dipolar Huisgen’s cycloaddition, the prototypic click reaction, presents a promising opportunity to develop a new paradigm for an orthogonal bioorganic and intramolecular side chain-to-side chain cyclization. In fact, the proteolytic stable 1,4- or 4,1-disubstituted [1,2,3]triazolyl moiety is isosteric with the peptide bond and can function as a surrogate of the classical side chain-to-side chain lactam forming bridge. Herein we report the design, synthesis, conformational analysis, and functional biological activity of a series of i-to-i+5 1,4- and 4,1-disubstituted [1,2,3]triazole-bridged cyclopeptides derived from MT-II, the homodetic Asp5 to Lys10 side chain-to-side chain bridged heptapeptide, an extensively studied agonist of melanocortin receptors. PMID:25347033

  13. Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

    PubMed

    Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin

    2015-10-14

    The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

  14. [Regulation of cell activity by the extracellular matrix: the concept of matrikines].

    PubMed

    Maquart, F X; Siméon, A; Pasco, S; Monboisse, J C

    1999-01-01

    The activity of connective tissue cells is modulated by a number of factors present in their environment. In addition to the soluble factors such as hormones, cytokines or growth factors, cells also receive signals from the surrounding extracellular matrix (ECM) macromolecules. Moreover, they may degrade the ECM proteins and liberate peptides which may by themselves constitute new signals for the surrounding cells. Therefore, an actual regulation loop exists in connective tissue, constituted by peptides generated by ECM degradation and connective tissue cells. The term of "matrikine" has been proposed to designate such ECM-derived peptides able to regulate cell activity. In this review, we summarize some data obtained in our laboratory with two different matrikines: the tripeptide glycyl-histidyl-lysine (GHK) and the heptapeptide cysteinyl-asparaginyl-tyrosyl-tyrosyl-seryl-asparaginyl-serine (CNYYSNS). GHK is a potent activator of ECM synthesis and remodeling, whereas CNYYSNS is able to inhibit polymorphonuclear leukocytes activation and decrease the invasive capacities of cancer cells. PMID:10689625

  15. Primary structures of skin antimicrobial peptides indicate a close, but not conspecific, phylogenetic relationship between the leopard frogs Lithobates onca and Lithobates yavapaiensis (Ranidae).

    PubMed

    Conlon, J Michael; Coquet, Laurent; Leprince, Jérôme; Jouenne, Thierry; Vaudry, Hubert; King, Jay D

    2010-04-01

    The phylogenetic relationship between the relict leopard frog Lithobates (Rana) onca (Cope, 1875) and the lowland leopard frog Lithobates (Rana) yavapaiensis (Platz and Frost, 1984) is unclear. Chromatographic analysis of norepinephrine-stimulated skin secretions from L. onca led to the identification of six peptides with antimicrobial activity. Determination of their primary structures indicated that four of the peptides were identical to brevinin-1Ya, brevinin-1Yb, brevinin-1Yc and ranatuerin-2Ya previously isolated from skin secretions of L. yavapaiensis. However, a peptide belonging to the temporin family (temporin-ONa: FLPTFGKILSGLF.NH(2)) and an atypical member of the ranatuerin-2 family containing a C-terminal cyclic heptapeptide domain (ranatuerin-2ONa: GLMDTVKNAAKNLAGQMLDKLKCKITGSC) were isolated from the L. onca secretions but were not present in the L. yavapaiensis secretions. Ranatuerin-2ONa inhibited the growth of Escherichia coli (MIC=50muM) and Candida albicans (MIC=100muM ) and showed hemolytic activity (LC(50)=90muM) but was inactive against Staphylococcus aureus. The data indicate a close phylogenetic relationship between L. onca and L. yavapaiensis but suggest that they are not conspecific species. PMID:20044030

  16. Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells

    PubMed Central

    Dias, João D; Rito, Tiago; Torlai Triglia, Elena; Kukalev, Alexander; Ferrai, Carmelo; Chotalia, Mita; Brookes, Emily; Kimura, Hiroshi; Pombo, Ana

    2015-01-01

    Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels. DOI: http://dx.doi.org/10.7554/eLife.11215.001 PMID:26687004

  17. Label-Free Electrical Immunosensor for Highly Sensitive and Specific Detection of Microcystin-LR in Water Samples.

    PubMed

    Tan, Feng; Saucedo, Nuvia Maria; Ramnani, Pankaj; Mulchandani, Ashok

    2015-08-01

    Microcystin-LR (MCLR) is one of the most commonly detected and toxic cyclic heptapeptide cyanotoxins released by cyanobacterial blooms in surface waters, for which sensitive and specific detection methods are necessary to carry out its recognition and quantification. Here, we present a single-walled carbon nanotube (SWCNTs)-based label-free chemiresistive immunosensor for highly sensitive and specific detection of MCLR in different source waters. MCLR was initially immobilized on SWCNTs modified interdigitated electrode, followed by incubation with monoclonal anti-MCLR antibody. The competitive binding of MCLR in sample solutions induced departure of the antibody from the antibody-antigen complexes formed on SWCNTs, resulting in change in the conductivity between source and drain of the sensor. The displacement assay greatly improved the sensitivity of the sensor compared with direct immunoassay on the same device. The immunosensor exhibited a wide linear response to log value of MCLR concentration ranging from 1 to 1000 ng/L, with a detection limit of 0.6 ng/L. This method showed good reproducibility, stability and recovery. The proposed method provides a powerful tool for rapid and sensitive monitoring of MCLR in environmental samples.

  18. RFamide neuropeptide actions on molluscan proboscis smooth muscle: interactions with primary neurotransmitters.

    PubMed

    Moulis, A; Huddart, H

    2004-07-01

    The potency (muscle force-generated) of a number of long-chain RFamide neuropeptides was examined in mechanical experiments with the radular-retractor and radular-sac muscles of gastropods Buccinum undatum and Neptunea antiqua. Many of the heptapeptides, octapeptides and the decapeptide LMS were found to induce greater contraction than FMRFamide in both smooth muscles and in both species. RFamide neuropeptides interacted with the neurotransmitter acetylcholine in an additive way and RFamide-induced contractions were inhibited by the neuromodulator serotonin. Pre-treatment with a calcium-free saline completely abolished acetylcholine-induced responses but only partially inhibited RFamide responses in the muscles, suggesting that acetylcholine acts to cause influx of extracellular calcium for contraction. In contrast, RFamide neuropeptides may mobilise intracellular calcium to maintain sustained tonic force in calcium-free conditions. This suggests that an additional involvement of a fast calcium channel may be present in the RFamide responses, since loss of the usual superimposed twitch activity is observed. Force regulation in these muscles appears to result from a complex interaction of RFamide neuropeptides with the primary transmitter acetylcholine and the neuromodulator serotonin.

  19. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

    NASA Astrophysics Data System (ADS)

    Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

    2012-11-01

    The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-β peptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

  20. Chain length, temperature and solvent effects on the structural properties of α-aminoisobutyric acid homooligopeptides.

    PubMed

    Grubišić, Sonja; Chandramouli, Balasubramanian; Barone, Vincenzo; Brancato, Giuseppe

    2016-07-27

    Non-coded α-amino acids, originally exploited by nature, have been successfully reproduced by recent synthetic strategies to confer special structural and functional properties to small peptides. The most known and well-studied atypical residue is α-aminoisobutyric acid (Aib), which is contained in a fairly large number of peptides with known antibiotic effects. Here, we report on a molecular dynamics (MD) study of a series of homooligopeptides based on α-aminoisobutyric acid (Aib) with increasing length (Ac-(Aib)n-NMe, n = 5, 6, 7 and 10) and at various temperatures, employing a recent extension of the AMBER force field tailored for the Aib residue. Solvent effects have been analyzed by comparative MD simulations of a heptapeptide in water and dimethylsulfoxide at different temperatures. Our results show that the preference for the 310- and/or α-helix structures, which typically characterize Aib based peptides, is finely tuned by several factors including the chain length, temperature and solvent nature. While the transitions between intra-molecular i → i + 3 and i → i + 4 hydrogen bonds characterizing 310 and α-helices, respectively, are rather fast in small peptides (in the picosecond timescale), our analysis shows that the above physical and chemical factors modulate the relative equilibrium populations of the two helical structures. The obtained results nicely agree with available experimental data and support the use of the new force field for modeling Aib containing peptides. PMID:27402118

  1. Cyanobacterial Cyclopeptides as Lead Compounds to Novel Targeted Cancer Drugs

    PubMed Central

    Sainis, Ioannis; Fokas, Demosthenes; Vareli, Katerina; Tzakos, Andreas G.; Kounnis, Valentinos; Briasoulis, Evangelos

    2010-01-01

    Cyanobacterial cyclopeptides, including microcystins and nodularins, are considered a health hazard to humans due to the possible toxic effects of high consumption. From a pharmacological standpoint, microcystins are stable hydrophilic cyclic heptapeptides with a potential to cause cellular damage following uptake via organic anion-transporting polypeptides (OATP). Their intracellular biological effects involve inhibition of catalytic subunits of protein phosphatase 1 (PP1) and PP2, glutathione depletion and generation of reactive oxygen species (ROS). Interestingly, certain OATPs are prominently expressed in cancers as compared to normal tissues, qualifying MC as potential candidates for cancer drug development. In the era of targeted cancer therapy, cyanotoxins comprise a rich source of natural cytotoxic compounds with a potential to target cancers expressing specific uptake transporters. Moreover, their structure offers opportunities for combinatorial engineering to enhance the therapeutic index and resolve organ-specific toxicity issues. In this article, we revisit cyanobacterial cyclopeptides as potential novel targets for anticancer drugs by summarizing existing biomedical evidence, presenting structure-activity data and discussing developmental perspectives. PMID:20411119

  2. Degradation and transport of AVP by proximal tubule

    SciTech Connect

    Carone, F.A.; Christensen, E.I.; Flouret, G. Univ. of Aarhus )

    1987-12-01

    High-performance liquid chromatography (HPLC) analysis revealed that (3,4,5-{sup 3}H-Phe{sup 3},Arg{sup 8})vasopressin (({sup 3}H)AVP) was not degraded by isolated renal brush-border membranes or by a cortical lysosomal fraction in vitro; however, in the presence of 1 mM reduced glutathione, ({sup 3}H)AVP was degraded by both preparations. Renal cortical homogenates in vitro and luminal peptidases of proximal tubule in vivo degraded ({sup 3}H)AVP and in both instances yielded phenylalanine, hexapeptide AVP 1-6, heptapeptide AVP 1-7, octapeptide AVP 1-8, and two uncharacterized products X and Y. These data suggest that filtered AVP is reduced in the proximal tubule by a reduced glutathione-dependent transhydrogenase and subsequently cleaved to ({sup 3}H)Phe by tubular aminopeptidases. Following microinfusion of ({sup 3}H)AVP into proximal tubules, 15.7% of the label was absorbed. Five and fifteen minutes after infusion of ({sup 3}H)AVP, sequestration of total label in proximal tubules was 4.5 and 2.1%, respectively, and quantitative electron microscope autoradiography revealed accumulation of grains over apical endocytic vacuoles and lysosomes consistent with endocytic uptake and rapid lysosomal degradation of AVP and/or a large metabolite. Thus, enzymatic cleavage of AVP by luminal and lysosomal peptidases in proximal tubules could involve disulfide bond, C-terminal, and N-terminal loci.

  3. Electrochemical behavior of phytochelatins and related peptides at the hanging mercury drop electrode in the presence of cobalt(II) ions.

    PubMed

    Dorcák, Vlastimil; Sestáková, Ivana

    2006-01-01

    Direct current voltammetry and differential pulse voltammetry have been used to investigate the electrochemical behaviour of two phytochelatins: heptapeptide (gamma-Glu-Cys)3-Gly and pentapeptide (gamma-Glu-Cys)2-Gly, tripeptide glutathione gamma-Glu-Cys-Gly and its fragments: dipeptides Cys-Gly and gamma-Glu-Cys at the hanging mercury drop electrode in the presence of cobalt(II) ions. Most interesting results were obtained with direct current voltammetry in the potential region of -0.80 V up to -1.80 V. Differential pulse voltammetry of the same solutions of Co(II) with peptides gives more complicated voltammograms with overlapping peaks, probably in connection with the influence of adsorption at slow scan rates necessarily used in this method. However, in using Brdicka catalytic currents for analytical purposes, differential pulse voltammograms seem to be more helpful. Presented investigations have shown that particularly the prewave of cobalt(II) allows distinguishing among phytochelatins, glutathione, and its fragments.

  4. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling.

    PubMed

    Rodrigues, Sylvie; Van Aken, Elisabeth; Van Bocxlaer, Saskia; Attoub, Samir; Nguyen, Quang-Dé; Bruyneel, Erik; Westley, Bruce R; May, Felicity E B; Thim, Lars; Mareel, Marc; Gespach, Christian; Emami, Shahin

    2003-01-01

    We previously established that the trefoil peptides (TFFs) pS2, spasmolytic polypeptide, and intestinal trefoil factor are involved in cellular scattering and invasion in kidney and colonic cancer cells. Using the chorioallantoic membrane (CAM) assay and the formation of tube-like structures by human umbilical vein endothelial cells (HUVEC) plated on the Matrigel matrix substratum, we report here that TFFs are proangiogenic factors. Angiogenic activity of TFFs is comparable to that induced by vascular endothelial growth factor, leptin, and transforming growth factor-alpha. Stimulation of angiogenesis by pS2 in the CAM assay is blocked by pharmacological inhibitors of cyclooxygenase COX-2 (NS-398) and epidermal growth factor receptor (EGF-R) tyrosine kinase (ZD1839), but is independent of KDR/Flk-1 and thromboxane A2 receptors. In contrast, the morphogenic switch induced by pS2 in HUVEC cells could be inhibited by the specific KDR heptapeptide antagonist ATWLPPR and by inhibitors of COX-2 and EGF-R signaling. These results implicate TFFs in the formation of new blood vessels during normal and pathophysiological processes linked to wound healing, inflammation, and cancer progression in the digestive mucosa and other human solid tumors associated with aberrant expression of TFFs.

  5. Site-Directed Conjugation of Antibodies to Apoferritin Nanocarrier for Targeted Drug Delivery to Prostate Cancer Cells.

    PubMed

    Dostalova, Simona; Cerna, Tereza; Hynek, David; Koudelkova, Zuzana; Vaculovic, Tomas; Kopel, Pavel; Hrabeta, Jan; Heger, Zbynek; Vaculovicova, Marketa; Eckschlager, Tomas; Stiborova, Marie; Adam, Vojtech

    2016-06-15

    Herein, we describe a novel approach for targeting of ubiquitous protein apoferritin (APO)-encapsulating doxorubicin (DOX) to prostate cancer using antibodies against prostate-specific membrane antigen (PSMA). The conjugation of anti-PSMA antibodies and APO was carried out using HWRGWVC heptapeptide, providing their site-directed orientation. The prostate-cancer-targeted and nontargeted nanocarriers were tested using LNCaP and HUVEC cell lines. A total of 90% of LNCaP cells died after treatment with DOX (0.25 μM) or DOX in nontargeted and prostate-cancer-targeted APO, proving that the encapsulated DOX toxicity for LNCaP cells remained the same. Free DOX showed higher toxicity for nonmalignant cells, whereas the toxicity was lower after treatment with the same dosage of APO-encapsulated DOX (APODOX) and even more in prostate-cancer-targeted APODOX. Hemolytic assay revealed exceptional hemocompatibility of the entire nanocarrier. The APO encapsulation mechanism ensures applicability using a wide variety of chemotherapeutic drugs, and the presented surface modification enables targeting to various tumors.

  6. Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase.

    PubMed Central

    Reiss, Y; Stradley, S J; Gierasch, L M; Brown, M S; Goldstein, J L

    1991-01-01

    We tested 42 tetrapeptides for their ability to bind to the rat brain p21ras protein farnesyltransferase as estimated by their ability to compete with p21Ha-ras in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-A1-A2-X, where positions A1 and A2 are occupied by aliphatic amino acids and position X is occupied by a COOH-terminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the A1 position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CAIL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) protein gamma subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21Ki-rasB was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the protein farnesyltransferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids. PMID:1992464

  7. Highly sensitive electrochemical lead ion sensor harnessing peptide probe molecules on porous gold electrodes.

    PubMed

    Su, Wenqiong; Cho, Misuk; Nam, Jae-Do; Choe, Woo-Seok; Lee, Youngkwan

    2013-10-15

    Lead ion is one of the most hazardous and ubiquitous heavy metal pollutants and poses an increasing threat to the environment and human health. This necessitates rapid and selective detection and/or removal of lead ions from various soil and water resources. Recently, we identified several Pb²⁺ binding peptides via phage display technique coupled with chromatographic biopanning (Nian et al., 2010) where a heptapeptide (TNTLSNN) capable of recognizing Pb²⁺ with high affinity and specificity evolved. In the present study, an electrochemical sensor harnessing this Pb²⁺ affinity peptide as a probe on a porous gold electrode was developed. The three dimensional porous gold electrode was obtained from electrochemical deposition using the dynamic hydrogen bubble template method. A thin layer of poly(thiophene acetic acid) (PTAA) was coated on the porous gold surface. The Pb²⁺ recognizing peptide was immobilized via amide linkage on the PTAA. The developed biosensor was demonstrated to be fast, selective and reproducible in Pb²⁺ etection, exhibiting Pb²⁺-specific peak current values around -0.15 V in a broad concentration range (1-1×10⁷ nM) in 10 min despite the repeated use after regeneration. PMID:23707872

  8. Insights From Atomic-Resolution X-Ray Structures Of Chemically-Synthesized Hiv-1 Protease In Complex With Inhibitors

    PubMed Central

    Johnson, Erik C.B.; Malito, Enrico; Shen, Yuequan; Pentelute, Brad; Rich, Dan; Florián, Jan; Tang, Wei-Jen; Kent, Stephen B.H.

    2007-01-01

    Summary The HIV-1 protease is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 protease has one catalytic site formed by the homodimeric enzyme. We have chemically synthesized fully active HIV-1 protease using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 protease formed crystals that diffracted to 1.04 and 1.2Å resolution, respectively. These atomic resolution structures revealed additional structural details of the HIV-1 protease interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process. PMID:17869270

  9. Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors.

    PubMed

    Johnson, Erik C B; Malito, Enrico; Shen, Yuequan; Pentelute, Brad; Rich, Dan; Florián, Jan; Tang, Wei-Jen; Kent, Stephen B H

    2007-10-26

    The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.

  10. Occurrence of articulins and epiplasmins in protists.

    PubMed

    Huttenlauch, Irm; Stick, Reimer

    2003-01-01

    The cortex of ciliates. dinoflagellates, and euglenoids comprises a unique structure called the epiplasm, implicated in pattern-forming processes of the cell cortex and in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm in the flagellate Euglena gracilis and the ciliate Pseudomicrothorax dubius. The hallmark of articulins is a core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. The VPV-motif repeats are 12 residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. Using molecular and immunological tools we show that articulins are also present in the dinoflagellate Amphidinium carterae and the ciliates Paramecium tetraurelia and Paramecium caudatum, Tetrahymena pyriformis, and Euplotes aediculatus. Our analysis further shows that epiplasmins, a group of epiplasmic proteins first characterized in Paramecium, are also present in all these species. Moreover, we present evidence that epiplasmins and articulins represent two distinct classes of cytoskeletal proteins.

  11. Characterisation of two articulins, the major epiplasmic proteins comprising the membrane skeleton of the ciliate Pseudomicrothorax.

    PubMed

    Huttenlauch, I; Peck, R K; Plessmann, U; Weber, K; Stick, R

    1998-07-30

    Most protists possess a unique membrane skeleton, the epiplasm, which is involved in pattern forming processes of the cell cortex and functions in maintaining cell shape. Articulins, a novel class of cytoskeletal proteins, are major constituents of the epiplasm. We have isolated cDNAs encoding the two major articulins of the ciliate Pseudomicrothorax dubius. Peptide sequence data confirm the identity of the cloned cDNAs encoding articulins 1 and 4. With the data presented here sequence information for all major articulins of ciliates as well as the distantly related euglenoids is available. Sequence comparison of the two newly characterised ciliate articulins with the previously determined sequences of p60, a minor articulin of the same species, and the two euglenoid articulins reveals general sequence principles and uncovers new features of this protein family. The hallmark of articulins is a central core domain of repetitive motifs of alternating valine and proline residues, the VPV-motif. These VPV-motif repeats are either 12-residues, or in some places, six residues long. Positively and negatively charged residues segregate in register with valine and proline positions. The VPV-motif is unique to articulins. The terminal domains flanking the core are generally hydrophobic and contain a series of hexa- or heptapeptide repeats rich in glycine and hydrophobic residues. The sequences of these short repeats are very similar in articulins of the same species but are not conserved between euglenoids and ciliates.

  12. Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex.

    PubMed

    Bourbon, Henri-Marc

    2008-07-01

    The multisubunit Mediator (MED) complex bridges DNA-bound transcriptional regulators to the RNA polymerase II (PolII) initiation machinery. In yeast, the 25 MED subunits are distributed within three core subcomplexes and a separable kinase module composed of Med12, Med13 and the Cdk8-CycC pair thought to control the reversible interaction between MED and PolII by phosphorylating repeated heptapeptides within the Rpb1 carboxyl-terminal domain (CTD). Here, MED conservation has been investigated across the eukaryotic kingdom. Saccharomyces cerevisiae Med2, Med3/Pgd1 and Med5/Nut1 subunits are apparent homologs of metazoan Med29/Intersex, Med27/Crsp34 and Med24/Trap100, respectively, and these and other 30 identified human MED subunits have detectable counterparts in the amoeba Dictyostelium discoideum, indicating that none is specific to metazoans. Indeed, animal/fungal subunits are also conserved in plants, green and red algae, entamoebids, oomycetes, diatoms, apicomplexans, ciliates and the 'deep-branching' protists Trichomonas vaginalis and Giardia lamblia. Surprisingly, although lacking CTD heptads, T. vaginalis displays 44 MED subunit homologs, including several CycC, Med12 and Med13 paralogs. Such observations have allowed the identification of a conserved 17-subunit framework around which peripheral subunits may be assembled, and support a very ancient eukaryotic origin for a large, four-module MED. The implications of this comprehensive work for MED structure-function relationships are discussed.

  13. Assessment of detoxification of microcystin extracts using electrochemical oxidation.

    PubMed

    Liang, Wenyan; Chen, Li; Sui, Lili; Yu, Jian; Wang, Li; Shi, Hongxing

    2011-01-01

    Microcystins, cyclic heptapeptide toxins produced by cyanobacteria, possess tumor-promoting activity, which act through inhibition of protein phosphatases 1 and 2A. In this study, the variation in toxicity of microcystins from Microcystis aeruginosa during electrooxidation was assessed using bioassays. The microcystin-LR samples (MCLR) were prepared by crude extracts dissolved in electrolytes, e.g., 0.02 mol L(-1) Na(2)SO(4), 0.02 mol L(-1) Na(2)SO(4) containing 0.5 mmol L(-1) NaCl, and tap water. Electrooxidation was conducted at a current density of 4 mA cm(-2) at room temperature (20-26°C), using Ti/RuO(2) anodes. Toxicological profiles for acute toxic effects (Vibrio fischeri) and genotoxic effects (Vicia faba micronucleus assay and single cell gel electrophoresis assay of mice lymphocytes) were determined for both untreated and treated MCLR samples. Results showed that acute toxicity during treatment was caused mainly by residual oxidants from electrooxidation. The by-products from the degradation of MCLR samples showed very weak acute toxicity to V. fischeri. Before electrooxidation, MCLR samples could induce obvious cell damage to V. faba root tips and mice lymphocytes. Electrooxidation degradation significantly decreased the genotoxicity of MCLR samples until the final by-products showed no toxicity. Thus, electrooxidation can detoxify MCLR samples via degradation processes. PMID:21806454

  14. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

    PubMed Central

    Descostes, Nicolas; Heidemann, Martin; Spinelli, Lionel; Schüller, Roland; Maqbool, Muhammad Ahmad; Fenouil, Romain; Koch, Frederic; Innocenti, Charlène; Gut, Marta; Gut, Ivo; Eick, Dirk; Andrau, Jean-Christophe

    2014-01-01

    In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001 PMID:24842994

  15. Amyloid-β Binds to the Extracellular Cysteine-rich Domain of Frizzled and Inhibits Wnt/β-Catenin Signaling*S⃞

    PubMed Central

    Magdesian, Margaret H.; Carvalho, Milena M. V. F.; Mendes, Fabio A.; Saraiva, Leonardo M.; Juliano, Maria A.; Juliano, Luiz; Garcia-Abreu, José; Ferreira, Sérgio T.

    2008-01-01

    The amyloid-β peptide (Aβ) plays a major role in neuronal dysfunction and neurotoxicity in Alzheimer disease. However, the signal transduction mechanisms involved in Aβ-induced neuronal dysfunction remain to be fully elucidated. A major current unknown is the identity of the protein receptor(s) involved in neuronal Aβ binding. Using phage display of peptide libraries, we have identified a number of peptides that bind Aβ and are homologous to neuronal receptors putatively involved in Aβ interactions. We report here on a cysteine-linked cyclic heptapeptide (denominated cSP5) that binds Aβ with high affinity and is homologous to the extracellular cysteine-rich domain of several members of the Frizzled (Fz) family of Wnt receptors. Based on this homology, we investigated the interaction between Aβ and Fz. The results show that Aβ binds to the Fz cysteine-rich domain at or in close proximity to the Wnt-binding site and inhibits the canonical Wnt signaling pathway. Interestingly, the cSP5 peptide completely blocks Aβ binding to Fz and prevents inhibition of Wnt signaling. These results indicate that the Aβ-binding site in Fz is homologous to cSP5 and that this is a relevant target for Aβ-instigated neurotoxicity. Furthermore, they suggest that blocking the interaction of Aβ with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in Alzheimer disease. PMID:18234671

  16. A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae.

    PubMed

    Kobor, M S; Simon, L D; Omichinski, J; Zhong, G; Archambault, J; Greenblatt, J

    2000-10-01

    Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo. PMID:11003641

  17. A Motif Shared by TFIIF and TFIIB Mediates Their Interaction with the RNA Polymerase II Carboxy-Terminal Domain Phosphatase Fcp1p in Saccharomyces cerevisiae

    PubMed Central

    Kobor, Michael S.; Simon, Lisa D.; Omichinski, Jim; Zhong, Guoqing; Archambault, Jacques; Greenblatt, Jack

    2000-01-01

    Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo. PMID:11003641

  18. Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I.

    PubMed

    Oksanen, Ilona; Jokela, Jouni; Fewer, David P; Wahlsten, Matti; Rikkinen, Jouko; Sivonen, Kaarina

    2004-10-01

    The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.

  19. Sulforaphane protects Microcystin-LR-induced toxicity through activation of the Nrf2-mediated defensive response

    SciTech Connect

    Gan Nanqin; Mi Lixin; Sun Xiaoyun; Dai Guofei; Chung Funglung; Song Lirong

    2010-09-01

    Microcystins (MCs), a cyclic heptapeptide hepatotoxins, are mainly produced by the bloom-forming cyanobacerium Microcystis, which has become an environmental hazard worldwide. Long term consumption of MC-contaminated water may induce liver damage, liver cancer, and even human death. Therefore, in addition to removal of MCs in drinking water, novel strategies that prevent health damages are urgently needed. Sulforaphane (SFN), a natural-occurring isothiocyanate from cruciferous vegetables, has been reported to reduce and eliminate toxicities from xenobiotics and carcinogens. The purpose of the present study was to provide mechanistic insights into the SFN-induced antioxidative defense system against MC-LR-induced cytotoxicity. We performed cell viability assays, including MTS assay, colony formation assay and apoptotic cell sorting, to study MC-LR-induced cellular damage and the protective effects by SFN. The results showed that SFN protected MC-LR-induced damages at a nontoxic and physiological relevant dose in HepG2, BRL-3A and NIH 3 T3 cells. The protection was Nrf2-mediated as evident by transactivation of Nrf2 and activation of its downstream genes, including NQO1 and HO-1, and elevated intracellular GSH level. Results of our studies indicate that pretreatment of cells with 10 {mu}M SFN for 12 h significantly protected cells from MC-LR-induced damage. SFN-induced protective response was mediated through Nrf2 pathway.

  20. Localization of the genes encoding the melanocortin-2 (Adrenocorticotropic hormone) and melanocortin-3 receptors to chromosomes 18p11. 2 and 20q13. 2-q13. 3 by fluorescence in situ hybridization

    SciTech Connect

    Gantz, I.; Tashiro, Takao; Konda, Yoshitaka; Shimoto, Yoshimasa; Miwa, Hiroto; Munzert, G.; Barcroft, C.; Glover, T.; Yamada, Tadataka )

    1993-10-01

    Adrenocorticotropic hormone (ACTH) and [alpha]-, [beta]-, and [gamma]-melanocyte-stimulating hormone (MSH) are products of propiomelanocortin post-translational processing. These compounds are collectively labeled as melanocortins (MC). Aside from their established effects on the regulation of the adrenal cortex (ACTH) and melanocytes ([alpha]-MSH), the melanocortins have been implicated in a broad array of physiological events. Melanocortins mediate their effects through cell membrane receptors belonging to the superfamily of seven transmembrane G-protein-linked receptors. Using the technique of polymerase chain reaction with primers based on conserved areas of the seven transmembrane G-protein-linked receptor family, the authors recently isolated an [open quotes]orphan[close quotes] subfamily of this receptor group. Within the past year, two of these receptors were identified as specific for [alpha]-MSH (MC1) and ACTH (MC2). They have recently described a third melanocortin receptor (MC3) that appears to recognize the core heptapeptide sequence of melanocortins with equal potency and efficacy and identified its presence in the brain, placenta, and gut. Using the FISH technique, they localized the ACTH and the melanocortin-3 receptors to chromosome loci 18p11.2 and 20q12.3-q13.2, respectively. 19 refs., 1 fig.

  1. UV-B Exposure Affects the Biosynthesis of Microcystin in Toxic Microcystis aeruginosa Cells and Its Degradation in the Extracellular Space

    PubMed Central

    Yang, Zhen; Kong, Fanxiang

    2015-01-01

    Microcystins (MCs) are cyclic hepatotoxic heptapeptides produced by cyanobacteria that can be toxic to aquatic and terrestrial organisms. MC synthesis and degradation are thought to be influenced by several different physical and environmental parameters. In this study, the effects of different intensities of UV-B radiation on MC biosynthesis in Microcystis cells and on its extracellular degradation were investigated by mRNA analysis and degradation experiments. Exposure to UV-B at intensities of 1.02 and 1.45 W/m2 not only remarkably inhibited the growth of Microcystis, but also led to a decrease in the MC concentration. In addition, mcyD transcription was decreased under the same UV-B intensities. These results demonstrated that the effects of UV-B exposure on the biosynthesis of MCs in Microcystis cells could be attributed to the regulation of mcy gene transcription. Moreover, the MC concentration was decreased significantly after exposure to different intensities of UV-B radiation. Of the three MC variants (MC-LR, -RR and -YR, L, R and Y are abbreviations of leucine, arginine and tyrosine), MC-LR and MC-YR were sensitive to UV-B radiation, whereas MC-RR was not. In summary, our results showed that UV-B radiation had a negative effect on MC production in Microcystis cells and MC persistence in the extracellular space. PMID:26492272

  2. Isolation and characterization of microcystins from a river nile strain of Oscillatoria tenuis Agardh ex Gomont.

    PubMed

    Brittain, S; Mohamed, Z A; Wang, J; Lehmann, V K; Carmichael, W W; Rinehart, K L

    2000-12-01

    The River Nile is the major source of drinking water in Egypt, however, increased eutrophication due to agricultural, municipal and industrial runoff has contributed to the growth of toxin producing cyanobacteria. This study describes the isolation and characterization of microcystins (MCYSTs), cyclic heptapeptide hepatotoxins, from a rare strain of Oscillatoria tenuis, isolated from the River Nile at Sohag province in July 1995. The MCYST concentration of laboratory-cultured O. tenuis strain E6 was found to be 0.3 mg/g freeze-dried weight determined by enzyme-linked immunosorbent assay (ELISA). Two microcystins, 1 and 2, were isolated from lyophilized cells using solid phase extraction and reversed-phase high performance liquid chromatography (HPLC). Structures were assigned based upon their amino acid analyses, electrospray ionization mass spectrometry (ESIMS, ESIMS-CID-MS), high resolution fast atom bombardment mass spectrometry, and nuclear magnetic resonance data ((1)H and (1)H COSY NMR). Toxin 1 was identified as MCYST-LR, and toxin 2, a new MCYST, as MCYST-LHArg ([L-homoarginine(4)]). Previous studies indicate that Oscillatoria agardhii strains produce demethylated MCYSTs (containing D-Asp and/or dehydroalanine). This is the first report of a toxic O. tenuis, strain E6, one which produces a fully methylated MCYST, MCYST-LR and a new L-homoarginine containing MCYST, MCYST-LHArg.

  3. AVE 0991, a non-peptide Mas-receptor agonist, facilitates penile erection.

    PubMed

    da Costa Gonçalves, Andrey C; Fraga-Silva, Rodrigo A; Leite, Romulo; Santos, Robson A S

    2013-03-01

    The renin-angiotensin system plays a crucial role in erectile function. It has been shown that elevated levels of angiotensin II contribute to the development of erectile dysfunction both in humans and in aminals. On the contrary, the heptapeptide angiotensin-(1-7) appears to mediate penile erection by activation of the Mas receptor. Recently, we have shown that the erectile function of Mas gene-deleted mice was substantially reduced, which was associated with a marked increase in fibrous tissue in the corpus cavernosum. We have hypothesized that the synthetic non-peptide Mas agonist, AVE 0991, would potentiate penile erectile function. We showed that intracavernosal injection of AVE 0991 potentiated the erectile response of anaesthetized Wistar rats, measured as the ratio between corpus cavernosum pressure and mean arterial pressure, upon electrical stimulation of the major pelvic ganglion. The facilitatory effect of AVE 0991 on erectile function was dose dependent and completely blunted by the nitric oxide synthesis inhibitor, l-NAME. Importantly, concomitant intracavernosal infusion of the specific Mas receptor blocker, A-779, abolished the effect of AVE 0991. We demonstrated that AVE 0991 potentiates the penile erectile response through Mas in an NO-dependent manner. Importantly, these results suggest that Mas agonists, such as AVE 0991, might have significant therapeutic benefits for the treatment of erectile dysfunction.

  4. Are fish fed with cyanobacteria safe, nutritious and delicious? A laboratory study.

    PubMed

    Liang, Hualei; Zhou, Wenshan; Zhang, Yulei; Qiao, Qin; Zhang, Xuezhen

    2015-01-01

    Toxic cyanobacterial blooms, which produce cyclic heptapeptide toxins known as microcystins, are worldwide environmental problems. On the other hand, the cyanobacteria protein (30-50%) has been recommended as substitute protein for aquaculture. The present laboratory study verified the feasibility of cyanobacteria protein substitution and risk assessment. Goldfish were fed diets supplemented lyophilised cyanobacteria powder for 16 weeks with the various doses: 0% (control), 10%, 20%, 30% and 40%. Low doses (10% and 20%) promoted growth whereas high doses (30% and 40%) inhibited growth. In cyanobacteria treated fish, the proximate composition of ash, crude fat content and crude protein content decreased in 16 weeks; the saturated fatty acid (SFA) content significantly increased; the n-3 polyunsaturated fatty acid content, collagen content and muscle pH significantly decreased; cooking loss percents increased significantly. Muscle fiber diameter and myofibril length were negatively correlation. Additionally, flavour compounds (e.g., amino acids, nucleotides, organic acids and carnosine) changed significantly in the treated fish, and odour compounds geosmin and 2-methylisoborneol increased significantly. The estimated daily intake (EDI) of microcystins in muscle was close to or exceeded the World Health Organization (WHO) tolerable daily intake (TDI), representing a great health risk. Cyanobacterie is not feasible for protein sources use in aquaculture. PMID:26470644

  5. Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor.

    PubMed Central

    Denzer, K; Weber, B; Hille-Rehfeld, A; Figura, K V; Pohlmann, R

    1997-01-01

    The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant. PMID:9291124

  6. Gold nanoparticles-peptide based gas sensor arrays for the detection of food aromas.

    PubMed

    Compagnone, D; Fusella, G C; Del Carlo, M; Pittia, P; Martinelli, E; Tortora, L; Paolesse, R; Di Natale, C

    2013-04-15

    A gas sensor array based on peptide modified gold nanoparticles deposited onto 20MHz quartz crystal microbalances has been realized. Glutathione and its constituting aminoacids and dipeptides have been used as ligands. A great increase in sensitivity (2 orders of magnitude) was achieved using gold nanoparticles versus monolayer modified QCMs. The sensors have been characterised in terms of sensitivity for hexane, water, trimethylammine and ethanol. Highest sensitivity was found for water. The ability to discriminate typical food aromas as cis-3-hexenol, isopentylacetate, ethylacetate, and terpinen-4-ol dissolved in different solvents was studied using a gas sensor array constituted by gold nanoparticles modified with the glutathione peptides, thioglycolic acid and an heptapeptide. The array was found able to discriminate the food aromas, the response being dependent on the polarity of the solvent used. Tests on real olive oil samples gave a satisfactory separation among samples having defects versus non defected samples demonstrating that this approach has high potential for the development of gas sensor arrays to be used in real samples. PMID:23261699

  7. Crystal Structure of Vancosaminyltransferase GtfD from the Vancomycin Biosynthetic Pathway: Interactions with Acceptor and Nucleotide Ligands

    SciTech Connect

    Mulichak, A.M.; Lu, W.; Losey, H.C.; Walsh, C.T.; Garavito, R.M.

    2010-03-08

    The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that are effective against vancomycin resistant bacterial strains. We have determined the X-ray crystal structure of GtfD as a complex with TDP and the natural glycopeptide substrate at 2.0 {angstrom} resolution. GtfD, a member of the bidomain GT-B glycosyltransferase superfamily, binds TDP in the interdomain cleft, while the aglycone acceptor binds in a deep crevice in the N-terminal domain. However, the two domains are more interdependent in terms of substrate binding and overall structure than was evident in the structures of closely related glycosyltransferases GtfA and GtfB. Structural and kinetic analyses support the identification of Asp13 as a catalytic general base, with a possible secondary role for Thr10. Several residues have also been identified as being involved in donor sugar binding and recognition.

  8. A Fhit-mimetic peptide suppresses annexin A4-mediated chemoresistance to paclitaxel in lung cancer cells

    PubMed Central

    Ngankeu, Apollinaire; Ortuso, Francesco; Lovat, Francesca; Pinton, Sandra; D'Agostino, Sabrina; Zanesi, Nicola; Aqeilan, Rami I.; Campiglia, Pietro; Novellino, Ettore; Alcaro, Stefano; Croce, Carlo M.; Trapasso, Francesco

    2016-01-01

    We recently reported that Fhit is in a molecular complex with annexin A4 (ANXA4); following to their binding, Fhit delocalizes ANXA4 from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. Here, we demonstrate that Fhit physically interacts with A4 through its N-terminus; molecular dynamics simulations were performed on a 3D Fhit model to rationalize its mechanism of action. This approach allowed for the identification of the QHLIKPS heptapeptide (position 7 to 13 of the wild-type Fhit protein) as the smallest Fhit sequence still able to preserve its ability to bind ANXA4. Interestingly, Fhit peptide also recapitulates the property of the native protein in inhibiting Annexin A4 translocation from cytosol to plasma membrane in A549 and Calu-2 lung cancer cells treated with paclitaxel. Finally, the combination of Tat-Fhit peptide and paclitaxel synergistically increases the apoptotic rate of cultured lung cancer cells and blocks in vivo tumor formation. Our findings address to the identification of chemically simplified Fhit derivatives that mimic Fhit tumor suppressor functions; intriguingly, this approach might lead to the generation of novel anticancer drugs to be used in combination with conventional therapies in Fhit-negative tumors to prevent or delay chemoresistance. PMID:27166255

  9. Are fish fed with cyanobacteria safe, nutritious and delicious? A laboratory study

    NASA Astrophysics Data System (ADS)

    Liang, Hualei; Zhou, Wenshan; Zhang, Yulei; Qiao, Qin; Zhang, Xuezhen

    2015-10-01

    Toxic cyanobacterial blooms, which produce cyclic heptapeptide toxins known as microcystins, are worldwide environmental problems. On the other hand, the cyanobacteria protein (30-50%) has been recommended as substitute protein for aquaculture. The present laboratory study verified the feasibility of cyanobacteria protein substitution and risk assessment. Goldfish were fed diets supplemented lyophilised cyanobacteria powder for 16 weeks with the various doses: 0% (control), 10%, 20%, 30% and 40%. Low doses (10% and 20%) promoted growth whereas high doses (30% and 40%) inhibited growth. In cyanobacteria treated fish, the proximate composition of ash, crude fat content and crude protein content decreased in 16 weeks; the saturated fatty acid (SFA) content significantly increased; the n-3 polyunsaturated fatty acid content, collagen content and muscle pH significantly decreased; cooking loss percents increased significantly. Muscle fiber diameter and myofibril length were negatively correlation. Additionally, flavour compounds (e.g., amino acids, nucleotides, organic acids and carnosine) changed significantly in the treated fish, and odour compounds geosmin and 2-methylisoborneol increased significantly. The estimated daily intake (EDI) of microcystins in muscle was close to or exceeded the World Health Organization (WHO) tolerable daily intake (TDI), representing a great health risk. Cyanobacterie is not feasible for protein sources use in aquaculture.

  10. Phenological and liver antioxidant profiles of adult Nile tilapia (Oreochromis niloticus) exposed to toxic live cyanobacterium (Microcystis aeruginosa Kützing) cells.

    PubMed

    Khairy, Hanan M; Ibrahim, Marwa A; Ibrahem, Mai D

    2012-01-01

    Blue-green algae (cyanobacteria) constitute the greater part of the phytoplankton. Microcystis aeruginosa is amongst the most ubiquitously distributed cyanobacterial species, and almost invariably produces cyclic heptapeptide toxins called microcystins (MCs). The present study was designed to investigate the phenological and liver antioxidant profiles of the Nile tilapia Oreochromis niloticus chronically exposed to toxic live M. aeruginosa cells. Fish were grown in the absence and presence of M. aeruginosa in three different concentrations for seven days, and subsequently reared for another 30 days in the absence of the cyanobacteria. While cyanobacteria did not cause any fish mortality, there was a progressive development of yellowish discolouration in the livers of exposed fish. In the livers, the activities and levels of superoxide dismutase (SOD), lactate dehydrogenase (LDH), glutathione (GSH), and lipid peroxidation products like malondialdehyde (MDA) were elevated in response to the concentration of M. aeruginosa. Moreover, DNA fragmentation and DNA-protein crosslinks were measured. These parameters can thus be considered potential biomarkers for the fish exposure to M. aeruginosa. The present study sheds light on cyanobacterial blooms like health, environmental, and economic problem, respectively. PMID:23413757

  11. Characterisation of biotoxins produced by a cyanobacteria bloom in Lake Averno using two LC-MS-based techniques.

    PubMed

    Ferranti, Pasquale; Fabbrocino, Serena; Cerulo, Maria Grazia; Bruno, Milena; Serpe, Luigi; Gallo, Pasquale

    2008-12-01

    Cyanobacteria (blue-green algae) cause blooms in eutrophic lakes and drinking water reservoirs. They also produce biotoxins, including microcystins (MCs), highly toxic cyclic heptapeptides that cause poisoning in animals and human. In this paper, we present a method for the analysis of four MCs by ion trap LC-MS and MALDI-TOF/MS. The data are compared to evaluate the performance and reliability of the different MS detection systems. The method was applied to the analysis of water and algae samples from Lake Averno, near Naples, as a consequence of a cyanobacteria bloom. The analysis of algae cell extracts showed no contamination by known microcystins, but the three main substances were detected. MALDI-TOF/MS was successful for screening of the biotoxins in the samples, identifying anabaenopeptin B and anabaenopeptin F as the major contaminants on the basis of literature mass spectrometry data. The structure of the third compound was not identified and is under further investigation. The method could characterise the biotoxins produced in Lake Averno for evaluating health risks related to their presence. PMID:19680862

  12. Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells.

    PubMed

    Zhan, Li; Sakamoto, Hiroko; Sakuraba, Mayumi; Wu, De Sheng; Zhang, Li Shi; Suzuki, Takayoshi; Hayashi, Makoto; Honma, Masamitsu

    2004-01-10

    Toxic cyanobacteria (blue-green algae) water blooms have become a serious problem in several industrialized areas of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacteria. In the present study, we used human lymphoblastoid cell line TK6 to investigate the in vitro genotoxicity of MCLR. In a standard 4h treatment, MCLR did not induce a significant cytotoxic response at <80 microg/ml. In a prolonged 24h treatment, in contrast, it induced cytotoxic as well as mutagenic responses concentration-dependently starting at 20 microg/ml. At the maximum concentration (80 microg/ml), the micronucleus frequency and the mutation frequency at the heterozygous thymidine kinase (TK) locus were approximately five-times the control values. Molecular analysis of the TK mutants revealed that MCLR specifically induced loss of heterozygosity at the TK locus, but not point mutations or other small structural changes. These results indicate that MCLR had a clastogenic effect. We discuss the mechanisms of MCLR genotoxicity and the possibility of its being a hepatocarcinogen. PMID:14706513

  13. Charting the trajectory of the ATLAS stream

    NASA Astrophysics Data System (ADS)

    de Boer, Thomas; Belokurov, Vasily; Koposov, Sergey; Irwin, Mike; Erkal, Denis

    2014-08-01

    Stellar streams provide dramatic confirmation that large systems accrete smaller systems, in the context of a hierarchical merging cosmology, and therefore contain important clues about the formation mechanism of the Galactic halo. By studying the detailed properties of streams we can determine how stars are stripped from their hosts due to the Galactic tidal field and how the formation of the Galactic halo may have proceeded. Here we propose to trace the full visible extent of the recently discovered ATLAS stream using deep, wide-field photometry, to determine its path across the sky in 3 dimensions. By utilising the very wide-field capabilities of DECam, we will determine the deep, MW decontaminated CMD in a 30 degree long portion of the stream, allowing us to determine the distance, density profile and stellar population makeup of the stream. The position and density on the sky of kinematically cold structures like the ATLAS stream provides powerful, unbiased constraints on the distribution of dark matter in the Galaxy. Furthermore, deep photometry of the stellar content of the stream will tell us what type of system was the likely progenitor: globular cluster, ultra-faint dwarf or dSph galaxy.

  14. Identification of Coupling and Repulsion Phase DNA Marker Associated With an Allele of a Gene Conferring Host Plant Resistance to Pigeonpea sterility mosaic virus (PPSMV) in Pigeonpea (Cajanus cajan L. Millsp.).

    PubMed

    Daspute, Abhijit; Fakrudin, B

    2015-03-01

    Pigeonpea Sterility Mosaic Disease (PSMD) is an important foliar disease caused by Pigeonpea sterility mosaic virus (PPSMV) which is transmitted by eriophyid mites (Aceria cajani Channabasavanna). In present study, a F2 mapping population comprising 325 individuals was developed by crossing PSMD susceptible genotype (Gullyal white) and PSMD resistant genotype (BSMR 736). We identified a set of 32 out of 300 short decamer random DNA markers that showed polymorphism between Gullyal white and BSMR 736 parents. Among them, eleven DNA markers showed polymorphism including coupling and repulsion phase type of polymorphism across the parents. Bulked Segregant Analysis (BSA), revealed that the DNA marker, IABTPPN7, produced a single coupling phase marker (IABTPPN7414) and a repulsion phase marker (IABTPPN7983) co-segregating with PSMD reaction. Screening of 325 F2 population using IABTPPN7 revealed that the repulsion phase marker, IABTPPN7983, was co-segregating with the PSMD responsive SV1 at a distance of 23.9 cM for Bidar PPSMV isolate. On the other hand, the coupling phase marker IABTPPN7414 did not show any linkage with PSMD resistance. Additionally, single marker analysis both IABTPPN7983 (P<0.0001) and IABTPPN 7414 (P<0.0001) recorded a significant association with the PSMD resistance and explained a phenotypic variance of 31 and 36% respectively in F2 population. The repulsion phase marker, IABTPPN7983, could be of use in Marker-Assisted Selection (MAS) in the PPSMV resistance breeding programmes of pigeonpea. PMID:25774108

  15. The background rate of false positives: Combining simulations of gravitational wave events with an unsupervised algorithm for transient identification in crowded image-subtracted data

    NASA Astrophysics Data System (ADS)

    Ackley, Kendall; Eikenberry, Stephen; Klimenko, Sergey; LIGO Collaboration

    2016-03-01

    We are now entering the era of multimessenger gravitational wave (GW) astronomy with the completion of the first observing run of Advanced LIGO. Multiwavelength electromagnetic (EM) emission is expected to accompany gravitational radiation from compact object binary mergers, such as those between neutron stars and stellar-mass black holes, where Advanced LIGO is most sensitive to their detection. Attempting to perform EM follow-up over the 10-100s deg2 error regions will be faced with many challenges, including the identification and removal of O (105) false positive transients that appear as a commotion of background events and as image artifacts in crowded image-subtracted fields. We present an update to our automated unsupervised algorithm including how our pipeline uses the existing coherent WaveBurst pipeline in an attempt to develop optimized EM follow-up schema. Our end-to-end pipeline combines simulated GW events with actual observational data from a number of ground-based optical observatories, including PTF, ROTSE, and DECam. Our performance is reported both in terms of the number of coincident false positives as well as the efficiency of recovery.

  16. VizieR Online Data Catalog: 1889-2015 photometry of Stingray nebula (Schaefer+, 2015)

    NASA Astrophysics Data System (ADS)

    Schaefer, B. E.; Edwards, Z. I.

    2016-02-01

    To get broad-band magnitudes for the Stingray, we have pulled from a wide variety of sources --the Harvard photographic plate collection from 1889 to 1989, the visual magnitude estimates of Albert Jones as archived by the American Association of Variable Star Observers (AAVSO) from 1994 to 2007, the All Sky Automated Survey (ASAS) from 2001 to 2009, the AAVSO telescopes going into the AAVSO Photometric All-sky Survey (APASS) from 2011 to 2015, plus our own photometry from CCD images with DECam on the Cerro Tololo 4-m Blanco telescope from 2014. We have added 15 mag from the literature or derived by us from the literature, all on six nights from 1969 to 1996. At our request, A. Henden has put the Stingray in the queue for time series photometry on the 0.61-m Optical Craftsmen Telescope at the Mount John Observatory in New Zealand. The 1-minute CCD integrations were through a Johnson V filter on the nights of 2015 March 23, 26, and 27. (1 data file).

  17. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

    DOE PAGES

    Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; Dauter, Zbigniew

    2015-10-30

    The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refinedmore » using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.« less

  18. SDSS-IV eBOSS emission-line galaxy pilot survey

    NASA Astrophysics Data System (ADS)

    Comparat, J.; Delubac, T.; Jouvel, S.; Raichoor, A.; Kneib, J.-P.; Yèche, C.; Abdalla, F. B.; Le Cras, C.; Maraston, C.; Wilkinson, D. M.; Zhu, G.; Jullo, E.; Prada, F.; Schlegel, D.; Xu, Z.; Zou, H.; Bautista, J.; Bizyaev, D.; Bolton, A.; Brownstein, J. R.; Dawson, K. S.; Escoffier, S.; Gaulme, P.; Kinemuchi, K.; Malanushenko, E.; Malanushenko, V.; Mariappan, V.; Newman, J. A.; Oravetz, D.; Pan, K.; Percival, W. J.; Prakash, A.; Schneider, D. P.; Simmons, A.; Abbott, T. M. C.; Allam, S.; Banerji, M.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Capozzi, D.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Castander, F. J.; Cunha, C. E.; da Costa, L. N.; Desai, S.; Doel, P.; Eifler, T. F.; Estrada, J.; Flaugher, B.; Fosalba, P.; Frieman, J.; Gaztanaga, E.; Gerdes, D. W.; Gruen, D.; Gruendl, R. A.; Gutierrez, G.; Honscheid, K.; James, D. J.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Lima, M.; Maia, M. A. G.; March, M.; Marshall, J. L.; Miquel, R.; Plazas, A. A.; Reil, K.; Roe, N.; Romer, A. K.; Roodman, A.; Rykoff, E. S.; Sako, M.; Sanchez, E.; Scarpine, V.; Sevilla-Noarbe, I.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarle, G.; Thaler, J.; Thomas, D.; Walker, A. R.; Zhang, Y.

    2016-08-01

    The Sloan Digital Sky Survey IV extended Baryonic Oscillation Spectroscopic Survey (SDSS-IV/eBOSS) will observe 195 000 emission-line galaxies (ELGs) to measure the baryonic acoustic oscillation (BAO) standard ruler at redshift 0.9. To test different ELG selection algorithms, 9000 spectra were observed with the SDSS spectrograph as a pilot survey based on data from several imaging surveys. First, using visual inspection and redshift quality flags, we show that the automated spectroscopic redshifts assigned by the pipeline meet the quality requirements for a reliable BAO measurement. We also show the correlations between sky emission, signal-to-noise ratio in the emission lines, and redshift error. Then we provide a detailed description of each target selection algorithm we tested and compare them with the requirements of the eBOSS experiment. As a result, we provide reliable redshift distributions for the different target selection schemes we tested. Finally, we determine an target selection algorithms that is best suited to be applied on DECam photometry because they fulfill the eBOSS survey efficiency requirements. The catalog is only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/592/A121

  19. In Silico RAPD Priming Sites in Expressed Sequences and iSCAR Markers for Oil Palm

    PubMed Central

    Premkrishnan, Balakrishnan Vasanthakumari; Arunachalam, Vadivel

    2012-01-01

    RAPD is a simple dominant marker system widely used in biology. Effectiveness of RAPD can be improved by selecting and redesigning primers whose priming sites occur in target sequence(s) of gene or organism at optimum distance. We developed software that uses sequences of random decamer primers and nucleotide sequence(s) as two input files. It locates the priming sites in input sequences and generates output files listing frequency and distance between priming sites. When the priming sites of a single primer occur more than once in a sequence with a distance of 200 to 2000 bp, the software also designs pairs of iSCAR primers. An input of 387 RAPD primers and 42,432 expressed sequences of oil palm are used as test. Wet-lab PCR results from a publication that used the same set of primers were compared with software output on priming sites. In the test sequences of oil palm covering 1.4% of genome, we found that at least 60% the primers chosen using software are sure of giving PCR amplification. We designed 641 iSCAR primers suitable for amplification of oil palm DNA. The software successfully predicted 92% (67 out of 73) of published polymorphic RAPD primers in oil palm. PMID:22474414

  20. Exploring the influence of EGCG on the β-sheet-rich oligomers of human islet amyloid polypeptide (hIAPP1-37) and identifying its possible binding sites from molecular dynamics simulation.

    PubMed

    Wang, Qianqian; Guo, Jingjing; Jiao, Pingzu; Liu, Huanxiang; Yao, Xiaojun

    2014-01-01

    EGCG possesses the ability of disaggregating the existing amyloid fibrils which were associated with many age-related degenerative diseases. However, the molecular mechanism of EGCG to disaggregate these fibrils is poorly known. In this work, to study the influence of EGCG on the full-length human islet amyloid polypeptide 1-37 (hIAPP1-37) oligomers, molecular dynamics simulations of hIAPP1-37 pentamer and decamer with EGCG were performed, respectively. The obtained results indicate that EGCG indeed destabilized the hIAPP1-37 oligomers. The nematic order parameter and secondary structure calculations coupled with the free-energy landscape indicate that EGCG broke the initial ordered pattern of two polymers, greatly reduced their β-sheet content and enlarged their conformational space. On this basis, three possible target sites were identified with the binding capacity order of S1>S2>S3. After a deeper analysis of each site, we found that S1 was the most possible site on which residues B-Ile26/Ala25, A-Phe23, B/C-Leu27 and E-Tyr37 played an important role for their binding. The proposal of this molecular mechanism can not only provide a prospective interaction figure between EGCG and β-sheet-rich fibrils of hIAPP1-37, but also is useful for further discovering other potential inhibitors.

  1. Cooling the dark energy camera instrument

    SciTech Connect

    Schmitt, R.L.; Cease, H.; DePoy, D.; Diehl, H.T.; Estrada, J.; Flaugher, B.; Kuhlmann, S.; Onal, Birce; Stefanik, A.; /Fermilab

    2008-06-01

    DECam, camera for the Dark Energy Survey (DES), is undergoing general design and component testing. For an overview see DePoy, et al in these proceedings. For a description of the imager, see Cease, et al in these proceedings. The CCD instrument will be mounted at the prime focus of the CTIO Blanco 4m telescope. The instrument temperature will be 173K with a heat load of 113W. In similar applications, cooling CCD instruments at the prime focus has been accomplished by three general methods. Liquid nitrogen reservoirs have been constructed to operate in any orientation, pulse tube cryocoolers have been used when tilt angles are limited and Joule-Thompson or Stirling cryocoolers have been used with smaller heat loads. Gifford-MacMahon cooling has been used at the Cassegrain but not at the prime focus. For DES, the combined requirements of high heat load, temperature stability, low vibration, operation in any orientation, liquid nitrogen cost and limited space available led to the design of a pumped, closed loop, circulating nitrogen system. At zenith the instrument will be twelve meters above the pump/cryocooler station. This cooling system expected to have a 10,000 hour maintenance interval. This paper will describe the engineering basis including the thermal model, unbalanced forces, cooldown time, the single and two-phase flow model.

  2. Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.

    PubMed

    Nowicka, Anna; Grzebelus, Ewa; Grzebelus, Dariusz

    2012-03-01

    Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot.

  3. X-ray Crystal Structure of a Locked Nucleic Acid (LNA) Duplex Composed of a Palindromic 10-mer DNA Strand Containing One LNA Thymine Monomer

    SciTech Connect

    Egli, M.; Minasov, G.; Teplova, M.; Kumar, R.; Wengel, J.

    2010-03-05

    Locked nucleic acid (LNA), a recently introduced nucleic acid analogue with a bicyclic 2'-O,4'-C-methylene linked furanose sugar, exhibits enhanced affinities for DNA and RNA relative to the corresponding oligodeoxyribonucleotides and oligoribonucleotides; we report the first crystal structure of an LNA unit incorporated in an oligonucleotide duplex. The structure at 1.4 {angstrom} resolution of the DNA-LNA decamer duplex with one LNA thymine monomer per strand provides a detailed view of the conformation and hydration of locked nucleic acid residues in a duplex A-form. Our study provides a first look at the conformational properties of LNA in a crystal structure at relatively high resolution. The main characteristics of the structure are the standard A-type conformation induced by LNA residues and the capacity of the 2'-oxygen that is part of the bicyclic sugar framework to engage in a least two hydrogen bonds to water molecules. Circular dichroism spectra (CD) of LNA-LNA duplexes in solution indicated that such duplexes appear to adopt a conformation that closely resembles the A-form geometry of RNA-RNA duplexes. However, these spectra also manifested subtle differences between the two species (data not shown). The present analysis of a duplex with only a single LNA residue per strand does not provide any insight into potential conformational differences between LNA and RNA duplexes. Attempts to determine a crystal structure of a completely modified LNA-LNA duplex are underway.

  4. An atypical AAA+ ATPase assembly controls efficient transposition through DNA remodeling and transposase recruitment

    PubMed Central

    Arias-Palomo, Ernesto; Berger, James M.

    2015-01-01

    Transposons are ubiquitous genetic elements that drive genome rearrangements, evolution, and the spread of infectious disease and drug-resistance. Many transposons, such as Mu, Tn7 and IS21, require regulatory AAA+ ATPases for function. We use x-ray crystallography and cryo-electron microscopy to show that the ATPase subunit of IS21, IstB, assembles into a clamshell-shaped decamer that sandwiches DNA between two helical pentamers of ATP-associated AAA+ domains, sharply bending the duplex into a 180° U-turn. Biochemical studies corroborate key features of the structure, and further show that the IS21 transposase, IstA, recognizes the IstB•DNA complex and promotes its disassembly by stimulating ATP hydrolysis. Collectively, these studies reveal a distinct manner of higher-order assembly and client engagement by a AAA+ ATPase and suggest a mechanistic model where IstB binding and subsequent DNA bending primes a selected insertion site for efficient transposition. PMID:26276634

  5. Detection of genetic diversity and selective gene introgression in coffee using RAPD markers.

    PubMed

    Orozco-Castillo, C; Chalmers, K J; Waugh, R; Powell, W

    1994-03-01

    RAPD (randomly amplified polymorphic DNA) markers generated by arbitary decamers have been successfully employed to detect genetic polymorphisms between coffee species and between Coffea arabica genotypes. The RAPD profiles were used to construct dendrograms and these were consistent with the known history and evolution of Coffea arabica. Material originating from Ethiopia and the arabica sub-groups - C. arabica var. typica and C. arabica var. bourbon - were clearly distinguished. RAPD analysis therefore reflects morphological differences between the sub-groups and the geographical origin of the coffee material. Species-specific amplification products were also identified, but, more importantly, amplification products specific to C. canephora were identified in two C. arabica genotypes, Rume Sudan and Catimor 5175. This diagnostic product is therefore indicative of interspecific gene flow in coffee and has biological implications for selective introgressive hybridisation in coffee. Our study demonstrates the power of the polymerase chain reaction technology for the generation of genetic markers for long-lived perennial tree and bush crops. PMID:24190527

  6. Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation

    PubMed Central

    Petrovska, Ivana; Nüske, Elisabeth; Munder, Matthias C; Kulasegaran, Gayathrie; Malinovska, Liliana; Kroschwald, Sonja; Richter, Doris; Fahmy, Karim; Gibson, Kimberley; Verbavatz, Jean-Marc; Alberti, Simon

    2014-01-01

    One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell. DOI: http://dx.doi.org/10.7554/eLife.02409.001 PMID:24771766

  7. Structural Basis for Recognition of Guanosine by a Synthetic Tricyclic Cytosine Analogue: Guanidinium G-Clamp

    SciTech Connect

    Wilds, C.J.; Maier, M.A.; Manoharan, M.; Egli, M.

    2010-03-08

    An oligonucleotide analogue containing a novel heterocyclic analogue, the guanidinium G-clamp, was designed to allow formation of five H-bonds to guanosine. The guanidinium group was introduced postsynthetically by treatment of the deprotected oligonucleotide containing a free amino group with a solution of 1H-pyrazole-1-carboxamidine and purified by a combination of size-exclusion chromatography and reversed-phase HPLC. A single incorporation of this modification into an oligodeoxynucleotide sequence was found to increase duplex stability by 13{sup o} and 16{sup o} per modification to RNA and DNA, respectively. Crystals of a self-complementary decamer sequence containing this modification were grown and diffracted to 1-{angstrom} resolution. The structure was solved by molecular replacement and revealed that the modification forms additional H-bonds to O(6) and N(7) of guanosine through the amino and imino N-atoms, respectively. The origins of enhanced duplex stability are also attributed to increased stacking interactions mediated by the phenoxazine moiety of the G-clamp and formation of H-bond networks between the positively charged guanidinium group, H{sub 2}O molecules, and negatively charged O-atoms from phosphates on the adjacent strand.

  8. Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe Ç

    PubMed Central

    Edwards, Thomas E.; Cekan, Pavol; Reginsson, Gunnar W.; Shelke, Sandip A.; Ferré-D’Amaré, Adrian R.; Schiemann, Olav; Sigurdsson, Snorri Th.

    2011-01-01

    Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies. PMID:21252294

  9. Detecting Mass Loss in Main Belt Asteroids

    NASA Astrophysics Data System (ADS)

    Sandberg, Erik; Rajagopal, Jayadev; Ridgway, Susan E.; Kotulla, Ralf C.; Valdes, Francisco; Allen, Lori

    2016-01-01

    Sandberg, E., Rajagopal, J., Ridgway, S.E, Kotulla, R., Valdes, F., Allen, L.The Dark Energy Camera (DECam) on the 4m Blanco telescope at the Cerro Tololo Inter-American Observatory (CTIO) is being used for a survey of Near Earth Objects (NEOs). Here we attempt to identify mass loss in main belt asteroids (MBAs) from these data. A primary motivation is to understand the role that asteroids may play in supplying dust and gas for debris disks. This work focuses on finding methods to automatically pick out asteroids that have qualities indicating possible mass loss. Two methods were chosen: looking for flux above a certain threshold in the asteroid's radial profile, and comparing its PSF to that of a point source. After sifting through 490 asteroids, several have passed these tests and should be followed up with a more rigorous analysis.Sandberg was supported by the NOAO/KPNO Research Experience for Undergraduates (REU) Program which is funded by the National Science Foundation Research Experiences for Undergraduates Program (AST-1262829)

  10. VizieR Online Data Catalog: Magellan/M2FS spectroscopy of Tucana 2 and Grus 1 (Walker+, 2016)

    NASA Astrophysics Data System (ADS)

    Walker, M. G.; Mateo, M.; Olszewski, E. W.; Koposov, S.; Belokurov, V.; Jethwa, P.; Nidever, D. L.; Bonnivard, V.; Bailey, J. I., III; Bell, E. F.; Loebman, S. R.

    2016-05-01

    We observed Tuc 2 and Gru 1 with the Michigan/Magellan Fiber System (M2FS) at the 6.5m Magellan/Clay telescope at Las Campanas Observatory, Chile, on the nights of July 17 (Tuc 2) and July 18 (Gru 1) 2015. We obtained repeat observations of the Tuc 2 field on 2015 September 12. We selected spectroscopic targets from the photometric catalogs that K15 (Koposov+ 2015ApJ...805..130K) generated from public images taken as part of the Dark Energy Survey (DES; Bechtol+, 2015ApJ...807...50B). DES is a wide-field optical imaging survey in the grizY bands performed with the Dark Energy Camera (DECam) installed at the prime focus of the 4m Blanco telescope at Cerro Tololo Inter-American Observatory. The full DES survey is scheduled for 525 nights distributed over 5 years. Uncertainties in the offsets between the DES photometric system and the AB system are estimated to be ~1%. (1 data file).

  11. The Role of Initial Oligomers in Amyloid Fibril Formation by Human Stefin B

    PubMed Central

    Taler-Verčič, Ajda; Kirsipuu, Tiina; Friedemann, Merlin; Noormägi, Andra; Polajnar, Mira; Smirnova, Julia; Žnidarič, Magda Tušek; Žganec, Matjaž; Škarabot, Miha; Vilfan, Andrej; Staniforth, Rosemary A.; Palumaa, Peep; Žerovnik, Eva

    2013-01-01

    Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation. PMID:24013380

  12. Random amplified polymorphic DNA (RAPD) detection of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) bananas.

    PubMed

    Damasco, O P; Graham, G C; Henry, R J; Adkins, S W; Smiths, M K; Godwin, I D

    1996-11-01

    A RAPD marker specific to the dwarf off-type (hereafter known as dwarf) from micropropagation of Cavendish banana (Musa spp. AAA) cultivars New Guinea Cavendish and Williams was identified following an analysis of 57 normal (true-to-type) and 59 dwarf plants generated from several different micropropagation events. Sixty-six random decamer primers were used in the initial screen, of which 19 (28.8%) revealed polymorphisms between normal and dwarf plants. Primer OPJ-04 (5'-CCGAACACGG-3') was found to amplify an approx. 1.5 kb band which was consistently present in all normal but absent in all dwarf plants of both cultivars. Reliable detection of dwarf plants was achieved using this marker, providing the only available means ofin vitro detection of dwarfs. The use of this marker could facilitate early detection and elimination of dwarfs from batches of micropropagated bananas, and may be a useful tool in determining what factors in the tissue culture process lead to this off type production.Other micropropagation-induced RAPD polymorphisms were observed but were not associated with the dwarf trait. PMID:24178669

  13. Effect of Protonation State on the Stability of Amyloid Oligomers Assembled from TTR(105-115).

    PubMed

    Porrini, Massimiliano; Zachariae, Ulrich; Barran, Perdita E; MacPhee, Cait E

    2013-04-18

    Amyloid fibrils are self-assembled aggregates of polypeptides that are implicated in the development of several human diseases. A peptide derived from amino acids 105-115 of the human plasma protein transthyretin forms homogeneous and well-defined fibrils and, as a model system, has been the focus of a number of studies investigating the formation and structure of this class of aggregates. Self-assembly of TTR(105-115) occurs at low pH, and this work explores the effect of protonation on the growth and stability of small cross-β aggregates. Using molecular dynamics simulations of structures up to the decamer in both protonated and deprotonated states, we find that, whereas hexamers are more stable for protonated peptides, higher order oligomers are more stable when the peptides are deprotonated. Our findings imply a change in the acid pK of the protonated C-terminal group during the formation of fibrils, which leads to stabilization of higher-order oligomers through electrostatic interactions. PMID:26282135

  14. Molecular characterization and biological response to respiration inhibitors of Pyricularia isolates from ctenanthe and rice plants.

    PubMed

    Paplomatas, Epaminondas J; Pappas, Athanasios C; Syranidou, Elene

    2005-07-01

    The molecular profile and the biological response of isolates of Pyricularia oryzae Cavara obtained from ctenanthe to two strobilurins (azoxystrobin, kresoxim-methyl) and the phenylpyridinamine fungicide fluazinam were characterized, and compared with isolates from rice plants. Five different isozymes (alpha-esterase, lactate, malate, isocitrate and sorbitol dehydrogenases) and five random decamer primers for RAPD-PCR were used to generate molecular markers. Using unweighted pair-group with arithmetic average analysis, ctenanthe isolates were found to form a separate group distinct from that of the rice isolates for both sets of markers. Amplified polymorphic sequences of mitochondrial cytochrome b that were digested with Fnu4HI or StyI revealed no differences among Pyricularia isolates at amino acid positions 143 or 129 which confer resistance to strobilurins in several fungi. In absence of the alternative respiration inhibitor salicylhydroxamic acid (SHAM) the three fungicides showed inferior and variable efficacy, with a trend toward the rice isolate being less sensitive. The addition of SHAM enhanced the effectiveness of all fungicides against isolates regardless of their origin. Appressorium formation was the most vulnerable target of action of the respiration inhibitors and azoxystrobin the most effective. This is the first report of a comparison between the molecular profiles and sensitivities to respiration inhibitors for Pyricularia oryzae isolates from a non-gramineous host and from rice. PMID:15739234

  15. Identification of Peroxiredoxin 1 as a novel interaction partner for the lifespan regulator protein p66Shc.

    PubMed

    Gertz, Melanie; Fischer, Frank; Leipelt, Martina; Wolters, Dirk; Steegborn, Clemens

    2009-02-01

    Damage caused by reactive oxygen species (ROS) contributes to many aging processes and accompanying diseases. ROS are toxic side products of cellular respiration, but also function as signal, e.g. in the mitochondrial apoptosis pathway. The protein p66Shc, which has been implicated in life-span regulation and aging-related diseases, is a central player in stress-induced apoptosis and the associated ROS burst. Stress signals, such as UV radiation or ROS themselves, activate p66Shc, which was proposed to stimulate its H(2)O(2) forming activity, ultimately triggering mitochondrial disintegration. However, mechanistic details of H(2)O(2) formation and apoptosis induction by p66Shc and regulation of these activities remain to be revealed. Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain. Prx1 was identified in affinity experiments as dominant interaction partner. Complex formation leads to disassembly of Prx1 decamers, which is known to increase its peroxidase activity. The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture. Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels. PMID:20157513

  16. Crystallization and preliminary X-ray characterization of 1,3-propanediol dehydrogenase from the human pathogen Klebsiella pneumoniae

    SciTech Connect

    Marçal, D.; Rego, A. T.; Fogg, M. J.; Wilson, K. S.; Carrondo, M. A.; Enguita, F. J.

    2007-03-01

    1,3-Propanediol dehydrogenase from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.7 Å resolution. 1,3-Propanediol dehydrogenase (1,3-PD-DH), encoded by the dhaT gene, is a key enzyme in the dissimilation process for converting glycerol to 1,3-propanediol in the human pathogen Klebsiella pneumoniae. Single colourless crystals were obtained from a recombinant preparation of 1,3-propanediol dehydrogenase overexpressed in Escherichia coli. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 91.9, b = 226.6, c = 232.6 Å, β = 92.9°. The crystals probably contain two decamers in the asymmetric unit, with a V{sub M} value of 3.07 Å{sup 3} Da{sup −1} and an estimated solvent content of 59%. Diffraction data were collected to 2.7 Å resolution using synchrotron radiation at the ID14-4 beamline of the European Synchrotron Radiation Facility.

  17. Crystal structure of a DNA/Ba2+ G-quadruplex containing a water-mediated C-tetrad.

    PubMed

    Zhang, Diana; Huang, Terry; Lukeman, Philip S; Paukstelis, Paul J

    2014-12-01

    We have determined the 1.50 Å crystal structure of the DNA decamer, d(CCA(CNV)KGCGTGG) ((CNV)K, 3-cyanovinylcarbazole), which forms a G-quadruplex structure in the presence of Ba(2+). The structure contains several unique features including a bulged nucleotide and the first crystal structure observation of a C-tetrad. The structure reveals that water molecules mediate contacts between the divalent cations and the C-tetrad, allowing Ba(2+) ions to occupy adjacent steps in the central ion channel. One ordered Mg(2+) facilitates 3'-3' stacking of two quadruplexes in the asymmetric unit, while the bulged nucleotide mediates crystal contacts. Despite the high diffraction limit, the first four nucleotides including the (CNV)K nucleoside are disordered though they are still involved in crystal packing. This work suggests that the bulky hydrophobic groups may locally influence the formation of non-Watson-Crick structures from otherwise complementary sequences. These observations lead to the intriguing possibility that certain types of DNA damage may act as modulators of G-quadruplex formation. PMID:25389267

  18. Cultivar based selection and genetic analysis of strawberry fruits with high levels of health promoting compounds.

    PubMed

    Padula, Maria Carmela; Lepore, Laura; Milella, Luigi; Ovesna, Jaroslava; Malafronte, Nicola; Martelli, Giuseppe; de Tommasi, Nunziatina

    2013-10-15

    Twenty different strawberry genotypes from phenolic compound content and genetic diversity have been investigated. Twelve phenolic derivatives in the strawberry fruit extracts, their total phenolic content (TPC) and their radical scavenging activity have been quantified. In order to study the influence of the genetic basis of each cultivar (cv) on the chemical composition of fruits, Principal Component Analysis of the obtained data was also used. Significant differences in the content of individual anthocyanins among the 20cvs have been found. Pelargonidin 3-O-glucoside was the predominant anthocyanin in the strawberry extracts with 61.0% of the total anthocyanins in Salva cv, followed by cyanidin 3-O-glucoside. TPC values ranged from 129,96 (Laica cv) to 269,04 (Naiad cv) mg of gallic acid equivalent per 100g of fresh weight and it was congruent previous studies. Moreover RAPD markers have been applied in order to describe their genetic relationships. A total of 32decamer primers were used in RAPD analysis; 19 of them provided at least one polymorphic band, the remaining primers were monomorphic. A total of 124 bands were detected with the mean number of 11.53 accountable fragments per primer and 59.98% were polymorphic. The results of the present study highlighted the health-promoting compound content of strawberry fruits, and provided a good prospect for discriminating strawberries by phenolic content and genetic analysis.

  19. Fischer carbene mediated covalent grafting of a peptide nucleic acid on gold surfaces and IR optical detection of DNA hybridization with a transition metalcarbonyl label

    NASA Astrophysics Data System (ADS)

    Srivastava, Pratima; Ghasemi, Mahsa; Ray, Namrata; Sarkar, Amitabha; Kocabova, Jana; Lachmanova, Stepanka; Hromadova, Magdalena; Boujday, Souhir; Cauteruccio, Silvia; Thakare, Pramod; Licandro, Emanuela; Fosse, Céline; Salmain, Michèle

    2016-11-01

    Amine-reactive surfaces comprising N-hydroxysuccinimide ester groups as well as much more unusual Fischer alkoxymetallocarbene groups were generated on gold-coated surfaces via self-assembled monolayers of carboxy- and azido-terminated thiolates, respectively. These functions were further used to immobilize homothymine peptide nucleic acid (PNA) decamer in a covalent fashion involving the primary amine located at its N-terminus. These stepwise processes were monitored by polarization modulation reflection - absorption infrared spectroscopy (PM-RAIRS) that gave useful information on the molecular composition of the organic layers. PNA grafting and hybridization with complementary DNA strand were successfully transduced by quartz crystal microbalance (QCM) measurements. Unfortunately, attempts to transduce the hybridization optically by IR in a label-free fashion were inconclusive. Therefore we undertook to introduce an IR reporter group, namely a transition metalcarbonyl (TMC) entity at the 5‧ terminus of complementary DNA. Evidence for the formation of PNA-DNA heteroduplex was brought by the presence of ν(Ctbnd O) bands in the 2000 cm-1 region of the IR spectrum of the gold surface owing to the metalcarbonyl label.

  20. SOURCE EXPLORER: Towards Web Browser Based Tools for Astronomical Source Visualization and Analysis

    NASA Astrophysics Data System (ADS)

    Young, M. D.; Hayashi, S.; Gopu, A.

    2014-05-01

    As a new generation of large format, high-resolution imagers come online (ODI, DECAM, LSST, etc.) we are faced with the daunting prospect of astronomical images containing upwards of hundreds of thousands of identifiable sources. Visualizing and interacting with such large datasets using traditional astronomical tools appears to be unfeasible, and a new approach is required. We present here a method for the display and analysis of arbitrarily large source datasets using dynamically scaling levels of detail, enabling scientists to rapidly move from large-scale spatial overviews down to the level of individual sources and everything in-between. Based on the recognized standards of HTML5+JavaScript, we enable observers and archival users to interact with their images and sources from any modern computer without having to install specialized software. We demonstrate the ability to produce large-scale source lists from the images themselves, as well as overlaying data from publicly available source ( 2MASS, GALEX, SDSS, etc.) or user provided source lists. A high-availability cluster of computational nodes allows us to produce these source maps on demand and customized based on user input. User-generated source lists and maps are persistent across sessions and are available for further plotting, analysis, refinement, and culling.

  1. Profiles and α-amylase inhibition activity of proanthocyanidins in unripe Manilkara zapota (chiku).

    PubMed

    Wang, Hongyu; Liu, Tingting; Song, Lixia; Huang, Dejian

    2012-03-28

    Proanthocyanidins in unripe Manilkara zapota (chiku) were isolated using solvent extraction followed by Sephadex LH-20 fractionation with a yield of 0.9%. HPLC analysis using a diol column revealed well-resolved oligomers ranging from dimer to hexamer. The majority of the proanthocyanidins are composed of higher-degree oligomers appearing as one large peak in the chromatogram. Analysis of the proanthocyanidins using LC/MS showed that (epi)gallocatechins were the dominant extension unit in the proanthocyanidins. In agreement with this result, thiolysis treatment of the proanthocyanidins using mercaptoacetic acid produced thioether derivatives of (epi)gallocatechins as the major product and (epi)gallocatechin gallate derivatives as the minor product. The mean of the degree of polymerization was estimated to be 9.0. From MALDI-TOF MS, B-type gallocatechin oligomers up to decamer could be detected. The unripe chiku proanthocyanidins are thus good starting material for preparation of (epi)gallocatechin derivatives. The proanthocyanidins was shown to inhibit α-amylase with an IC(50) value of 4.2 ± 0.2 μg/mL and inhibit α-glucosidase with an IC(50) of 16.6 ± 0.3 μg/mL.

  2. Sex Determination in the Fly Megaselia Scalaris, a Model System for Primary Steps of Sex Chromosome Evolution

    PubMed Central

    Traut, W.

    1994-01-01

    The fly Megaselia scalaris Loew possesses three homomorphic chromosome pairs; 2 is the sex chromosome pair in two wild-type laboratory stocks of different geographic origin (designated ``original'' sex chromosome pair in this paper). The primary male-determining function moves at a very low rate to other chromosomes, thereby creating new Y chromosomes. Random amplified polymorphic DNA markers obtained by polymerase chain reaction with single decamer primers and a few available phenotypic markers were used in testcrosses to localize the sex-determining loci and to define the new sex chromosomes. Four cases are presented in which the primary male-determining function had been transferred from the original Y chromosome to a new locus either on one of the autosomes or on the original X chromosome, presumably by transposition. In these cases, the sex-determining function had moved to a different locus without an obvious cotransfer of other Y chromosome markers. Thus, with Megaselia we are afforded an experimental system to study the otherwise hypothetical primary stages of sex chromosome evolution. An initial molecular differentiation is apparent even in the new sex chromosomes. Molecular differences between the original X and Y chromosomes illustrate a slightly more advanced stage of sex chromosome evolution. PMID:8005417

  3. Peptide array on cellulose support--a screening tool to identify peptides with dipeptidyl-peptidase IV inhibitory activity within the sequence of α-lactalbumin.

    PubMed

    Lacroix, Isabelle M E; Li-Chan, Eunice C Y

    2014-11-13

    The inhibition of the enzyme dipeptidyl-peptidase IV (DPP-IV) is an effective pharmacotherapeutic approach for the management of type 2 diabetes. Recent findings have suggested that dietary proteins, including bovine α-lactalbumin, could be precursors of peptides able to inhibit DPP-IV. However, information on the location of active peptide sequences within the proteins is far from being comprehensive. Moreover, the traditional approach to identify bioactive peptides from foods can be tedious and long. Therefore, the objective of this study was to use peptide arrays to screen α-lactalbumin-derived peptides for their interaction with DPP-IV. Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using "SPOT" technology, and their binding to and inhibition of DPP-IV was studied. Among the 114 α-lactalbumin-derived decamers investigated, the peptides 60WCKDDQNPHS69 (αK(i) = 76 µM), 105LAHKALCSEK114 (K(i) = 217 µM) and 110LCSEKLDQWL119 (K(i) = 217 µM) were among the strongest DPP-IV inhibitors. While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides' binding behavior was not correlated to their ability to inhibit the enzyme. This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.

  4. The Dark Energy Survey and Operations: Years 1 to 3

    SciTech Connect

    Diehl, H. T.

    2016-01-01

    The Dark Energy Survey (DES) is an operating optical survey aimed at understanding the accelerating expansion of the universe using four complementary methods: weak gravitational lensing, galaxy cluster counts, baryon acoustic oscillations, and Type Ia supernovae. To perform the 5000 sq-degree wide field and 30 sq-degree supernova surveys, the DES Collaboration built the Dark Energy Camera (DECam), a 3 square-degree, 570-Megapixel CCD camera that was installed at the prime focus of the Blanco 4-meter telescope at the Cerro Tololo Inter-American Observatory (CTIO). DES has completed its third observing season out of a nominal five. This paper describes DES “Year 1” (Y1) to “Year 3” (Y3), the strategy, an outline of the survey operations procedures, the efficiency of operations and the causes of lost observing time. It provides details about the quality of the first three season's data, and describes how we are adjusting the survey strategy in the face of the El Niño Southern Oscillation

  5. Structural investigation into physiological DNA phosphorothioate modification

    PubMed Central

    Lan, Wenxian; Hu, Zhongpei; Shen, Jie; Wang, Chunxi; Jiang, Feng; Liu, Huili; Long, Dewu; Liu, Maili; Cao, Chunyang

    2016-01-01

    DNA phosphorothioate (PT) modification, with sulfur replacing a nonbridging phosphate oxygen in a sequence and stereo specific manner, is a novel physiological variation in bacteria. But what effects on DNA properties PT modification has is still unclear. To address this, we prepared three double-stranded (ds) DNA decamers, d(CGPXGCCGCCGA) with its complementary strand d(TCGGCGPXGCCG) (where X = O or S, i.e., PT-free dsDNA, [Sp, Sp]-PT dsDNA or [Rp, Rp]-PT dsDNA) located in gene of Streptomyces lividans. Their melting temperature (Tm) measurement indicates that [Rp, Rp]-PT dsDNA is most unstable. Their electron transfer potential detection presents an order of anti-oxidation properties: Sp-PT DNA > Rp-PT DNA > PT-free DNA. Their NMR structures demonstrate that PT modification doesn’t change their B-form conformation. The sulfur in [Rp, Rp]-PT dsDNA locates in the major groove, with steric effects on protons in the sugar close to modification sites, resulting in its unstability, and facilitating its selectively interactions with ScoMcrA. We thought that PT modification was dialectical to the bacteria. It protects the hosting bacteria by working as antioxidant against H2O2, and acts as a marker, directing restriction enzyme observed in other hosts, like ScoMcrA, to correctly cleave the PT modified DNA, so that bacteria cannot spread and survive. PMID:27169778

  6. Insight into the packing pattern of β2 fibrils: a model study of glutamic acid rich oligomers with 13C isotopic edited vibrational spectroscopy.

    PubMed

    Chi, Heng; Welch, William R W; Kubelka, Jan; Keiderling, Timothy A

    2013-11-11

    Polyglutamic acid at low pH forms aggregates and self-assembles into a spiral, fibril-like superstructure formed as a β2-type sheet conformation that has a more compact intersheet packing than commonly found. This is stabilized by three-centered bifurcated hydrogen bonding of the amide carbonyl involving the protonated glutamic acid side chain. We report vibrational spectroscopic results and analyses for oligopeptides rich in glutamic acid enhanced with (13)C isotope labeling in a study modeling low pH poly-Glu self-assembly. Our results indicate bifurcated H-bonding and β2 aggregation can be attained in these model decamers, confirming they have the same conformations as poly-Glu. We also prepared conventional β1-sheet aggregates by rapid precipitation from the residual peptides in the higher pH supernatant. By comparing the isotope-enhanced IR and VCD spectra with theoretical predictions, we deduced that the oligo-Glu β2 structure is based on stacked, twisted, antiparallel β-sheets. The best fit to theoretical predictions was obtained for the strands being out of register, sequentially stepped by one residue, in a ladder-like fashion. The alternate β1 conformer for this oligopeptide was similarly shown to be antiparallel but was less ordered and apparently had a different registry in its aggregate structure.

  7. Stabilization, Characterization, and Selective Removal of Cystatin C Amyloid Oligomers*

    PubMed Central

    Östner, Gustav; Lindström, Veronica; Hjort Christensen, Per; Kozak, Maciej; Abrahamson, Magnus; Grubb, Anders

    2013-01-01

    The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers. PMID:23629649

  8. Design and Development of a Cyclic Decapeptide Scaffold with Suitable Properties for Bioavailability and Oral Exposure.

    PubMed

    Fouché, Marianne; Schäfer, Michael; Berghausen, Jörg; Desrayaud, Sandrine; Blatter, Markus; Piéchon, Philippe; Dix, Ina; Martin Garcia, Aimar; Roth, Hans-Jörg

    2016-05-19

    Permeability and oral bioavailability of macrocyclic peptides still represent difficult challenges in drug discovery. Despite the recognized potential of macrocyclic peptides as therapeutics, their use is still restricted to extracellular targets and intravenous administration. Indeed, macrocyclic peptides generally suffer from limited proteolytic stability, high clearance, and poor membrane permeability, and this leads to the absence of systemic exposure after oral administration. To overcome these limitations, we started to investigate the development of a general cyclic decapeptide scaffold that possesses ideal features for cell permeability and oral exposure. On the basis of a rigid hairpin structure, the scaffold design aimed to decrease the overall polarity of the compound, thereby limiting the energetic cost of NH desolvation and the entropy penalty during cell penetration. The results of this study also demonstrate the importance of rigidity for the β-turn design regarding clearance. To stabilize the scaffold in the desired β-hairpin conformation, the introduction of d-proline at the i+1 turn position proved to be beneficial for both permeability and clearance. As a result, cyclopeptide decamers with unprecedented high values for oral bioavailability and exposure are reported herein. NMR spectroscopy conformation and dynamic analysis confirmed, for selected examples, the rigidity of the scaffold and the presence of transannular hydrogen bonds in polar and apolar environments. Furthermore, we showed, for one compound, that its transition from a polar environment to an apolar one was accompanied by an increased molecular motion, revealing an entropy contribution to membrane permeation. PMID:27154275

  9. Genetic diversity in mesoamerican populations of mahogany (Swietenia macrophylla), assessed using RAPDs.

    PubMed

    Gillies, A C; Navarro, C; Lowe, A J; Newton, A C; Hernández, M; Wilson, J; Cornelius, J P

    1999-12-01

    Swietenia macrophylla King, a timber species native to tropical America, is threatened by selective logging and deforestation. To quantify genetic diversity within the species and monitor the impact of selective logging, populations were sampled across Mesoamerica, from Mexico to Panama, and analysed for RAPD DNA variation. Ten decamer primers generated 102 polymorphic RAPD bands and pairwise distances were calculated between populations according to Nei, then used to construct a radial neighbour-joining dendrogram and examine intra- and interpopulation variance coefficients, by analysis of molecular variation (AMOVA). Populations from Mexico clustered closely together in the dendrogram and were distinct from the rest of the populations. Those from Belize also clustered closely together. Populations from Panama, Guatemala, Costa Rica, Nicaragua and Honduras, however, did not cluster closely by country but were more widely scattered throughout the dendrogram. This result was also reflected by an autocorrelation analysis of genetic and geographical distance. Genetic diversity estimates indicated that 80% of detected variation was maintained within populations and regression analysis demonstrated that logging significantly decreased population diversity (P = 0.034). This study represents one of the most wide-ranging surveys of molecular variation within a tropical tree species to date. It offers practical information for the future conservation of mahogany and highlights some factors that may have influenced the partitioning of genetic diversity in this species across Mesoamerica.

  10. The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

    PubMed Central

    Brochu, Denis; Vadeboncoeur, Christian

    1999-01-01

    In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg2+ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its Kms for HPr and ATP are 31 μM and 1 mM, respectively. PMID:9922231

  11. Crystal structure of a DNA/Ba2+ G-quadruplex containing a water-mediated C-tetrad.

    PubMed

    Zhang, Diana; Huang, Terry; Lukeman, Philip S; Paukstelis, Paul J

    2014-12-01

    We have determined the 1.50 Å crystal structure of the DNA decamer, d(CCA(CNV)KGCGTGG) ((CNV)K, 3-cyanovinylcarbazole), which forms a G-quadruplex structure in the presence of Ba(2+). The structure contains several unique features including a bulged nucleotide and the first crystal structure observation of a C-tetrad. The structure reveals that water molecules mediate contacts between the divalent cations and the C-tetrad, allowing Ba(2+) ions to occupy adjacent steps in the central ion channel. One ordered Mg(2+) facilitates 3'-3' stacking of two quadruplexes in the asymmetric unit, while the bulged nucleotide mediates crystal contacts. Despite the high diffraction limit, the first four nucleotides including the (CNV)K nucleoside are disordered though they are still involved in crystal packing. This work suggests that the bulky hydrophobic groups may locally influence the formation of non-Watson-Crick structures from otherwise complementary sequences. These observations lead to the intriguing possibility that certain types of DNA damage may act as modulators of G-quadruplex formation.

  12. Genetic diversity of eggplant (Solanum melongena) germplasm from Turkey assessed by SSR and RAPD markers.

    PubMed

    Demir, K; Bakir, M; Sarikamiş, G; Acunalp, S

    2010-08-10

    Eggplant is a major crop in Turkey, which produces more of this crop than all of Europe; consequently, germplasm resources are of concern for the country. Molecular characterization of eggplant genotypes collected from different geographical regions of Turkey was carried out using SSR and RAPD markers. With amplification of five SSR loci, the number of alleles per microsatellite locus ranged from 2 to 10, with a total of 24 alleles. The greatest number of alleles was found at the emf21H22 locus (10 alleles); followed by emh11O01 and emf21C11 as five and four alleles, respectively. The average number of alleles per locus was 4.8. Using 11 decamer RAPD primers, 100 bands were amplified, among which 29 were polymorphic. The number of bands per primer ranged from seven (OPH10, OPH19, OPH20, OPH03) to 14 (OPB07). Primer OPB07 was the most polymorphic, generating 64% polymorphic bands; the rest of the primers gave less than 50% polymorphism. UPGMA dendrograms were used to examine the genetic relatedness of the genotypes.

  13. Crystallization and X-ray diffraction analysis of the Trp/amber editing site of hepatitis delta virus (+)RNA: a case of rational design

    SciTech Connect

    MacElrevey, Celeste; Wedekind, Joseph E.

    2005-12-01

    Well diffracting decamer crystals of the hepatitis delta virus RNA-editing site were prepared, but exhibited merohedral twinning and base averaging owing to duplex symmetry. A longer asymmetric construct that includes additional flanking RNA sequences has been crystallized that does not appear to exhibit these defects. RNA editing by mammalian ADAR1 (Adenosine Deaminase Acting on RNA) is required for the life cycle of the hepatitis delta virus (HDV). Editing extends the single viral open reading frame to yield two protein products of alternate length. ADARs are believed to recognize double-stranded RNA substrates via a ‘structure-based’ readout mechanism. Crystals of 10-mer duplexes representing the HDV RNA-editing site diffracted to 1.35 Å resolution, but suffered from merohedral twinning and averaging of the base registry. Expansion of the construct to include two flanking 3 × 1 internal loops yielded crystals in the primitive tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. X-ray diffraction data were collected to 2.8 Å resolution, revealing a unit cell with parameters a = 62.5, c = 63.5 Å. The crystallization and X-ray analysis of multiple forms of the HDV RNA-editing substrate, encounters with common RNA crystal-growth defects and a strategy to overcome these problems are reported.

  14. Large Scale Structure in the Epoch of Reionization

    NASA Astrophysics Data System (ADS)

    Koekemoer, Anton; Mould, Jeremy; Cooke, Jeffrey; Wyithe, Stuart; Lidman, Christopher; Trenti, Michele; Abbott, Tim; Kunder, Andrea; Barone-Nugent, Robert; Tescari, Edoardo; Katsianis, Antonios

    2014-02-01

    We propose to capitalize on the high red sensitivity and large field of view of DECam to detect the brightest and rarest galaxies at z=6-7. Our 2012 results show the signature of large scale structure with wavenumber of order 0.1 inverse Mpc in line with expectations of primordial non-gaussianity. But the signal to noise in one deep field from two nights' data is insufficient for a robust conclusion. Ten nights' data will do the job. These data will also constrain the galaxy contribution to reionization by enabling a tighter constraint on the full galaxy luminosity function, including the faint end. The observations will be executed with a cadence and depth that will enable the detection of super-luminous supernovae at z=6-7. Super-luminous supernovae are a recently observed class of supernovae that are 10-100x more luminous than typical supernovae. This class includes pair- instability supernovae that are a rare, third type of supernova explosion in which only 3 events are known. The proposed observations will greatly extend the current reach of supernovae research, examining their occurrence rate and properties near the epoch of reionization.

  15. Molecular characterization and biological response to respiration inhibitors of Pyricularia isolates from ctenanthe and rice plants.

    PubMed

    Paplomatas, Epaminondas J; Pappas, Athanasios C; Syranidou, Elene

    2005-07-01

    The molecular profile and the biological response of isolates of Pyricularia oryzae Cavara obtained from ctenanthe to two strobilurins (azoxystrobin, kresoxim-methyl) and the phenylpyridinamine fungicide fluazinam were characterized, and compared with isolates from rice plants. Five different isozymes (alpha-esterase, lactate, malate, isocitrate and sorbitol dehydrogenases) and five random decamer primers for RAPD-PCR were used to generate molecular markers. Using unweighted pair-group with arithmetic average analysis, ctenanthe isolates were found to form a separate group distinct from that of the rice isolates for both sets of markers. Amplified polymorphic sequences of mitochondrial cytochrome b that were digested with Fnu4HI or StyI revealed no differences among Pyricularia isolates at amino acid positions 143 or 129 which confer resistance to strobilurins in several fungi. In absence of the alternative respiration inhibitor salicylhydroxamic acid (SHAM) the three fungicides showed inferior and variable efficacy, with a trend toward the rice isolate being less sensitive. The addition of SHAM enhanced the effectiveness of all fungicides against isolates regardless of their origin. Appressorium formation was the most vulnerable target of action of the respiration inhibitors and azoxystrobin the most effective. This is the first report of a comparison between the molecular profiles and sensitivities to respiration inhibitors for Pyricularia oryzae isolates from a non-gramineous host and from rice.

  16. Hexahydrated magnesium ions bind in the deep major groove and at the outer mouth of A-form nucleic acid duplexes.

    SciTech Connect

    Robinson, H.; Gao, Y.-G.; Sanishvili, R.; Joachimiak, A.; Wang, A. H.-J.; Univ. of Illinois; Northwestern Univ.

    2000-01-01

    Magnesium ions play important roles in the structure and function of nucleic acids. Whereas the tertiary folding of RNA often requires magnesium ions binding to tight places where phosphates are clustered, the molecular basis of the interactions of magnesium ions with RNA helical regions is less well understood. We have refined the crystal structures of four decamer oligonucleotides, d(ACCGGCCGGT), r(GCG)d(TATACGC), r(GC)d(GTATACGC) and r(G)d(GCGTATACGC) with bound hexahydrated magnesium ions at high resolution. The structures reveal that A-form nucleic acid has characteristic [Mg(H2O)6]2+ binding modes. One mode has the ion binding in the deep major groove of a GpN step at the O6/N7 sites of guanine bases via hydrogen bonds. Our crystallographic observations are consistent with the recent NMR observations that in solution [Co(NH3)6]3+, a model ion of [Mg(H2O)6]2+, binds in an identical manner. The other mode involves the binding of the ion to phosphates, bridging across the outer mouth of the narrow major groove. These [Mg(H2O)6]2+ ions are found at the most negative electrostatic potential regions of A-form duplexes. We propose that these two binding modes are important in the global charge neutralization, and therefore stability, of A-form duplexes.

  17. Probing Early Galaxy Growth and Dusty Star-Forming Systems Across Diverse Environments in the 28 deg2 Herschel/Stripe82/HETDEX Field

    NASA Astrophysics Data System (ADS)

    Larson, Rebecca; Jogee, Shardha; Watson, Nicholas; Viero, Marco; Weinzirl, Tim; Yorke, Harold W.; Finkelstein, Steven; Papovich, Casey; Casey, Caitlin M.; Ciardullo, Robin; Gronwall, Caryl; LaMassa, Stephanie; Urry, C. Meg

    2015-08-01

    In the next few years, we will embark on an unprecedented study of how a million galaxies grow their stars and dark matter halos over a large a huge comoving volume (0.5 Gpc^3) in the cosmic web at the critical epoch (z~1.9 - 3.5), where cosmic star formation and black hole activity peak, and proto-clusters start to collapse. This study is enabled by the powerful synergy of six photometric and spectroscopic surveys, which are providing Herschel SPIRE, Spitzer IRAC, NEWFIRM K-band, DECam ugriz, and XMM X-ray imaging data, along with optical spectroscopic data from HETDEX over a very large-area (28 sq. deg.) in the Stripe82/HETDEX field. In this poster, we illustrate the power of these combined datasets and focus on studying dusty, star-forming systems (DSFSs) identified with the Herschel Stripe 82 Survey (HerS). Using the 250, 350, and 500 micron SPIRE data over our 28 sq. deg. field, we identify a number of possible high redshift (z > 4) DSFSs which will be prime candidates for follow-up observations. We discuss their properties and possible association with galaxies and quasars detected at X-ray, IR, optical, and UV wavelengths. We present examples of SED fits to DSFSs to constrain their star formation rates, redshifts and dust properties, and discuss broader implications for galaxy growth at early cosmic times. We acknowledge support from NSF grant AST-1413652 andthe JPL/NASA SURP Program.

  18. Light Echoes of Galactic Explosions and Eruptions

    NASA Astrophysics Data System (ADS)

    Rest, Armin; Bianco, Federica; Chornock, Ryan; Clocchiatti, Alejandro; Foley, Ryan; James, David; Matheson, Thomas; Narayan, Gautham; Olsen, Knut; Points, Sean; Prieto, Jose Luis; Smith, Chris; Smith, Nathan; Suntzeff, Nick; Welch, Doug; Zenteno, Alfredo

    2014-08-01

    We propose to search for light echoes (LEs) from the historical brightening of the Luminous Blue Variable (LBV) P Cygni using the KPNO 4m Mosaic 1.1 imager. We also propose to us DECam to continue our search for LEs from the the Crab supernova SN 1054. In addition, we continue to monitor the LEs from the Cas A and Tycho supernovae in order to identify suitable LE candidates for 3D-spectroscopy and spectral time series. In previously granted NOAO time, we have discovered light echoes of three ancient SNe in the LMC as well as from the historic SN events of Cas A and Tycho [2, 3], which allowed their spectroscopic classification [6, 7, 10] and 3D spectroscopy [8, 9]. Most recently, we discovered light echoes of the mid-19th-century Great Eruption of η Carinae using CTIO 4m Mosaic images [11]. Subsequent spectroscopic follow-up of Eta Carinae revealed that its outburst spectral type was most similar to those of G-type supergiants, rather than reported LBV outburst spectral types of F-type (or earlier) [11]. Our extension of LE techniques to LBV outbursts promises to extend our ability to record outburst activity hundreds of years into the past - a timescale which is likely a significant fraction of the brief final phases of these probable core- collapse supernova precursors.

  19. Photometric Calibrations of Standard Star Fields for the Dark Energy Survey

    NASA Astrophysics Data System (ADS)

    Wyatt, Samuel; Tucker, D. L.; Smith, A.

    2014-01-01

    The Dark Energy Survey (DES) is a 5000 square deg grizY imaging survey to be conducted using the new 3 square deg (2. 2-diameter) wide-field mosaic camera (DECam) on the CTIO Blanco 4-m telescope. The primary scientific goal of the DES is to constrain dark energy cosmological parameters via four complementary methods: galaxy cluster counting, weak lensing, galaxy angular correlations, and Type Ia supernovae, supported by precision photometric redshifts. Here, we describe code developed to calibrate additional standard star fields in the DES natural grizY system, and we describe our initial results using the data obtained during the DES Science Verification Phase. Some of these standard stars will be used to supplement those currently being used for nightly calibrations. Others are to be used as local tertiary standards scattered throughout the DES footprint in order to anchor the DES global relative calibrations against large-scale spatial gradients, as well as to tie the whole DES data set to an initial absolute flux scale.

  20. Light Echoes of Galactic Explosions and Eruptions

    NASA Astrophysics Data System (ADS)

    Rest, Armin; Bianco, Federica; Chornock, Ryan; Foley, Ryan; Matheson, Thomas; Olsen, Knut; Prieto, Jose Luis; Sinnott, Brendan; Smith, Chris; Smith, Nathan; Welch, Doug

    2013-02-01

    We propose to continue our search for the first light echoes (LEs) associated with historical Galactic supernovae and LBV outbursts: SN 1006, Kepler's SN, RCW 86, Crab Nebula, and P Cygni. In previously granted NOAO time, we have discovered light echoes of three ancient SNe in the LMC as well as from the historic SN events of Cas A and Tycho [2, 3], which allowed their spectroscopic classification [6, 7, 10] and 3D spectroscopy [8, 9]. Most recently, we discovered light echoes of the mid-19th-century Great Eruption of eta Carinae using CTIO 4m Mosaic images [11]. Subsequent spectroscopic follow-up of Eta Carinae revealed that its outburst spectral type was most similar to those of G-type supergiants, rather than reported LBV outburst spectral types of F-type (or earlier) [11]. We propose to continue our search for light echoes of the remaining historical events. With DECam, we have a 10-15 fold improvement in efficiency over the retired Mosaic camera, which allows us to cover the bigger search areas of most of the remaining targets. The study of scattered-light echoes from these Galactic supernovae and eruptions will give us the opportunity to directly compare the original outburst and its current remnant, and in favorable cases (like Eta Carinae), it provides a three-dimensional view of the event and/or a spectral time series.

  1. Identification of an Extradiol Dioxygenase Involved in Tetralin Biodegradation: Gene Sequence Analysis and Purification and Characterization of the Gene Product

    PubMed Central

    Andújar, Eloísa; Hernáez, María José; Kaschabek, Stefan R.; Reineke, Walter; Santero, Eduardo

    2000-01-01

    A genomic region involved in tetralin biodegradation was recently identified in Sphingomonas strain TFA. We have cloned and sequenced from this region a gene designated thnC, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to define a subfamily of 1,2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7,8-tetrahydronaphthalene was found to be the metabolite accumulated by a thnC insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (Vmax, 40.5 U mg−1; Km, 18.6 μM). The enzyme shows even higher activity with 1,2-dihydroxynaphthalene and also significant activity toward 1,2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1,2-dihydroxynaphthalene dioxygenases. PMID:10633115

  2. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

    PubMed Central

    Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; Dauter, Zbigniew

    2015-01-01

    The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Furthermore, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors. PMID:26579138

  3. The octamer motif in immunoglobulin genes: extraction of structural constraints from two-dimensional NMR studies.

    PubMed

    Weisz, K; Shafer, R H; Egan, W; James, T L

    1992-08-25

    Phase-sensitive two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlated (2QF-COSY) spectra were recorded at 500 MHz for the DNA duplex d(CATTTGCATC).d(GATGCAAATG), which contains the octamer element of immunoglobulin genes. Exchangeable and nonexchangeable proton resonances including those of the H5' and H5" protons were assigned. Overall, the decamer duplex adopts a B-type DNA conformation. Scalar coupling constants for the sugar protons were determined by quantitative simulations of 2QF-COSY cross-peaks. These couplings are consistent with a two-state dynamic equilibrium between a minor N- and a major S-type conformer for all residues. The pseudorotation phase angle P of the major conformer is in the range 117-135 degrees for nonterminal pyrimidine nucleotides and 153-162 degrees for nonterminal purine nucleotides. Except for the terminal residues, the minor conformer comprises less than 25% of the population. Distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities with the MARDIGRAS algorithm confirm the dependence of the sugar pucker on pyrimidine and purine bases. Averaging by fast local motions has at most small effects on the NOE-derived interproton distances.

  4. Development of RAPD and SCAR markers linked to the Pvr4 locus for resistance to PVY in pepper ( Capsicum annuum L.).

    PubMed

    Arnedo-Andrés, S.; Gil-Ortega, R.; Luis-Arteaga, M.; Hormaza, I.

    2002-11-01

    Potato Virus Y (PVY) is the only potyvirus infecting pepper ( Capsicum annuum L.) in Europe. Currently, the development of pepper varieties resistant to PVY seems to be the most-efficient method to control PVY damage. Among the sources of resistance, a monogenic dominant gene Pvr4 confers resistance against all known PVY pathotypes. In this work, bulked segregant analysis (BSA) was used to search for randomly amplified polymorphic DNA (RAPD) markers linked to the Pvr4 gene, using segregating progenies obtained by crossing a homozygous resistant ('Serrano Criollo de Morelos-334') with a homozygous susceptible ('Yolo Wonder') cultivar. Eight hundred decamer primers were screened to identify one RAPD marker (UBC19(1432)) linked in repulsion phase to Pvr4. This marker was converted into a dominant sequence characterised amplified region (SCAR) marker (SCUBC19(1423)). This marker was mapped into a dense Capsicum genetic map in a region where several genes for resistance to different diseases are located. This marker can be useful to identify PVY-resistant genotypes in segregating progenies of pepper in marker-assisted selection (MAS) breeding programs. PMID:12582935

  5. Communication: variational many-body expansion: accounting for exchange repulsion, charge delocalization, and dispersion in the fragment-based explicit polarization method.

    PubMed

    Gao, Jiali; Wang, Yingjie

    2012-02-21

    A fragment-based variational many-body (VMB) expansion method is described to directly account for exchange repulsion, charge delocalization (charge transfer) and dispersion interactions in the explicit polarization (X-Pol) method. The present VMB/X-Pol approach differs from other fragment molecular orbital (FMO) techniques in two major aspects. First, the wave function for the monomeric system is variationally optimized using standard X-Pol method, as opposed to the iterative update procedure adopted in FMO. Second, the mutual polarizations in the dimeric terms are also variationally determined, whereas single-point energy calculations of the individual dimers embedded in a static monomer field are used in FMO. The second-order (two-body) VMB (VMB2) expansion method is illustrated on a series of water hexamer complexes and one decamer cluster, making use of Hartree-Fock theory, MP2, and the PBE1 and M06 density functionals to represent the monomer and dimer fragments. The computed binding energies are within 2 kcal/mol of the corresponding results from fully delocalized calculations. Energy decomposition analyses reveal specific dimeric contributions to exchange repulsion, charge delocalization, and dispersion. Since the wave functions for one-body and all two-body terms are variationally optimized in VMB2 and X-Pol, it is straightforward to obtain analytic gradient without the additional coupled-perturbed Hartree-Fock step. Thus, the method can be useful for molecular dynamics simulations.

  6. A closer look at the Canarias Einstein ring

    NASA Astrophysics Data System (ADS)

    Lee (), Chien-Hsiu

    2016-11-01

    Following the recently discovery of the Canarias Einstein ring by Bettinelli et al., we present further studies of this system. Searching the DECam data archive, we found i-band images of this system, in additional to the g-, and r-band images presented in Bettinelli et al. Using GALFIT, we are able to model the lens light contribution and derive the lens colour, g - r = 2.27 and r - i = 0.70 mag, indicating that the lens is a luminous red galaxy. After subtracting the lens light, we use LENSVIEW to model the Einstein ring image and obtain an angular Einstein radius of 2.25 arcsec, which translates into an enclosed lens mass of 2.02 × 1012 M⊙ and a velocity dispersion of 460 km s-1, assuming the lens has a singular isothermal ellipsoid mass profile. The best-fitting model yields a total magnification of 10.5 and enables us to derive the un-lensed star formation rate of the source to be ˜8 M⊙ yr-1.

  7. Calculations of band gaps in polyaniline from theoretical studies of oligomers

    SciTech Connect

    Kwon, O.; McKee, M.L.

    2000-03-02

    Geometries and band gaps of polyaniline oligomers up to decamer have been systematically calculated and analyzed using various computational techniques such as molecular mechanics, semiempirical, and ab initio methods. On the basis of fully optimized geometries of neural and charged forms of polyaniline oligomers, excitation energies are calculated at the semiempirical ZINDO (INDO/S) level and extrapolated to the band gap value of the infinite chain. Band gaps are also approximately by extrapolating the HOMO/LUMO difference calculated at the density functional level (B3LYP/6--31G*). The SINDO//AM1 band gaps in the reduced and oxidized form of polyaniline (4.3 and 2.7 eV) are in good agreement with experimental values (3.8 {+-} 2 and 1.8 {+-} 3 eV, respectively). The doped form of polyaniline (two positive charges per four aniline units) has been computed with a spin-unrestricted method (UAM1) and the band gap approximated from an extrapolation of the tetramer and octamer. The calculated band gap of 1.3 eV (UZINDO//UAM1) is in good agreement with experiment (1.5 eV). The influence of ring torsional angle and interchain interaction on the band gap of the polyaniline system are also discussed.

  8. Peptide-conjugated hapten groups are the major antigenic determinants for trinitrophenyl-specific cytotoxic T cells.

    PubMed

    von Bonin, A; Ortmann, B; Martin, S; Weltzien, H U

    1992-08-01

    Several trinitrophenyl (TNP)-specific mouse cytotoxic T cell (CTL) clones recognize TNP-conjugated peptides in association with class I MHC molecules ('hapten-peptide determinants'). However, cell modification with trinitrobenzene sulfonic acid (TNBS) also leads to the formation of TNP determinants covalently attached to MHC molecules ('altered self'). To determine the importance of 'peptide' versus 'altered self' determinants, we used the mutant cell line RMA-S which expresses peptide-free ('empty') Kb and Db molecules at 26 degrees C. Additionally, we stabilized Kb molecules on RMA-S cells at 37 degrees C using the Kb binding heptapeptide N53-59 derived from the vesicular stomatitis virus nucleoprotein. Lacking lysine, this peptide remains unmodified by TNBS and, therefore, only allows the formation of 'altered self' TNP determinants on occupied Kb molecules. RMA-S targets, pretreated or untreated with N53-59, upon TNBS modification were only lysed poorly or not at all by four different TNP-specific CTL. In contrast, all of these clones efficiently lysed TNBS-treated, unmutated RMA cells, and three of them strongly reacted with RMA or RMA-S cells in the presence of tryptic TNP-BSA peptides. Moreover, the clone unreactive for TNP-BSA peptides also recognized TNP self-peptides extracted from TNBS-treated syngeneic spleen cells. Taken together, these data clearly show that TNP residues linked to MHC via associated peptides but not by covalent bondage represent the dominant antigenic epitopes for class I MHC-restricted, hapten-specific T cells. PMID:1384686

  9. Calmodulin inhibition regulates morphological and functional changes related to the actin cytoskeleton in pure microglial cells.

    PubMed

    Szabo, Melinda; Dulka, Karolina; Gulya, Karoly

    2016-01-01

    The roles of calmodulin (CaM), a multifunctional intracellular calcium receptor protein, as concerns selected morphological and functional characteristics of pure microglial cells derived from mixed primary cultures from embryonal forebrains of rats, were investigated through use of the CaM antagonists calmidazolium (CALMID) and trifluoperazine (TFP). The intracellular localization of the CaM protein relative to phalloidin, a bicyclic heptapeptide that binds only to filamentous actin, and the ionized calcium-binding adaptor molecule 1 (Iba1), a microglia-specific actin-binding protein, was determined by immunocytochemistry, with quantitative analysis by immunoblotting. In unchallenged and untreated (control) microglia, high concentrations of CaM protein were found mainly perinuclearly in ameboid microglia, while the cell cortex had a smaller CaM content that diminished progressively deeper into the branches in the ramified microglia. The amounts and intracellular distributions of both Iba1 and CaM proteins were altered after lipopolysaccharide (LPS) challenge in activated microglia. CALMID and TFP exerted different, sometimes opposing, effects on many morphological, cytoskeletal and functional characteristics of the microglial cells. They affected the CaM and Iba1 protein expressions and their intracellular localizations differently, inhibited cell proliferation, viability and fluid-phase phagocytosis to different degrees both in unchallenged and in LPS-treated (immunologically challenged) cells, and differentially affected the reorganization of the actin cytoskeleton in the microglial cell cortex, influencing lamellipodia, filopodia and podosome formation. In summary, these CaM antagonists altered different aspects of filamentous actin-based cell morphology and related functions with variable efficacy, which could be important in deciphering the roles of CaM in regulating microglial functions in health and disease.

  10. SM50 repeat-polypeptides self-assemble into discrete matrix subunits and promote appositional calcium carbonate crystal growth during sea urchin tooth biomineralization.

    PubMed

    Mao, Yelin; Satchell, Paul G; Luan, Xianghong; Diekwisch, Thomas G H

    2016-01-01

    The two major proteins involved in vertebrate enamel formation and echinoderm sea urchin tooth biomineralization, amelogenin and SM50, are both characterized by elongated polyproline repeat domains in the center of the macromolecule. To determine the role of polyproline repeat polypeptides in basal deuterostome biomineralization, we have mapped the localization of SM50 as it relates to crystal growth, conducted self-assembly studies of SM50 repeat polypeptides, and examined their effect on calcium carbonate and apatite crystal growth. Electron micrographs of the growth zone of Strongylocentrotus purpuratus sea urchin teeth documented a series of successive events from intravesicular mineral nucleation to mineral deposition at the interface between tooth surface and odontoblast syncytium. Using immunohistochemistry, SM50 was detected within the cytoplasm of cells associated with the developing tooth mineral, at the mineral secreting front, and adjacent to initial mineral deposits, but not in muscles and ligaments. Polypeptides derived from the SM50 polyproline alternating hexa- and hepta-peptide repeat region (SM50P6P7) formed highly discrete, donut-shaped self-assembly patterns. In calcium carbonate crystal growth studies, SM50P6P7 repeat peptides triggered the growth of expansive networks of fused calcium carbonate crystals while in apatite growth studies, SM50P6P7 peptides facilitated the growth of needle-shaped and parallel arranged crystals resembling those found in developing vertebrate enamel. In comparison, SM50P6P7 surpassed the PXX24 polypeptide repeat region derived from the vertebrate enamel protein amelogenin in its ability to promote crystal nucleation and appositional crystal growth. Together, these studies establish the SM50P6P7 polyproline repeat region as a potent regulator in the protein-guided appositional crystal growth that occurs during continuous tooth mineralization and eruption. In addition, our studies highlight the role of species

  11. Toxic Peptides Occur Frequently in Pergid and Argid Sawfly Larvae

    PubMed Central

    Boevé, Jean-Luc; Rozenberg, Raoul; Shinohara, Akihiko; Schmidt, Stefan

    2014-01-01

    Toxic peptides containing D-amino acids are reported from the larvae of sawfly species. The compounds are suspected to constitute environmental contaminants, as they have killed livestock grazing in areas with congregations of such larvae, and related larval extracts are deleterious to ants. Previously, two octapeptides (both called lophyrotomin) and three heptapeptides (pergidin, 4-valinepergidin and dephosphorylated pergidin) were identified from three species in the family Pergidae and one in Argidae. Here, the hypothesis of widespread occurrence of these peptides among sawflies was tested by LC-MS analyses of single larvae from eight pergid and 28 argid species, plus nine outgroup species. At least two of the five peptides were detected in most sawfly species, whereas none in any outgroup taxon. Wherever peptides were detected, they were present in each examined specimen of the respective species. Some species show high peptide concentrations, reaching up to 0.6% fresh weight of 4-valinepergidin (1.75 mg/larva) in the pergid Pterygophorus nr turneri. All analyzed pergids in the subfamily Pterygophorinae contained pergidin and 4-valinepergidin, all argids in Arginae contained pergidin and one of the two lophyrotomins, whereas none of the peptides was detected in any Perginae pergid or Sterictiphorinae argid (except in Schizocerella pilicornis, which contained pergidin). Three of the four sawfly species that were previously known to contain toxins were reanalyzed here, resulting in several, often strong, quantitative and qualitative differences in the chemical profiles. The most probable ecological role of the peptides is defense against natural enemies; the poisoning of livestock is an epiphenomenon. PMID:25121515

  12. Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands.

    PubMed

    Auvynet, Constance; Seddiki, Nabila; Dunia, Irene; Nicolas, Pierre; Amiche, Mohamed; Lacombe, Claire

    2006-01-01

    The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.

  13. Gageostatins A–C, Antimicrobial Linear Lipopeptides from a Marine Bacillus subtilis

    PubMed Central

    Tareq, Fakir Shahidullah; Lee, Min Ah; Lee, Hyi-Seung; Lee, Jong-Seok; Lee, Yeon-Ju; Shin, Hee Jae

    2014-01-01

    Concerning the requirements of effective drug candidates to combat against high rising multidrug resistant pathogens, we isolated three new linear lipopeptides, gageostatins A–C (1–3), consisting of hepta-peptides and new 3-β-hydroxy fatty acids from the fermentation broth of a marine-derived bacterium Bacillus subtilis. Their structures were elucidated by analyzing a combination of extensive 1D, 2D NMR spectroscopic data and high resolution ESIMS data. Fatty acids, namely 3-β-hydroxy-11-methyltridecanoic and 3-β-hydroxy-9,11-dimethyltridecanoic acids were characterized in lipopeptides 1 and 2, respectively, whereas an unsaturated fatty acid (E)-7,9-dimethylundec-2-enoic acid was assigned in 3. The 3R configuration of the stereocenter of 3-β-hydroxy fatty acids in 1 and 2 was established by Mosher’s MTPA method. The absolute stereochemistry of amino acid residues in 1–3 was ascertained by acid hydrolysis followed by Marfey’s derivatization studies. Gageostatins 1–3 exhibited good antifungal activities with MICs values of 4–32 µg/mL when tested against pathogenic fungi (R. solani, B. cinerea and C. acutatum) and moderate antibacterial activity against bacteria (B. subtilis, S. aeureus, S. typhi and P. aeruginosa) with MICs values of 8–64 µg/mL. Futhermore, gageostatins 1–3 displayed cytotoxicity against six human cancer cell lines with GI50 values of 4.6–19.6 µg/mL. It is also noteworthy that mixed compounds 1+2 displayed better antifungal and cytotoxic activities than individuals. PMID:24492520

  14. Microbial enhanced oil recovery research. [Peptides

    SciTech Connect

    Sharma, M.M.; Georgiou, G. )

    1992-01-01

    The surface active lipopeptide produced by Bacillus licheniformis JF-2 was isolated to near apparent homogeneity. NMR experiments revealed that this compound consists of a heptapeptide with an amino acid sequence similar to surfactin and a heterogeneous fatty acid consisting of the normal-, anteiso-, and iso- branched isomers. The surface activity of the B. licheniformis JF-2 surfactant was shown to depend on the presence of fermentation products and is strongly affected by the pH. Under conditions of optimal salinity and pH the interfacial tension against decane was 6 [times] 10[sup 3] mN/m which is one of the lowest values ever obtained with a microbial surfactant. Microbial compounds which exhibit particularly high surface activity are classified as biosurfactants. Microbial biosurfactants include a wide variety of surface and interfacially active compounds, such as glycolipids, lipopeptides polysaccharideprotein complexes, phospholipids, fatty acids and neutral lipids. Biosurfactants are easily biodegradable and thus are particularly suited for environmental applications such as bioremediation and the dispersion of oil spills. Bacillus licheniformis strain JF-2 has been shown to be able to grow and produce a very effective biosurfactant under both aerobic and anaerobic conditions and in the presence of high salt concentrations. The production of biosurfactants in anaerobic, high salt environments is potentially important for a variety of in situ applications such as microbial enhanced oil recovery. As a first step towards evaluating the commercial utility of the B. licheniformis JF-2 surfactant, we isolated t-he active. compound from the culture supernatant, characterized its chemical structure and investigated its phase behavior. We found that the surface activity of the surfactant is strongly dependent on the pH of the aqueous. phase. This may be important for the biological function of the surfactant and is of interest for several applications in surfactancy.

  15. Microbial enhanced oil recovery research. Annex 5, Summary annual report, 1991--1992

    SciTech Connect

    Sharma, M.M.; Georgiou, G.

    1992-12-31

    The surface active lipopeptide produced by Bacillus licheniformis JF-2 was isolated to near apparent homogeneity. NMR experiments revealed that this compound consists of a heptapeptide with an amino acid sequence similar to surfactin and a heterogeneous fatty acid consisting of the normal-, anteiso-, and iso- branched isomers. The surface activity of the B. licheniformis JF-2 surfactant was shown to depend on the presence of fermentation products and is strongly affected by the pH. Under conditions of optimal salinity and pH the interfacial tension against decane was 6 {times} 10{sup 3} mN/m which is one of the lowest values ever obtained with a microbial surfactant. Microbial compounds which exhibit particularly high surface activity are classified as biosurfactants. Microbial biosurfactants include a wide variety of surface and interfacially active compounds, such as glycolipids, lipopeptides polysaccharideprotein complexes, phospholipids, fatty acids and neutral lipids. Biosurfactants are easily biodegradable and thus are particularly suited for environmental applications such as bioremediation and the dispersion of oil spills. Bacillus licheniformis strain JF-2 has been shown to be able to grow and produce a very effective biosurfactant under both aerobic and anaerobic conditions and in the presence of high salt concentrations. The production of biosurfactants in anaerobic, high salt environments is potentially important for a variety of in situ applications such as microbial enhanced oil recovery. As a first step towards evaluating the commercial utility of the B. licheniformis JF-2 surfactant, we isolated t-he active. compound from the culture supernatant, characterized its chemical structure and investigated its phase behavior. We found that the surface activity of the surfactant is strongly dependent on the pH of the aqueous. phase. This may be important for the biological function of the surfactant and is of interest for several applications in surfactancy.

  16. Identification of microcystin toxins from a strain of Microcystis aeruginosa by liquid chromatography introduction into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer.

    PubMed

    Diehnelt, Chris W; Dugan, Nicholas R; Peterman, Scott M; Budde, William L

    2006-01-15

    The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment ions produced by collision-activated dissociation of the [M + H]+ ions. The cyanobacteria B2666 strain was cultured in a standard growth medium, and the toxins were released from the cells, extracted from the aqueous phase, and concentrated using standard procedures. The microcystins were separated by reversed-phase microbore liquid chromatography and introduced directly into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer with electrospray ionization. The known microcystins (MC) MC-LR, MC-LA, [MeSer7]MC-LR, MC-LL, MC-LF, and MC-L(Aba) were identified along with the two previously unreported structural variants [Asp3]MC-LA and [Asp3]MC-LL. In addition to the [M + H]+ ions, accurate m/z measurements were made of 12-18 product ions for each identified microcystin. The mean difference between measured and calculated exact m/z was less than 2 parts per million, which often allowed assignment of unique compositions to the observed ions. A mechanism is presented that accounts for an important collision-activated dissociation process that gives valuable sequence ions from microcystins that do not contain arginine. The analytical technique used in this work is capable of supporting fairly rapid and very reliable identifications of known microcystins when standards are not available and of most structural variants independent of additional information from other analytical techniques.

  17. Rescue of amyloid-Beta-induced inhibition of nicotinic acetylcholine receptors by a peptide homologous to the nicotine binding domain of the alpha 7 subtype.

    PubMed

    Nery, Arthur A; Magdesian, Margaret H; Trujillo, Cleber A; Sathler, Luciana B; Juliano, Maria A; Juliano, Luiz; Ulrich, Henning; Ferreira, Sergio T

    2013-01-01

    Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

  18. A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags.

    PubMed

    Islam, Tuhidul; Aguilar-Yañez, José Manuel; Simental-Martínez, Jesús; Ortiz-Alcaraz, Cesar Ivan; Rito-Palomares, Marco; Fernandez-Lahore, Marcelo

    2014-04-25

    In recent years, affinity fusion-tag systems have become a popular technique for the purification of recombinant proteins from crude extracts. However, several drawbacks including the high expense and low stability of ligands, their leakage during operation, and difficulties in immobilization, make it important to further develop the method. The present work is concerned with the utilization of a ceramic fluorapatite (CFT)-based chromatographic matrix to overcome these drawbacks. A heptapeptide library exhibiting a range of properties have been synthesized and subjected to ceramic fluorapatite (CFT) chromatography to characterize their retention behavior as a function of pH and composition of the binding buffer. The specific binding and elution behavior demonstrates the possible application of CFT-binding peptides as tags for enhancing the selective recovery of proteins by CFT chromatography. To materialize this strategy, a phage-derived CFT-specific sequence KPRSVSG (Tag1) with/without a consecutive hexalysine sequence, KKKKKKKPRSVSG (Tag2), were fused at the C-terminus of an enhanced green fluorescent protein (eGFP). The resulting gene constructs H-eGFP, H-eGFP-Tag1 and H-eGFP-Tag2 were expressed in Escherichia coli strain BL-21, and the clarified cell lysate was applied to the CFT column equilibrated with binding buffer (20-50mM sodium phosphate, pH 6-8.4). Sodium phosphate (500mM) or 1M NaCl in the respective binding buffer was used to elute the fused proteins, and the chromatographic fractions were analyzed by gel electrophoresis. Both the yield and purity were over 90%, demonstrating the potential application of the present strategy.

  19. Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.

    PubMed Central

    Grant, B. D.; Tsigelny, I.; Adams, J. A.; Taylor, S. S.

    1996-01-01

    The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site. PMID:8819164

  20. High diversity of microcystins in a Microcystis bloom from an Algerian lake.

    PubMed

    Bouhaddada, Ratiba; Nélieu, Sylvie; Nasri, Hichem; Delarue, Ghislaine; Bouaïcha, Noureddine

    2016-09-01

    Microcystins (MCs) are cyanobacterial heptapeptides, produced by several genera and species of cyanobacteria, which have been involved in poisoning of animals throughout the world and have also been implicated in human health problems. They are regarded as the most frequently occurring and widespread of the cyanotoxins, with more than 100 MC variants reported to date including the present study. The lake des Oiseaux is a shallow permanent freshwater lake located in north-eastern Algeria. It is an important natural reserve playing a major role for the migratory birds after the crossing of the Mediterranean Sea and from the Sahara desert. In recent years, possibly related to increased eutrophication of the lake, massive blooms of cyanobacteria identified as Microcystis spp. have been observed. A bloom sample collected in September 2013 was analyzed by the serine/threonine phosphatase PP2A inhibition assay and liquid chromatography-mass spectrometry to determine respectively, the total concentration of MCs and the different variants of these toxins present. The results revealed that the Microcystis spp. bloom sample contained microcystins of which 21 putatively congeners were detected. Among these, 12 known microcystins (MC-RR, MC-LR, MC-FR, MC-WR, MC-YR, MC-LA, MC-(H4)YR, MC-HilR, [Asp(3)]MC-RAba, and [Glu(OCH3)(6)]MC-LR) and two new congeners ([Asp(3)]MC-HarAba and [Glu(OCH3)(6)]MC-FR) were characterized, considering their molecular mass and the fragment ions produced by collision-induced dissociation of the [M+H](+) ions. MC-RR was the major (43.4%) in the bloom sample. PMID:27394081

  1. Identification of a novel aFGF-binding peptide with anti-tumor effect on breast cancer from phage display library

    SciTech Connect

    Dai, Xiaoyong; Cai, Cuizan; Xiao, Fei; Xiong, Yaoling; Huang, Yadong; Zhang, Qihao; Xiang, Qi; Lou, Guofeng; Lian, Mengyang; Su, Zhijian; Zheng, Qing

    2014-03-21

    Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs have been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.

  2. ACE2 and vasoactive peptides: novel players in cardiovascular/renal remodeling and hypertension.

    PubMed

    Mendoza-Torres, Evelyn; Oyarzún, Alejandra; Mondaca-Ruff, David; Azocar, Andrés; Castro, Pablo F; Jalil, Jorge E; Chiong, Mario; Lavandero, Sergio; Ocaranza, María Paz

    2015-08-01

    The renin-angiotensin system (RAS) is a key component of cardiovascular physiology and homeostasis due to its influence on the regulation of electrolyte balance, blood pressure, vascular tone and cardiovascular remodeling. Deregulation of this system contributes significantly to the pathophysiology of cardiovascular and renal diseases. Numerous studies have generated new perspectives about a noncanonical and protective RAS pathway that counteracts the proliferative and hypertensive effects of the classical angiotensin-converting enzyme (ACE)/angiotensin (Ang) II/angiotensin type 1 receptor (AT1R) axis. The key components of this pathway are ACE2 and its products, Ang-(1-7) and Ang-(1-9). These two vasoactive peptides act through the Mas receptor (MasR) and AT2R, respectively. The ACE2/Ang-(1-7)/MasR and ACE2/Ang-(1-9)/AT2R axes have opposite effects to those of the ACE/Ang II/AT1R axis, such as decreased proliferation and cardiovascular remodeling, increased production of nitric oxide and vasodilation. A novel peptide from the noncanonical pathway, alamandine, was recently identified in rats, mice and humans. This heptapeptide is generated by catalytic action of ACE2 on Ang A or through a decarboxylation reaction on Ang-(1-7). Alamandine produces the same effects as Ang-(1-7), such as vasodilation and prevention of fibrosis, by interacting with Mas-related GPCR, member D (MrgD). In this article, we review the key roles of ACE2 and the vasoactive peptides Ang-(1-7), Ang-(1-9) and alamandine as counter-regulators of the ACE-Ang II axis as well as the biological properties that allow them to regulate blood pressure and cardiovascular and renal remodeling.

  3. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    PubMed Central

    Fewer, David P; Rouhiainen, Leo; Jokela, Jouni; Wahlsten, Matti; Laakso, Kati; Wang, Hao; Sivonen, Kaarina

    2007-01-01

    Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. PMID:17908306

  4. A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes.

    PubMed

    Velkov, Tony; Yun, Bo; Schneider, Elena K; Azad, Mohammad A K; Dolezal, Olan; Morris, Faye C; Nation, Roger L; Wang, Jiping; Chen, Ke; Yu, Heidi H; Wang, Lv; Thompson, Philip E; Roberts, Kade D; Li, Jian

    2016-05-13

    Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships.

  5. Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis

    SciTech Connect

    Shi,R.; Lamb, S.; Bhat, S.; Sulea, T.; Wright, G.; Matte, A.; Cygler, M.

    2007-01-01

    Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vanco-mycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934 [GenBank], unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 [GenBank] biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys{sup 12}, His{sup 67}, and Ser{sup 98}), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp{sup 34}, His{sup 43}, Phe{sup 77}, Trp{sup 132}, and Glu{sup 205}.

  6. The Interaction of FABP with Kapα

    PubMed Central

    Amber-Vitos, Ortal; Kucherenko, Nataly; Nachliel, Esther; Gutman, Menachem; Tsfadia, Yossi

    2015-01-01

    Gene-activating lipophilic compounds are carried into the nucleus when loaded on fatty-acid-binding proteins (FABP). Some of these proteins are recognized by the α-Karyopherin (Kapα) through its nuclear localization signal (NLS) consisting of three positive residues that are not in a continuous sequence. The Importin system can distinguish between FABP loaded with activating and non-activating compounds. In the present study, we introduced molecular dynamics as a tool for clarifying the mechanism by which FABP4, loaded with activating ligand (linoleate) is recognized by Kapα. In the first phase, we simulated the complex between KapαΔIBB (termed “Armadillo”) that was crystallized with two NLS hepta-peptides. The trajectory revealed that the crystal-structure orientation of the peptides is rapidly lost and new interactions dominate. Though, the NLS sequence of FABP4 is cryptic, since the functional residues are not in direct sequence, implicating more than one possible conformation. Therefore, four possible docked conformations were generated, in which the NLS of FABP4 is interacting with either the major or the minor sites of Kapα, and the N → C vectors are parallel or anti-parallel. Out of these four basic starting positions, only the FABP4-minor site complex exhibited a large number of contact points. In this complex, the FABP interacts with the minor and the major sites, suppressing the self-inhibitory interaction of the Kapα, rendering it free to react with Kapβ. Finally, we propose that the transportable conformation generated an extended hydrophobic domain which expanded out of the boundary of the FABP4, allowing the loaded linoleate to partially migrate out of the FABP into a joint complex in which the Kapα contributes part of a combined binding pocket. PMID:26284534

  7. Genome-wide identification and expression analysis of the WRKY gene family in common tobacco (Nicotiana tabacum L.).

    PubMed

    Xiaohua, Xiang; Xinru, Wu; Jiangtao, Chao; Minglei, Yang; Fan, Yang; Guo, Chen; Guanshan, Liu; Yuanying, Wang

    2016-09-01

    The coding products of WRKY gene family plays important roles in plant growth and development as well as in various stress responses. They have been identified in various plants, but only few in common tobacco (Nicotiana tabacum L.). In this study, 164 putative WRKY proteins in the common tobacco genome were identified by using the conserved WRKY sequence (PF03106) from the Pfam database. Phylogenetic trees, functional domain analysis, chromosomal localization, subcellular localization and tissue expression patterns were analyzed with the bioinformatics softwares, including DNAMAN 5.0, Weblogo 3, MEGA 5.1, MG2C and MEME. First of all, phylogenetic trees divided all the candidate genes into three subfamilies: Ⅰ, Ⅱ and Ⅲ, respectively, and subfamily Ⅱ could be further divided into five subgroups: group Ⅱ-a, -b, -c, -d and -e. Secondly, the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK followed by a zinc-finger motif. Most of the NtWRKY genes contained 2-5 exons and a highly conserved gene structure. Thirdly, 154 out of 164 NtWRKY genes were distributed with different densities on 24 chromosomes, and each subfamily with different patterns and frequency. The largest number of NtWRKY genes was found on chromosome VI, and only one on chromosome X. Fourthly, the majority of NtWRKY members located in the nucleus, with 74 percent of subfamily Ⅲ in the extracellular matrix. Lastly, the members in the same subfamily had different spatial and temporal expression profiles, with 11 NtWRKY genes in roots, stems and leaves expressed at various levels. The expression of genes NtWRKY26, NtWRKY30 and NtWRKY32 can be induced by Phytophthora nicotianae. Our research thus provides valuable information for NtWRKY gene cloning and functional characterization in common tobacco. PMID:27644745

  8. [Genome-wide identification and expression analysis of the WRKY gene family in peach].

    PubMed

    Yanbing, Gu; Zhirui, Ji; Fumei, Chi; Zhuang, Qiao; Chengnan, Xu; Junxiang, Zhang; Zongshan, Zhou; Qinglong, Dong

    2016-03-01

    The WRKY transcription factors are one of the largest families of transcriptional regulators and play diverse regulatory roles in biotic and abiotic stresses, plant growth and development processes. In this study, the WRKY DNA-binding domain (Pfam Database number: PF03106) downloaded from Pfam protein families database was exploited to identify WRKY genes from the peach (Prunus persica 'Lovell') genome using HMMER 3.0. The obtained amino acid sequences were analyzed with DNAMAN 5.0, WebLogo 3, MEGA 5.1, MapInspect and MEME bioinformatics softwares. Totally 61 peach WRKY genes were found in the peach genome. Our phylogenetic analysis revealed that peach WRKY genes were classified into three Groups: Ⅰ, Ⅱ and Ⅲ. The WRKY N-terminal and C-terminal domains of Group Ⅰ (group I-N and group I-C) were monophyletic. The Group Ⅱ was sub-divided into five distinct clades (groupⅡ-a, Ⅱ-b, Ⅱ-c, Ⅱ-d and Ⅱ-e). Our domain analysis indicated that the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK at its N-terminus followed by a zinc-finger motif. The chromosome mapping analysis showed that peach WRKY genes were distributed with different densities over 8 chromosomes. The intron-exon structure analysis revealed that structures of the WRKY gene were highly conserved in the peach. The conserved motif analysis showed that the conserved motifs 1, 2 and 3, which specify the WRKY domain, were observed in all peach WRKY proteins, motif 5 as the unknown domain was observed in group Ⅱ-d, two WRKY domains were assigned to GroupⅠ. SqRT-PCR and qRT-PCR results indicated that 16 PpWRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels. Our analysis thus identified the PpWRKY gene families, and future functional studies are needed to reveal its specific roles. PMID:27001479

  9. The RNAPII-CTD Maintains Genome Integrity through Inhibition of Retrotransposon Gene Expression and Transposition

    PubMed Central

    Aristizabal, Maria J.; Negri, Gian Luca; Kobor, Michael S.

    2015-01-01

    RNA polymerase II (RNAPII) contains a unique C-terminal domain that is composed of heptapeptide repeats and which plays important regulatory roles during gene expression. RNAPII is responsible for the transcription of most protein-coding genes, a subset of non-coding genes, and retrotransposons. Retrotransposon transcription is the first step in their multiplication cycle, given that the RNA intermediate is required for the synthesis of cDNA, the material that is ultimately incorporated into a new genomic location. Retrotransposition can have grave consequences to genome integrity, as integration events can change the gene expression landscape or lead to alteration or loss of genetic information. Given that RNAPII transcribes retrotransposons, we sought to investigate if the RNAPII-CTD played a role in the regulation of retrotransposon gene expression. Importantly, we found that the RNAPII-CTD functioned to maintaining genome integrity through inhibition of retrotransposon gene expression, as reducing CTD length significantly increased expression and transposition rates of Ty1 elements. Mechanistically, the increased Ty1 mRNA levels in the rpb1-CTD11 mutant were partly due to Cdk8-dependent alterations to the RNAPII-CTD phosphorylation status. In addition, Cdk8 alone contributed to Ty1 gene expression regulation by altering the occupancy of the gene-specific transcription factor Ste12. Loss of STE12 and TEC1 suppressed growth phenotypes of the RNAPII-CTD truncation mutant. Collectively, our results implicate Ste12 and Tec1 as general and important contributors to the Cdk8, RNAPII-CTD regulatory circuitry as it relates to the maintenance of genome integrity. PMID:26496706

  10. Photodegradation of microcystin-LR catalyzed by metal phthalocyanines immobilized on TiO2-SiO2 under visible-light irradiation.

    PubMed

    Peng, Guotao; Fan, Zhengqiu; Wang, Xiangrong; Sui, Xin; Chen, Chen

    2015-01-01

    Microcystins (MCs) are a group of monocyclic heptapeptide toxins produced by species of cyanobacteria. Since MCs exhibit acute and chronic effects on humans and wildlife by damaging the liver, they are of increasing concern worldwide. In this study, we investigated the ability of the phthalocyanine compound (ZnPc-TiO2-SiO2) to degrade microcystin-LR (MC-LR) in the presence of visible light. X-ray diffraction (XRD) and UV-Visible diffuse reflectance spectra (UV-Vis DRS) were utilized to characterize the crystalline phase and the absorption behavior of this catalyst. According to the results, XRD spectra of ZnPc-TiO2-SiO2 powders taken in the 2θ configuration exhibited the peaks characteristic of the anatase phase. UV-Vis DRS showed that the absorption band wavelength shifted to the visible range when ZnPc was supported on the surface of TiO2-SiO2. Subsequently, several parameters including catalyst dose, MC-LR concentrations and pH were investigated. The MC-LR was quantified in each sample through high-performance liquid chromatography (HPLC). The maximum MC-LR degradation rate of 80.2% can be obtained within 300 minutes under the following conditions: catalyst dose of 7.50 g/L, initial MC-LR concentration of 17.35 mg/L, pH 6.76 and the first cycling run of the photocatalytic reaction. Moreover, the degradation process fitted well with the pseudo-first-order kinetic model.

  11. The Interaction of FABP with Kapα.

    PubMed

    Amber-Vitos, Ortal; Kucherenko, Nataly; Nachliel, Esther; Gutman, Menachem; Tsfadia, Yossi

    2015-01-01

    Gene-activating lipophilic compounds are carried into the nucleus when loaded on fatty-acid-binding proteins (FABP). Some of these proteins are recognized by the α-Karyopherin (Kapα) through its nuclear localization signal (NLS) consisting of three positive residues that are not in a continuous sequence. The Importin system can distinguish between FABP loaded with activating and non-activating compounds. In the present study, we introduced molecular dynamics as a tool for clarifying the mechanism by which FABP4, loaded with activating ligand (linoleate) is recognized by Kapα. In the first phase, we simulated the complex between KapαΔIBB (termed "Armadillo") that was crystallized with two NLS hepta-peptides. The trajectory revealed that the crystal-structure orientation of the peptides is rapidly lost and new interactions dominate. Though, the NLS sequence of FABP4 is cryptic, since the functional residues are not in direct sequence, implicating more than one possible conformation. Therefore, four possible docked conformations were generated, in which the NLS of FABP4 is interacting with either the major or the minor sites of Kapα, and the N → C vectors are parallel or anti-parallel. Out of these four basic starting positions, only the FABP4-minor site complex exhibited a large number of contact points. In this complex, the FABP interacts with the minor and the major sites, suppressing the self-inhibitory interaction of the Kapα, rendering it free to react with Kapβ. Finally, we propose that the transportable conformation generated an extended hydrophobic domain which expanded out of the boundary of the FABP4, allowing the loaded linoleate to partially migrate out of the FABP into a joint complex in which the Kapα contributes part of a combined binding pocket. PMID:26284534

  12. The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells

    SciTech Connect

    Fischer, A.; Hoeger, S.J.; Stemmer, K.; Feurstein, D.J.; Knobeloch, D.; Nussler, A.; Dietrich, D.R.

    2010-05-15

    Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

  13. Chromium(III) Binding Phage Screening for the Selective Adsorption of Cr(III) and Chromium Speciation.

    PubMed

    Yang, Ting; Zhang, Xiao-Yu; Zhang, Xiao-Xiao; Chen, Ming-Li; Wang, Jian-Hua

    2015-09-30

    The screening of suitable sorption medium is the key for highly selective solid phase extraction (SPE) of heavy metals. Herein, we demonstrate a universal protocol for producing selective SPE adsorbent through an evolutional approach based on phage display peptide library. By choosing chromium(III) as the model target, immobilized Cr(III) resins are first prepared using Ni-NTA affinity resins for the interaction with NEB heptapeptide phage library. After three rounds of positive biopanning against target Cr(III) and negative biopanning against foreign metal species, Cr(III) binding phages with high selectivity are obtained. The binding affinity and selectivity are further assessed with ELISA. The phages bearing peptide (YKASLIT) is finally chosen and immobilized on cytopore beads for Cr(III) preconcentration. The retained Cr(III) is efficiently recovered by 0.10 mol L(-1) HNO3 and quantified with ICP-MS. By loading 4000 μL of sample solution at pH 7.0 for 2 h and stripping with 400 μL of 0.10 mol L(-1) HNO3, a linear range of 0.05-0.50 μg L(-1) is achieved along with an enrichment factor of 7.1. The limit of detection is derived to be 15 ng L(-1) (3σ, n = 7) with a RSD of 3.6% (0.25 μg L(-1), n = 7). The procedure is validated by analyzing chromium content in a certified reference material GBW08608 (simulate water). In addition, chromium speciation in real water samples is demonstrated. Cr(VI) is first converted into Cr(III), and the latter subjected to the sorption onto the Cr(III) binding phage, followed by elution and quantification of the total chromium amount, and finally speciation is achieved by difference.

  14. Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA

    PubMed Central

    Stoermer, Martin J.; Tay, Stephanie; McMahon, Róisín M.; Fairlie, David P.; Martin, Jennifer L.

    2015-01-01

    Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB - or peptides - complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors. PMID:26225423

  15. Evaluation of melanotan-II, a superpotent cyclic melanotropic peptide in a pilot phase-I clinical study.

    PubMed

    Dorr, R T; Lines, R; Levine, N; Brooks, C; Xiang, L; Hruby, V J; Hadley, M E

    1996-01-01

    A pilot phase I study was conducted with a cyclic heptapeptide analog of alpha-melanocyte stimulating hormone (alpha-MSH). The lactam-bridged molecule, called Melanotan-II (MT-II), has the structure Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10 alpha-MSH4-10-NH2 (MT-II) and has superpotent melanotropic activity in vitro. A single-blind, alternating day (saline or MT-II), placebo-controlled trial was conducted in 3 normal male volunteers at the starting dose of 0.01 mg/kg of MT-II. Subcutaneous injections of MT-II or saline were given daily (Monday-Friday) for 2 consecutive weeks. Two subjects were escalated by 0.005 mg/kg increments to 0.03 mg/kg and one to 0.025 mg/kg. The 0.03 mg/kg dose produced Grade II somnolence and fatigue in one of two subjects (WHO standards). Mild nausea, not requiring antiemetic treatment, was reported at most MT-II dose levels. A stretching and yawning complex appeared to correlate with the onset of spontaneous, penile erections which were intermittently experienced for 1-5 hours after MT-II dosing, depending on the MT-II dose. Two subjects had increased pigmentation in the face, upper body and buttock, as measured by quantitative reflectance and by visual perception 1 week after MT-II dosing ended. These results demonstrate that MT-II has tanning activity in humans given only 5 low doses every other day by subcutaneous injection. The recommended single MT-II dose for future Phase I studies is 0.025 mg/kg/day.

  16. Characterization of Biosurfactant Produced by Bacillus licheniformis TT42 Having Potential for Enhanced Oil Recovery.

    PubMed

    Suthar, Harish; Nerurkar, Anuradha

    2016-09-01

    Bacillus licheniformis TT42 produced a low-molecular weight anionic biosurfactant that reduced the surface tension of water from 72 to 27 mN/m and the interfacial tension from 12 to 0.05 mN/m against crude oil. We have earlier reported significant enhancement in oil recovery in laboratory sand pack columns and core flood studies, by biosurfactant-TT42 compared to standard strain, Bacillus mojavensis JF2. In the context of this application of the biosurfactant-TT42, its characterization was deemed important. In the preliminary studies, the biosurfactant-TT42 was found to be functionally stable at under conditions of temperature, pH, and salinity generally prevalent in oil reservoirs. Furthermore, the purified biosurfactant-TT42 was found to have a CMC of 22 mg/l. A newly developed activity staining TLC method was used for the purification of biosurfactant-TT42. Structural characterization of biosurfactant-TT42 using TLC, Fourier transform infrared spectroscopy (FTIR), GC-MS, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF)/TOF suggested that it was a mixture of lipopeptide species, all having a common hydrophilic cyclic heptapeptide head with the sequence, Gln-Leu/Ileu-Leu/Ileu-Val-Asp-Leu/Ileu-Leu/Ileu linked to hydrophobic tails of different lengths of 3β-OH-fatty acids bearing 1043, 1057 and 1071 Da molecular weight, where 3β-OH-C19 fatty acid was predominant. This is the longest chain length of fatty acids reported in a lipopeptide.

  17. A Genetic Determinant in Streptococcus gordonii Challis Encodes a Peptide with Activity Similar to That of Enterococcal Sex Pheromone cAM373, Which Facilitates Intergeneric DNA Transfer▿

    PubMed Central

    Vickerman, M. M.; Flannagan, S. E.; Jesionowski, A. M.; Brossard, K. A.; Clewell, D. B.; Sedgley, C. M.

    2010-01-01

    Enterococcus faecalis strains secrete multiple peptides representing different sex pheromones that induce mating responses by bacteria carrying specific conjugative plasmids. The pheromone cAM373, which induces a response by the enterococcal plasmid pAM373, has been of interest because a similar activity is also secreted by Streptococcus gordonii and Staphylococcus aureus. The potential to facilitate intergeneric DNA transfer from E. faecalis is of concern because of extensive multiple antibiotic resistance, including vancomycin resistance, that has emerged among enterococci in recent years. Here, we characterize the related pheromone determinant in S. gordonii and show that the peptide it encodes, gordonii-cAM373, does indeed induce transfer of plasmid DNA from E. faecalis into S. gordonii. The streptococcal determinant camG encodes a lipoprotein with a leader sequence, the last 7 residues of which represent the gordonii-cAM373 heptapeptide SVFILAA. Synthetic forms of the peptide had activity similar to that of the enterococcal cAM373 AIFILAS. The lipoprotein moiety bore no resemblance to the lipoprotein encoded by E. faecalis. We also identified determinants in S. gordonii encoding a signal peptidase and an Eep-like zinc metalloprotease (lspA and eep, respectively) similar to those involved in processing certain pheromone precursors in E. faecalis. Mutations generated in camG, lspA, and eep each resulted in the ablation of gordonii-cAM373 activity in culture supernatants. This is the first genetic analysis of a potential sex pheromone system in a commensal oral streptococcal species, which may have implications for intergeneric gene acquisition in oral biofilms. PMID:20233933

  18. Lack of angiotensin II conversion to angiotensin III increases water but not alcohol consumption in aminopeptidase A-deficient mice.

    PubMed

    Faber, Franziska; Gembardt, Florian; Sun, Xiaoou; Mizutani, Shigehiko; Siems, Wolf-Eberhard; Walther, Thomas

    2006-09-11

    Elevated central concentrations of the vasopressor octapeptide angiotensin (Ang) II increase the water intake in mammals. Recently, we showed that central AngII is also crucial in alcohol-consuming behavior. Since the heptapeptide AngIII, an AngII metabolite, is discussed to mediate AngII-related effects, we investigated water and alcohol consumption in mice, genetically deficient in aminopeptidase A (APA), a peptidase responsible for AngII conversion to AngIII. Sixteen male APA-deficient mice and their age matched wild-type controls were monitored on their water intake under basal conditions and total fluid and alcohol intake before and after social stress in a two-bottle free-choice paradigm. Alterations were connected to the regulation in activity of Ang-related peptidases (APA, ACE; ACE2) in brain regions involved in alcohol intake and peripheral organs. In comparison to their wild-type controls, APA-deficient mice drank significantly more water but not more alcohol at all investigated time points. A reduction in water intake, as observed in wild-type animals after social stress, did not occur in knockout mice. However, the reduction in alcohol consumption after social stress was significantly reduced in both strains. Alcohol consumption upregulated all three peptidases in the kidney, but not in lung. Notable, renal ACE2 activity was significantly higher in APA-deficient mice under basal condition. While the inhibition of AngII metabolism to AngIII does not influence the alcohol intake, water consumption in mice deficient for APA was significantly elevated. These differences induced by an altered AngII/AngIII ratio oppose the hypothesis that central AngII and AngIII act in a congruent pattern.

  19. Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization

    NASA Astrophysics Data System (ADS)

    Pepin, Robert; Laszlo, Kenneth J.; Marek, Aleš; Peng, Bo; Bush, Matthew F.; Lavanant, Helène; Afonso, Carlos; Tureček, František

    2016-10-01

    Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions.

  20. Splicing Factors Associate with Hyperphosphorylated RNA Polymerase II in the Absence of Pre-mRNA

    PubMed Central

    Kim, Euikyung; Du, Lei; Bregman, David B.; Warren, Stephen L.

    1997-01-01

    The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains multiple tandem copies of the consensus heptapeptide, TyrSerProThrSerProSer. Concomitant with transcription initiation the CTD is phosphorylated. Elongating polymerase has a hyperphosphorylated CTD, but the role of this modification is poorly understood. A recent study revealed that some hyperphosphorylated polymerase molecules (Pol IIo) are nonchromosomal, and hence transcriptionally unengaged (Bregman, D.B., L. Du, S. van der Zee, S.L. Warren. 1995. J. Cell Biol. 129: 287–298). Pol IIo was concentrated in discrete splicing factor domains, suggesting a possible relationship between CTD phosphorylation and splicing factors, but no evidence beyond immunolocalization data was provided to support this idea. Here, we show that Pol IIo co-immunoprecipitates with members of two classes of splicing factors, the Sm snRNPs and non-snRNP SerArg (SR) family proteins. Significantly, Pol IIo's association with splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged. We also provide definitive evidence that hyperphosphorylation of Pol II's CTD is poorly correlated with its transcriptional activity. Using monoclonal antibodies (mAbs) H5 and H14, which are shown here to recognize phosphoepitopes on Pol II's CTD, we have quantitated the level of Pol IIo at different stages of the cell cycle. The level of Pol IIo is similar in interphase and mitotic cells, which are transcriptionally active and inactive, respectively. Finally, complexes containing Pol IIo and splicing factors can be prepared from mitotic as well as interphase cells. The experiments reported here establish that hyperphosphorylation of the CTD is a good indicator of polymerase's association with snRNP and SR splicing factors, but not of its transcriptional activity. Most importantly, the present study suggests that splicing factors may associate with the

  1. Combined Use of Post-Ion Mobility/Collision-Induced Dissociation and Chemometrics for b Fragment Ion Analysis

    NASA Astrophysics Data System (ADS)

    Zekavat, Behrooz; Miladi, Mahsan; Becker, Christopher; Munisamy, Sharon M.; Solouki, Touradj

    2013-09-01

    Although structural isomers may yield indistinguishable ion mobility (IM) arrival times and similar fragment ions in tandem mass spectrometry (MS), it is demonstrated that post-IM/collision-induced dissociation MS (post-IM/CID MS) combined with chemometrics can enable independent study of the IM-overlapped isomers. The new approach allowed us to investigate the propensity of selected b type fragment ions from AlaAlaAlaHisAlaAlaAla-NH2 (AAA(His)AAA) heptapeptide to form different isomers. Principle component analysis (PCA) of the unresolved post-IM/CID profiles indicated the presence of two different isomer types for b4 +, b5 +, and b6 + and a single isomer type for b7 + fragments of AAA(His)AAA. We employed a simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) to calculate the total IM profiles and CID mass spectra of b fragment isomers. The deconvoluted CID mass spectra showed discernible fragmentation patterns for the two isomers of b4 +, b5 +, and b6 + fragments. Under our experimental conditions, calculated percentages of the "cyclic" isomers (at the 95 % confidence level for n = 3) for b4 +, b5 +, and b6 + were 61 (± 5) %, 36 (± 5) %, and 48 (± 2) %, respectively. Results from the SIMPLISMA deconvolution of b5 + species resembled the CID MS patterns of fully resolved IM profiles for the two b5 + isomers. The "cyclic" isomers for each of the two-component b fragment ions were less susceptible to ion fragmentation than their "linear" counterparts.

  2. A parallel semisynthetic approach for structure-activity relationship studies of peptide YY.

    PubMed

    Albertsen, Louise; Østergaard, Søren; Paulsson, Johan F; Norrild, Jens Chr; Strømgaard, Kristian

    2013-09-01

    The gut hormone peptide YY (PYY) is postprandially secreted from enteroendocrine L cells and is involved in the regulation of energy homeostasis. The N-terminal truncated version PYY(3-36) decreases food intake and has potential as an anti-obesity agent. The anorectic effect of PYY(3-36) is mediated through Y₂ receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C-terminal tetrapeptide sequence of PYY(3-36) is crucial for Y2 receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C-terminally modified PYY(3-36) analogues. By using an intein-based expression system, PYY(3-29) was generated as a C-terminal peptide α-thioester. Heptapeptides bearing an N-terminal cysteine and modifications at one of the four C-terminal positions were synthesized in a 96-well plate by parallel solid-phase synthesis. In the plate format, an array of [Ala30]PYY(3-36) analogues were generated by ligation, desulfurization, and subsequent solid-phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non-proteinogenic amino acids, were tested in a functional Y₂ receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y₂ receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.

  3. Archael phosphoproteins. Identification of a hexosephosphate mutase and the alpha-subunit of succinyl-CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus.

    PubMed Central

    Solow, B.; Bischoff, K. M.; Zylka, M. J.; Kennelly, P. J.

    1998-01-01

    When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [gamma-32P]ATP, several radiolabeled polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35-kDa one, whose protein-phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35- and 50-kDa phosphoproteins were purified and their amino-terminal sequences determined. The former polypeptide's amino-terminal sequence closely matched a conserved portion of the alpha-subunit of succinyl-CoA synthetase, which forms an acid-labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50-kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro-Gly-Thr-Asp/Ser-Gly-Val/Leu-Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6-phosphate to donate its phosphoryl group to the protein. PMID:9514265

  4. Chromium(III) Binding Phage Screening for the Selective Adsorption of Cr(III) and Chromium Speciation.

    PubMed

    Yang, Ting; Zhang, Xiao-Yu; Zhang, Xiao-Xiao; Chen, Ming-Li; Wang, Jian-Hua

    2015-09-30

    The screening of suitable sorption medium is the key for highly selective solid phase extraction (SPE) of heavy metals. Herein, we demonstrate a universal protocol for producing selective SPE adsorbent through an evolutional approach based on phage display peptide library. By choosing chromium(III) as the model target, immobilized Cr(III) resins are first prepared using Ni-NTA affinity resins for the interaction with NEB heptapeptide phage library. After three rounds of positive biopanning against target Cr(III) and negative biopanning against foreign metal species, Cr(III) binding phages with high selectivity are obtained. The binding affinity and selectivity are further assessed with ELISA. The phages bearing peptide (YKASLIT) is finally chosen and immobilized on cytopore beads for Cr(III) preconcentration. The retained Cr(III) is efficiently recovered by 0.10 mol L(-1) HNO3 and quantified with ICP-MS. By loading 4000 μL of sample solution at pH 7.0 for 2 h and stripping with 400 μL of 0.10 mol L(-1) HNO3, a linear range of 0.05-0.50 μg L(-1) is achieved along with an enrichment factor of 7.1. The limit of detection is derived to be 15 ng L(-1) (3σ, n = 7) with a RSD of 3.6% (0.25 μg L(-1), n = 7). The procedure is validated by analyzing chromium content in a certified reference material GBW08608 (simulate water). In addition, chromium speciation in real water samples is demonstrated. Cr(VI) is first converted into Cr(III), and the latter subjected to the sorption onto the Cr(III) binding phage, followed by elution and quantification of the total chromium amount, and finally speciation is achieved by difference. PMID:26346061

  5. Selection of ceramic fluorapatite-binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography.

    PubMed

    Islam, Tuhidul; Bibi, Noor Shad; Vennapusa, Rami Reddy; Fernandez-Lahore, Marcelo

    2013-08-01

    Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.

  6. Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy.

    PubMed Central

    Polans, A S; Witkowska, D; Haley, T L; Amundson, D; Baizer, L; Adamus, G

    1995-01-01

    Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:7568096

  7. Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization

    NASA Astrophysics Data System (ADS)

    Pepin, Robert; Laszlo, Kenneth J.; Marek, Aleš; Peng, Bo; Bush, Matthew F.; Lavanant, Helène; Afonso, Carlos; Tureček, František

    2016-07-01

    Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions.

  8. Photodegradation of microcystin-LR catalyzed by metal phthalocyanines immobilized on TiO2-SiO2 under visible-light irradiation.

    PubMed

    Peng, Guotao; Fan, Zhengqiu; Wang, Xiangrong; Sui, Xin; Chen, Chen

    2015-01-01

    Microcystins (MCs) are a group of monocyclic heptapeptide toxins produced by species of cyanobacteria. Since MCs exhibit acute and chronic effects on humans and wildlife by damaging the liver, they are of increasing concern worldwide. In this study, we investigated the ability of the phthalocyanine compound (ZnPc-TiO2-SiO2) to degrade microcystin-LR (MC-LR) in the presence of visible light. X-ray diffraction (XRD) and UV-Visible diffuse reflectance spectra (UV-Vis DRS) were utilized to characterize the crystalline phase and the absorption behavior of this catalyst. According to the results, XRD spectra of ZnPc-TiO2-SiO2 powders taken in the 2θ configuration exhibited the peaks characteristic of the anatase phase. UV-Vis DRS showed that the absorption band wavelength shifted to the visible range when ZnPc was supported on the surface of TiO2-SiO2. Subsequently, several parameters including catalyst dose, MC-LR concentrations and pH were investigated. The MC-LR was quantified in each sample through high-performance liquid chromatography (HPLC). The maximum MC-LR degradation rate of 80.2% can be obtained within 300 minutes under the following conditions: catalyst dose of 7.50 g/L, initial MC-LR concentration of 17.35 mg/L, pH 6.76 and the first cycling run of the photocatalytic reaction. Moreover, the degradation process fitted well with the pseudo-first-order kinetic model. PMID:26540545

  9. Eptifibatide: The evidence for its role in the management of acute coronary syndromes

    PubMed Central

    Shah, Ibrahim; Khan, Shakeel O; Malhotra, Surender; Fischell, Tim

    2010-01-01

    Introduction: Acute coronary syndromes and non-Q-wave myocardial infarction are often initiated by platelet activation. Eptifibatide is a cyclic heptapeptide and is the third inhibitor of glycoprotein (Gp) IIb/IIIa that has found broad acceptance after the specific antibody abciximab and the nonpeptide tirofiban entered the global market. Gp IIb/IIIa inhibitors act by inhibiting the final common pathway of platelet aggregation, and play an important role in the management of acute coronary syndromes. Aims: This review assesses the evidence for therapeutic value of eptifibatide as a Gp IIb/IIIa inhibitor in patients with acute coronary syndromes. Evidence review: Several large, randomized controlled trials show that eptifibatide as adjunctive therapy to standard care in patients with non-ST segment elevation acute coronary syndrome is associated with a significant reduction in the incidence of death or myocardial infarction. Data are limited regarding the use of eptifibatide in patients with ST segment elevation myocardial infarction. Cost-effectiveness analysis indicates that eptifibatide is associated with a favorable cost-effectiveness ratio relative to standard care. According to US cost-effectiveness analysis about 70% of the acquisition costs of eptifibatide are offset by the reduced medical resource consumption during the first year. Eptifibatide was well tolerated in most of the trials. Bleeding is the most commonly reported adverse event, with most major bleeding episodes occurring at the vascular access site. Major intracranial bleeds, stroke, or profound thrombocytopenia rarely occurred during eptifibatide treatment. Place in therapy: Eptifibatide has gained widespread acceptance as an adjunct to standard anticoagulation therapy in patients with acute coronary syndromes, and may be particularly useful in the management of patients with elevated troponin or undergoing percutaneous coronary interventions. PMID:20694065

  10. Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae.

    PubMed

    Zhou, Bang-Jun; Jia, Pei-Song; Gao, Feng; Guo, Hui-Shan

    2012-07-01

    Verticillium dahliae Kleb. is a hemibiotrophic, phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. Successful host colonization by hemibiotrophic pathogens requires the induction of plant cell death to provide the saprophytic nutrition for the transition from the biotrophic to the necrotrophic stage. In this study, we identified a necrosis-inducing Phytophthora protein (NPP1) domain-containing protein family containing nine genes in a virulent, defoliating isolate of V. dahliae (V592), named the VdNLP genes. Functional analysis demonstrated that only two of these VdNLP genes, VdNLP1 and VdNLP2, encoded proteins that were capable of inducing necrotic lesions and triggering defense responses in Nicotiana benthamiana, Arabidopsis, and cotton plants. Both VdNLP1 and VdNLP2 induced the wilting of cotton seedling cotyledons. However, gene-deletion mutants targeted by VdNLP1, VdNLP2, or both did not affect the pathogenicity of V. dahliae V592 in cotton infection. Similar expression and induction patterns were found for seven of the nine VdNLP transcripts. Through a comparison of the conserved amino acid residues of VdNLP with different necrosis-inducing activities, combined with mutagenesis-based analyses, we identified several novel conserved amino acid residues, in addition to the known conserved heptapeptide GHRHDWE motif and the cysteine residues of the NPP domain-containing protein, that are indispensable for the necrosis-inducing activity of the VdNLP2 protein.

  11. Construction of differentially expressed genes library of bighead carp (Aristichthys nobilis) exposed to microcystin-lr using ssh and expression profile of related genes.

    PubMed

    Cui, Zhihui; Zhang, Kaiyue; Qu, Xiancheng; Liu, Qigen

    2011-12-01

    Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp (Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins.

  12. Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization.

    PubMed

    Pepin, Robert; Laszlo, Kenneth J; Marek, Aleš; Peng, Bo; Bush, Matthew F; Lavanant, Helène; Afonso, Carlos; Tureček, František

    2016-10-01

    Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions. Graphical Abstract ᅟ.

  13. [Studying specific effects of nootropic drugs on glutamate receptors in the rat brain].

    PubMed

    Firstova, Iu Iu; Vasil'eva, E V; Kovalev, G I

    2011-01-01

    The influence of nootropic drugs of different groups (piracetam, phenotropil, nooglutil, noopept, semax, meclofenoxate, pantocalcine, and dimebon) on the binding of the corresponding ligands to AMPA, NMDA, and mGlu receptors of rat brain has been studied by the method of radio-ligand binding in vitro. It is established that nooglutil exhibits pharmacologically significant competition with a selective agonist of AMPA receptors ([G-3H]Ro 48-8587) for the receptor binding sites (with IC50 = 6.4 +/- 0.2 microM), while the competition of noopept for these receptor binding sites was lower by an order of magnitude (IC50 = 80 +/- 5.6 microM). The heptapeptide drug semax was moderately competitive with [G-3H]LY 354740 for mGlu receptor sites (IC50 = 33 +/- 2.4 microM). Dimebon moderately influenced the specific binding of the ligand of NMDA receptor channel ([G-3H]MK-801) at IC50 = 59 +/- 3.6 microM. Nootropic drugs of the pyrrolidone group (piracetam, phenotropil) as well as meclofenoxate, pantocalcine (pantogam) in a broad rage of concentrations (10(-4)-10(-10) M) did not affect the binding of the corresponding ligands to glutamate receptors (IC50 100 pM). Thus, the direct neurochemical investigation was used for the first time to qualitatively characterize the specific binding sites for nooglutil and (to a lower extent) noopept on AMPA receptors, for semax on metabotropic glutamate receptors, and for dimebon on the channel region of NMDA receptors. The results are indicative of a selective action of some nootropes on the glutamate family. PMID:21476267

  14. Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

    SciTech Connect

    Rodi, D. J.; Soares, A. S.; Makowski, L.; Biosciences Division; BNL

    2002-01-01

    Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.

  15. Double-sided magnetic molecularly imprinted polymer modified graphene oxide for highly efficient enrichment and fast detection of trace-level microcystins from large-volume water samples combined with liquid chromatography-tandem mass spectrometry.

    PubMed

    Pan, Sheng-Dong; Chen, Xiao-Hong; Li, Xiao-Ping; Cai, Mei-Qiang; Shen, Hao-Yu; Zhao, Yong-Gang; Jin, Mi-Cong

    2015-11-27

    Microcystins (MCs), a group of cyclic heptapeptide heaptoxins and tumor promoters, are generated by cyanobacteria occurring in surface waters, such as eutrophic lakes, rivers, and reservoirs. In this present study, a novel double-sided magnetic molecularly imprinted polymer modified graphene oxide (DS-MMIP@GO) based magnetic solid-phase extraction (MSPE) method was developed for fast, effective and selective enrichment, and recognition of trace MCs in environmental water samples combined with high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The synthesized novel DS-MMIP@GO was used as the adsorbents in this work and was carefully characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and Raman spectra. The adsorption and desorption conditions of DS-MMIP@GO toward MCs were optimized in detail to obtain the highest binding capacity, selectivity, and release efficiency. Under the optimum conditions, the enrichment factors of the method for eight target MCs were found to be 2000. The limits of quantitation (LOQs) of the method for eight MCs were in range of 0.1-2.0ngL(-1). The double-sided MMIP modified structure provided DS-MMIP@GO with abundant adsorption sites and permitted it to exhibit excellent enrichment and selectivity toward trace-level MCs. The proposed method was successfully applied for the analysis of environmental water samples with recoveries ranging from 84.1 to 98.2%. Compared to conventional methods for MCs detection reported in literatures, the one developed in this work based on DS-MMIP@GO and LC-MS/MS showed much faster, more sensitive, and more convenient.

  16. Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization.

    PubMed

    Pepin, Robert; Laszlo, Kenneth J; Marek, Aleš; Peng, Bo; Bush, Matthew F; Lavanant, Helène; Afonso, Carlos; Tureček, František

    2016-10-01

    Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions. Graphical Abstract ᅟ. PMID:27400696

  17. A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes.

    PubMed

    Velkov, Tony; Yun, Bo; Schneider, Elena K; Azad, Mohammad A K; Dolezal, Olan; Morris, Faye C; Nation, Roger L; Wang, Jiping; Chen, Ke; Yu, Heidi H; Wang, Lv; Thompson, Philip E; Roberts, Kade D; Li, Jian

    2016-05-13

    Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships. PMID:27627202

  18. Protein farnesyltransferase and protein prenylation in Plasmodium falciparum.

    PubMed

    Chakrabarti, Debopam; Da Silva, Thiago; Barger, Jennifer; Paquette, Steve; Patel, Hetal; Patterson, Shelley; Allen, Charles M

    2002-11-01

    Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.

  19. A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

    PubMed Central

    Bourquin, J P; Stagljar, I; Meier, P; Moosmann, P; Silke, J; Baechi, T; Georgiev, O; Schaffner, W

    1997-01-01

    The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription. PMID:9153302

  20. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

    SciTech Connect

    Feurstein, D.; Holst, K.; Fischer, A.; Dietrich, D.R.

    2009-01-15

    Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brain-barrier (BBB) and blood-cerebrospinal-fluid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses, the presence of murine Oatp (mOatp) in primary murine whole brain cells (mWBC) was investigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LF) with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 {mu}M) and BSP (50 {mu}M). MC-Western blot analysis demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vitro data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells.

  1. Organic anion transporting polypeptides expressed in liver and brain mediate uptake of microcystin

    SciTech Connect

    Fischer, W.J.; Altheimer, S.; Cattori, V.; Meier, P.J.; Dietrich, D.R. . E-mail: Daniel.Dietrich@uni-konstanz.de; Hagenbuch, B.

    2005-03-15

    Microcystins are toxins produced by freshwater cyanobacteria. They are cyclic heptapeptides that exhibit hepato- and neurotoxicity. However, the transport systems that mediate uptake of microcystins into hepatocytes and across the blood-brain barrier have not yet been identified. Using the Xenopus laevis oocyte expression system we tested whether members of the organic anion transporting polypeptide superfamily (rodent: Oatps; human: OATPs) are involved in transport of the most common microcystin variant microcystin-LR by measuring uptake of a radiolabeled derivative dihydromicrocystin-LR. Among the tested Oatps/OATPs, rat Oatp1b2, human OATP1B1, human OATP1B3, and human OATP1A2 transported microcystin-LR 2- to 5-fold above water-injected control oocytes. This microcystin-LR transport was inhibited by co-incubation with the known Oatp/OATP substrates taurocholate (TC) and bromosulfophthalein (BSP). Microcystin-LR transport mediated by the human OATPs was further characterized and showed saturability with increasing microcystin-LR concentrations. The apparent K{sub m} values amounted to 7 {+-} 3 {mu}M for OATP1B1, 9 {+-} 3 {mu}M for OATP1B3, and 20 {+-} 8 {mu}M for OATP1A2. No microcystin-LR transport was observed in oocytes expressing Oatp1a1, Oatp1a4, and OATP2B1. These results may explain some of the observed organ-specific toxicity of microcystin-LR. Oatp1b2, OATP1B1, and OATP1B3 are responsible for microcystin transport into hepatocytes, whereas OATP1A2 mediates microcystin-LR transport across the blood-brain barrier.

  2. Induction of apoptosis in mouse liver by microcystin-LR: a combined transcriptomic, proteomic, and simulation strategy.

    PubMed

    Chen, Ting; Wang, Qingsong; Cui, Jun; Yang, Wei; Shi, Qian; Hua, Zichun; Ji, Jianguo; Shen, Pingping

    2005-07-01

    Microcystins (MCs) are a family of cyclic heptapeptide hepatotoxins produced by freshwater species of cyanobacteria that have been implicated in the development of liver cancer, necrosis, and even deadly intrahepatic bleeding. MC-LR, the most toxic MC variant, is also the most commonly encountered in a contaminated aquatic system. This study presents the first data in the toxicological research of MCs that combines the use of standard apoptotic assays with transcriptomics, proteomic technologies, and computer simulations. By using histochemistry, DNA fragmentation assays, and flow cytometry analysis, we determined that MC-LR causes rapid, dose-dependent apoptosis in mouse liver when BALB/c mice are treated with MC-LR for 24 h at doses of either 50, 60, or 70 microg/kg of body weight. We then used gene expression profiling to demonstrate differential expressions (>2-fold) of 61 apoptosis-related genes in cells treated with MC-LR. Further proteomic analysis identified a total of 383 proteins of which 35 proteins were up-regulated and 30 proteins were down-regulated more than 2.5-fold when compared with controls. Combining computer simulations with the transcriptomic and proteomic data, we found that low doses (50 microg/kg) of MC-LR lead to apoptosis primarily through the BID-BAX-BCL-2 pathway, whereas high doses of MC-LR (70 microg/kg) caused apoptosis via a reactive oxygen species pathway. These results indicated that MC-LR exposure can cause apoptosis in mouse liver and revealed two independent pathways playing a major regulatory role in MC-LR-induced apoptosis, thereby contributing to a better understanding of the hepatotoxicity and the tumor-promoting mechanisms of MCs.

  3. Construction of differentially expressed genes library of bighead carp (Aristichthys nobilis) exposed to microcystin-lr using ssh and expression profile of related genes.

    PubMed

    Cui, Zhihui; Zhang, Kaiyue; Qu, Xiancheng; Liu, Qigen

    2011-12-01

    Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp (Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins. PMID:21803161

  4. CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

    PubMed Central

    Santos, B; Duran, A; Valdivieso, M H

    1997-01-01

    The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating. PMID:9111317

  5. Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II.

    PubMed

    Stiller, J W; Duffield, E C; Hall, B D

    1998-09-29

    Unlike parasitic protist groups that are defined by the absence of mitochondria, the Pelobiontida is composed mostly of free-living species. Because of the presence of ultrastructural and cellular features that set them apart from all other eukaryotic organisms, it has been suggested that pelobionts are primitively amitochondriate and may represent the earliest-evolved lineage of extant protists. Analyses of rRNA genes, however, have suggested that the group arose well after the diversification of the earliest-evolved protists. Here we report the sequence of the gene encoding the largest subunit of DNA-dependent RNA polymerase II (RPB1) from the pelobiont Mastigamoeba invertens. Sequences within RPB1 encompass several of the conserved catalytic domains that are common to eubacterial, archaeal, and eukaryotic nuclear-encoded RNA polymerases. In RNA polymerase II, these domains catalyze the transcription of all nuclear pre-mRNAs, as well as the majority of small nuclear RNAs. In contrast with rDNA-based trees, phylogenetic analyses of RPB1 sequences indicate that Mastigamoeba represents an early branch of eukaryotic evolution. Unlike sequences from parasitic amitochondriate protists that were included in our study, there is no indication that Mastigamoeba RPB1 is attracted to the base of the eukaryotic tree artifactually. In addition, the presence of introns and a heptapeptide C-terminal repeat in the Mastigamoeba RPB1 sequence, features that are typically associated with more recently derived eukaryotic groups, raise provocative questions regarding models of protist evolution that depend almost exclusively on rDNA sequence analyses.

  6. A Virtual Mixture Approach to the Study of Multistate Equilibrium: Application to Constant pH Simulation in Explicit Water

    PubMed Central

    Wu, Xiongwu; Brooks, Bernard R.

    2015-01-01

    Chemical and thermodynamic equilibrium of multiple states is a fundamental phenomenon in biology systems and has been the focus of many experimental and computational studies. This work presents a simulation method to directly study the equilibrium of multiple states. This method constructs a virtual mixture of multiple states (VMMS) to sample the conformational space of all chemical states simultaneously. The VMMS system consists of multiple subsystems, one for each state. The subsystem contains a solute and a solvent environment. The solute molecules in all subsystems share the same conformation but have their own solvent environments. Transition between states is implicated by the change of their molar fractions. Simulation of a VMMS system allows efficient calculation of relative free energies of all states, which in turn determine their equilibrium molar fractions. For systems with a large number of state transition sites, an implicit site approximation is introduced to minimize the cost of simulation. A direct application of the VMMS method is for constant pH simulation to study protonation equilibrium. Applying the VMMS method to a heptapeptide of 3 ionizable residues, we calculated the pKas of those residues both with all explicit states and with implicit sites and obtained consistent results. For mouse epidermal growth factor of 9 ionizable groups, our VMMS simulations with implicit sites produced pKas of all 9 ionizable groups and the results agree qualitatively with NMR measurement. This example demonstrates the VMMS method can be applied to systems of a large number of ionizable groups and the computational cost scales linearly with the number of ionizable groups. For one of the most challenging systems in constant pH calculation, SNase Δ+PHS/V66K, our VMMS simulation shows that it is the state-dependent water penetration that causes the large deviation in lysine66’s pKa. PMID:26506245

  7. Occurrence of microcystin-producing cyanobacteria in Ugandan freshwater habitats

    PubMed Central

    Okello, William; Portmann, Cyril; Erhard, Marcel; Gademann, Karl; Kurmayer, Rainer

    2011-01-01

    Microcystins (MCs) are cyclic heptapeptides that are the most abundant toxins produced by cyanobacteria in freshwater. The phytoplankton of many freshwater lakes in Eastern Africa is dominated by cyanobacteria. Less is known, however, on the occurrence of MC producers and the production of MCs. Twelve Ugandan freshwater habitats ranging from mesotrophic to hypertrophic conditions were sampled in May and June of 2004 and April of 2008 and were analyzed for their physico-chemical parameters, phytoplankton composition, and MC concentrations. Among the group of the potential MC-producing cyanobacteria, Anabaena (0 - 107 cells ml−1) and Microcystis (103 - 107 cells ml−1) occurred most frequently and dominated in eutrophic systems. A significant linear relationship (n = 31, r2 = 0.38, p < 0.001) between the Microcystis cell numbers and MC concentration (1.3-93 fg of MC cell−1) was observed. Beside [MeAsp3, Mdha7]-MC-RR two new microcystins, [Asp3]-MC-RY and [MeAsp3]-MC-RY were isolated and their constitution assigned by LC-MS2. In order to identify the MC-producing organism in the water samples (i), the conserved aminotransferase domain part of the mcyE gene that is indicative of MC production was amplified by general primers and cloned and sequenced, and (ii), genus-specific primers were used to amplify the mcyE gene of the genera Microcystis, Anabaena, and Planktothrix. Only mcyE genotypes that are indicative of Microcystis sp. were obtained via the environmental cloning approach (337 bp, 96.1%-96.7% similarity to the Microcystis aeruginosa strain PCC7806). Accordingly, only the mcyE primers, which are specific for Microcystis, revealed PCR products. We concluded that Microcystis is the major MC-producer in Ugandan freshwater. PMID:19609871

  8. [Genome-wide identification and expression analysis of the WRKY gene family in peach].

    PubMed

    Yanbing, Gu; Zhirui, Ji; Fumei, Chi; Zhuang, Qiao; Chengnan, Xu; Junxiang, Zhang; Zongshan, Zhou; Qinglong, Dong

    2016-03-01

    The WRKY transcription factors are one of the largest families of transcriptional regulators and play diverse regulatory roles in biotic and abiotic stresses, plant growth and development processes. In this study, the WRKY DNA-binding domain (Pfam Database number: PF03106) downloaded from Pfam protein families database was exploited to identify WRKY genes from the peach (Prunus persica 'Lovell') genome using HMMER 3.0. The obtained amino acid sequences were analyzed with DNAMAN 5.0, WebLogo 3, MEGA 5.1, MapInspect and MEME bioinformatics softwares. Totally 61 peach WRKY genes were found in the peach genome. Our phylogenetic analysis revealed that peach WRKY genes were classified into three Groups: Ⅰ, Ⅱ and Ⅲ. The WRKY N-terminal and C-terminal domains of Group Ⅰ (group I-N and group I-C) were monophyletic. The Group Ⅱ was sub-divided into five distinct clades (groupⅡ-a, Ⅱ-b, Ⅱ-c, Ⅱ-d and Ⅱ-e). Our domain analysis indicated that the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK at its N-terminus followed by a zinc-finger motif. The chromosome mapping analysis showed that peach WRKY genes were distributed with different densities over 8 chromosomes. The intron-exon structure analysis revealed that structures of the WRKY gene were highly conserved in the peach. The conserved motif analysis showed that the conserved motifs 1, 2 and 3, which specify the WRKY domain, were observed in all peach WRKY proteins, motif 5 as the unknown domain was observed in group Ⅱ-d, two WRKY domains were assigned to GroupⅠ. SqRT-PCR and qRT-PCR results indicated that 16 PpWRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels. Our analysis thus identified the PpWRKY gene families, and future functional studies are needed to reveal its specific roles.

  9. Nostocyclopeptide-M1: a potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and OATP1B1.

    PubMed

    Herfindal, Lars; Myhren, Lene; Kleppe, Rune; Krakstad, Camilla; Selheim, Frode; Jokela, Jouni; Sivonen, Kaarina; Døskeland, Stein O

    2011-04-01

    We have isolated a novel cyanobacterial cyclic peptide (nostocyclopeptide M1; Ncp-M1) that blocks the hepatotoxic action of microcystin (MC) and nodularin (Nod). We show here that Ncp-M1 is nontoxic to primary hepatocytes in long-term culture. Ncp-M1 does not affect any known intracellular targets or pathways involved in MC action, like protein phosphatases, CaM-KII, or ROS-dependent cell death effectors. In support of this conclusion Ncp-M1 had no protective effect when microinjected into cells. Rather, the antitoxin effect was solely due to blocked hepatocyte uptake of MC and Nod. The hepatic uptake of MC and Nod is mainly via the closely related organic anion transporters OATP1B1 and OATP1B3, which also mediate hepatic transport of endogenous metabolites and hormones as well as drugs. OATP1B3 is also expressed in some aggressive cancers, where it confers apoptosis resistance. We show that Ncp-M1 inhibits transport through OATP1B3 and OATP1B1 expressed in HEK293 cells. The Ncp-M1 molecule has several nonproteinogenic amino acids and an imino bond, which hamper its synthesis. Moreover, a cyclic all L-amino acid heptapeptide analogue of Ncp-M1 also inhibits the OATP1B1/1B3 transporters, and with higher OATP1B3 preference than Ncp-M1 itself. The nontoxic Ncp-M1 and its synthetic cyclic peptide analogues thus provide new tools to probe the role of OATB1B1/1B3 mediated drug and metabolite transport in liver and cancer cells. They can also serve as scaffolds to design new, exopeptidase resistant OATP1B3-specific modulators.

  10. One-sided polymerase chain reaction: the amplification of cDNA.

    PubMed Central

    Ohara, O; Dorit, R L; Gilbert, W

    1989-01-01

    We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin. Images PMID:2788276

  11. LC/ESI/MS method development for the analysis of hepatotoxic cyclic peptide microcystins in animal tissues.

    PubMed

    Ott, Jennifer L; Carmichael, Wayne W

    2006-06-01

    Microcystins (MCYSTs) are a family of related cyclic heptapeptides produced by several genera and species of blue-green algae (cyanobacteria). MCYSTs are potent and specific inhibitors of the serine threonine family of protein phosphatases, especially PP1 and PP2A. MCYSTs inhibit a liver's protein phosphatase by forming a covalent linkage between MCYSTs' Mdha residue and the phosphatase's cysteine residue. Due to the covalent linkage, analysis of MCYSTs in animal tissues has been limited to determination of unbound MCYST concentration. The MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) oxidation procedure allows for the detection of total MCYST burden by releasing the carboxylic acid MMPB from MCYST's Adda amino acid. An internal standard 4-phenylbutyric acid (4PB) accounts for losses during the method. LC/MS conditions were developed using a ThermoFinnigan LCQDuo ion trap in negative electrospray ionization (ESI). Since both compounds produce the [M-H](-) ion, analysis occurs in selected ion monitoring (SIM) mode for both MMPB (m/z 207.1) and 4PB (m/z 163.1). Complete oxidation of MCYST-LR in liver tissues occurs in 3h. A solid phase extraction (SPE) cartridge removes MMPB and 4PB from the oxidant solution. The process efficiency for the SPE procedure is only 51.3%; however, suppression experiments indicate a 41.8% loss in signal strength due to matrix interferences. Therefore, the extraction efficiency for the SDB-XC cartridge procedure is 93.1%. This research has been successful in developing an LC/MS method for the analysis of total MCYST burden in animal tissues.

  12. THE TOXIC CYANOBACTERIUM NOSTOC SP. STRAIN 152 PRODUCES HIGHEST AMOUNTS OF MICROCYSTIN AND NOSTOPHYCIN UNDER STRESS CONDITIONS

    PubMed Central

    Kurmayer, Rainer

    2012-01-01

    The understanding of how environmental factors regulate toxic secondary metabolite production in cyanobacteria is important to guarantee water quality. Very little is known on the regulation of toxic secondary metabolite production in benthic cyanobacteria. In this study the physiological regulation of the production of the toxic heptapeptide microcystin (MC) and the non-toxic related peptide nostophycin (NP) in the benthic cyanobacterium Nostoc sp. strain 152 was studied under contrasting environmental conditions. I used a 2k levels factorial design, where k is the number of four factors that have been tested: Reduction in temperature (20 vs. 12°C), irradiance (50 vs. 1 μmol · m−2 · s−1), P-PO4 (144 vs. 0.14 μM P-PO4), N-NO3 (5.88 mM vs. N-NO3 free). While the growth rate was reduced more than hundred fold under most severe conditions of temperature, irradiance, and phosphate reduction the production of MC and NP never ceased. The MC and NP contents per cell varied at maximum 5- and 10.6-fold each, however the physiological variation did not outweigh the highly significant linear relationship between the daily cell division rate and the MC and NP net production rates. Surprisingly the MC and NP contents per cell showed a maximum under P-PO4 reduced and irradiance reduced conditions. Both intra- and extracellular MC and NP concentrations were negatively related to P-PO4 and irradiance. It is concluded that the proximate factor behind maximal cellular MC and NP contents is physiological stress. PMID:22723716

  13. A Virtual Mixture Approach to the Study of Multistate Equilibrium: Application to Constant pH Simulation in Explicit Water.

    PubMed

    Wu, Xiongwu; Brooks, Bernard R

    2015-10-01

    Chemical and thermodynamic equilibrium of multiple states is a fundamental phenomenon in biology systems and has been the focus of many experimental and computational studies. This work presents a simulation method to directly study the equilibrium of multiple states. This method constructs a virtual mixture of multiple states (VMMS) to sample the conformational space of all chemical states simultaneously. The VMMS system consists of multiple subsystems, one for each state. The subsystem contains a solute and a solvent environment. The solute molecules in all subsystems share the same conformation but have their own solvent environments. Transition between states is implicated by the change of their molar fractions. Simulation of a VMMS system allows efficient calculation of relative free energies of all states, which in turn determine their equilibrium molar fractions. For systems with a large number of state transition sites, an implicit site approximation is introduced to minimize the cost of simulation. A direct application of the VMMS method is for constant pH simulation to study protonation equilibrium. Applying the VMMS method to a heptapeptide of 3 ionizable residues, we calculated the pKas of those residues both with all explicit states and with implicit sites and obtained consistent results. For mouse epidermal growth factor of 9 ionizable groups, our VMMS simulations with implicit sites produced pKas of all 9 ionizable groups and the results agree qualitatively with NMR measurement. This example demonstrates the VMMS method can be applied to systems of a large number of ionizable groups and the computational cost scales linearly with the number of ionizable groups. For one of the most challenging systems in constant pH calculation, SNase Δ+PHS/V66K, our VMMS simulation shows that it is the state-dependent water penetration that causes the large deviation in lysine66's pKa.

  14. ACE2 and vasoactive peptides: novel players in cardiovascular/renal remodeling and hypertension.

    PubMed

    Mendoza-Torres, Evelyn; Oyarzún, Alejandra; Mondaca-Ruff, David; Azocar, Andrés; Castro, Pablo F; Jalil, Jorge E; Chiong, Mario; Lavandero, Sergio; Ocaranza, María Paz

    2015-08-01

    The renin-angiotensin system (RAS) is a key component of cardiovascular physiology and homeostasis due to its influence on the regulation of electrolyte balance, blood pressure, vascular tone and cardiovascular remodeling. Deregulation of this system contributes significantly to the pathophysiology of cardiovascular and renal diseases. Numerous studies have generated new perspectives about a noncanonical and protective RAS pathway that counteracts the proliferative and hypertensive effects of the classical angiotensin-converting enzyme (ACE)/angiotensin (Ang) II/angiotensin type 1 receptor (AT1R) axis. The key components of this pathway are ACE2 and its products, Ang-(1-7) and Ang-(1-9). These two vasoactive peptides act through the Mas receptor (MasR) and AT2R, respectively. The ACE2/Ang-(1-7)/MasR and ACE2/Ang-(1-9)/AT2R axes have opposite effects to those of the ACE/Ang II/AT1R axis, such as decreased proliferation and cardiovascular remodeling, increased production of nitric oxide and vasodilation. A novel peptide from the noncanonical pathway, alamandine, was recently identified in rats, mice and humans. This heptapeptide is generated by catalytic action of ACE2 on Ang A or through a decarboxylation reaction on Ang-(1-7). Alamandine produces the same effects as Ang-(1-7), such as vasodilation and prevention of fibrosis, by interacting with Mas-related GPCR, member D (MrgD). In this article, we review the key roles of ACE2 and the vasoactive peptides Ang-(1-7), Ang-(1-9) and alamandine as counter-regulators of the ACE-Ang II axis as well as the biological properties that allow them to regulate blood pressure and cardiovascular and renal remodeling. PMID:26275770

  15. Expression of class II beta-tubulin by proliferative myoepithelial cells in canine mammary mixed tumors.

    PubMed

    Arai, K; Nakano, H; Shibutani, M; Naoi, M; Matsuda, H

    2003-11-01

    Benign mammary mixed tumors in dogs resemble human salivary pleomorphic adenomas with regard to their histogenesis, including the occurrence of cartilaginous or bony metaplasia as well as the expression pattern of cytoskeletal proteins in proliferative myoepithelial cells. Recently, a monoclonal antibody specific for class II beta-tubulin has been developed. The epitope it recognizes was determined to be the heptapeptide Glu-Glu-Glu-Glu-Gly-Glu-Asp, which is the common sequence found among the canine, rat, mouse, and human class II beta-tubulin-specific regions. We carried out immunohistochemical studies on mammary mixed tumors obtained from three female dogs using this the monoclonal antibody. The antibody to class II beta-tubulin reacted intensely with proliferative myoepithelial cells in canine mammary mixed tumors, whereas staining was barely detectable in normal myoepithelial cells surrounding alveoli and alveolar ducts within the tumor and adjacent normal tissue. Proliferative myoepithelial cells also expressed vimentin, but alpha-smooth muscle actin (alphaSMA) staining was barely detectable. Immunoblot analysis showed that class II beta-tubulin and vimentin were expressed in myoepithelial cell lines prepared from the three mammary mixed tumors. On the other hand, only one cell line, which was negative for alphaSMA, produced cartilage-specific type II collagen. These results suggest that class II beta-tubulin could be a new molecular marker of proliferating myoepithelial cells in canine mammary mixed tumors and that differential expression of cytoskeletal components is associated with cartilaginous metaplasia of proliferative myoepithelial cells in mixed mammary tumors.

  16. Use of Synthetic Peptides to Study Structure-Function Relationships of Matrix Metalloproteinases and Their Substrates.

    NASA Astrophysics Data System (ADS)

    Netzel-Arnett, Sarah Joann

    The matrix metalloproteinases (MMPs) are a family of zinc proteinases that is collectively capable of degrading the major components of the extracellular matrix. A variety of synthetic peptides has been prepared which are models for the human MMP and their substrates to study structure -function relationships in this enzyme-substrate system. To elucidate the sequence specificity of the MMP, the k _{cat}/K_ M values for the hydrolysis of over 50 synthetic octapeptides has been investigated. Similarities, as well as distinct differences have been found between the individual MMP with the largest differences occurring at subsites P_1, P_1^' and P_3 ^'. Based on these data, quenched -fluorescence substrates with optimized sequences have been developed for five human MMP. The key features of these heptapeptides are a tryptophan on the P_ n^' side and a dinitrophenol quenching group on the amino terminus. To assess the role of the triple helical conformation in the collagenase-collagen system, a series of triple helical peptides has been prepared and shown to compete with collagen in collagenase assays. This provides evidence for the existence of a triple helical recognition site distinct from the active site. All of the MMP are secreted as zymogens and it has been postulated that the portion of the propeptide surrounding a critical cysteine is responsible for maintaining latency. Conformational energy calculations and mutagenesis studies have suggested that this region adopts a specific conformation that stabilizes the latent form. Peptide models of this region of the propeptide have been prepared and shown to inhibit the MMP. CD and NMR studies, however, have failed to provide evidence for the predicted peptide conformation. Thus, the observed inhibition may reflect their propensity to adopt the propeptide conformation upon binding to the enzyme.

  17. Evaluation of potential chemoprotectants against microcystin-LR hepatotoxicity in mice.

    PubMed

    Hermansky, S J; Stohs, S J; Eldeen, Z M; Roche, V F; Mereish, K A

    1991-02-01

    Microcystin-LR (MCLR) is a potent cyclic heptapeptide hepatotoxin produced by the blue-green algae, Microcystis aeruginosa. Toxic blooms of this cyanobacteria have been reported throughout the temperate world. In spite of the potential economic loss and health hazard posed by this toxin, few studies on the development of an antidote have been conducted. Thus, a number of biologically active compounds were tested in mice for effectiveness in preventing the toxicity of a lethal dose of MCLR (100 micrograms kg-1). Efficacy was evaluated based upon the percentage of surviving mice, time to death and serum lactate dehydrogenase activity 45 min after treatment with the toxin. The biologically active compounds were separated into groups based upon proposed mechanisms of action. Enzyme induction by phenobarbital but not by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in partial protection against toxicity. Calcium channel blockers, free-radical scavengers and water-soluble antioxidants produced little protection against toxicity. The membrane-active antioxidants vitamin E and silymarin, as well as glutathione and the monoethyl ester of glutathione, produced significant protection from lethality. Rifampin and cyclosporin-A, both immunosuppressive and membrane-active agents, which also block the bile acid uptake system of hepatocytes, produced complete protection from the toxicity of MCLR. Thus, lipophilic antioxidants provide partial protection against MCLR toxicity while cyclosporin-A and rifampin are highly effective and potentially useful antidotes. The toxicity of MCLR may depend upon stimulation of the immune system and may be mediated by membrane alterations. PMID:1902496

  18. The three-dimensional structure of calcium-depleted human C-reactive protein from perfectly twinned crystals.

    PubMed

    Ramadan, Mohamed A M; Shrive, Annette K; Holden, David; Myles, Dean A A; Volanakis, John E; DeLucas, Larry J; Greenhough, Trevor J

    2002-06-01

    C-reactive protein is a member of the pentraxin family of oligomeric serum proteins which has been conserved through evolution, homologues having been found in every species in which they have been sought. Human C-reactive protein (hCRP) is the classical acute-phase reactant produced in large amounts in response to tissue damage and inflammation and is used almost universally as a clinical indicator of infection and inflammation. The role of hCRP in host defence and the calcium-dependent ligand-binding specificity of hCRP for phosphocholine moieties have long been recognized. In order to clarify the structural rearrangements associated with calcium binding, the reported affinity of calcium-depleted hCRP for polycations and other ligands, and the role of calcium in protection against denaturation and proteolysis, the structure of calcium-depleted hCRP has been determined by X-ray crystallography. Crystals of calcium-depleted hCRP are invariably twinned and those suitable for analysis are merohedral type II twins of point group 4 single crystals. The structure has been solved by molecular replacement using the calcium-bound hCRP structure [Shrive et al. (1996), Nature Struct. Biol. 3, 346-354]. It reveals two independent pentamers which form a face-to-face decamer across a dyad near-parallel to the twinning twofold axis. Cycles of intensity deconvolution, density modification (tenfold NCS) and model building, eventually including refinement, give a final R factor of 0.19 (R(free) = 0.20). Despite poor definition in some areas arising from the limited resolution of the data and from the twinning and disorder, the structure reveals the probable mode of twinning and the conformational changes, localized in one of the calcium-binding loops, which accompany calcium binding. PMID:12037301

  19. The hemocyanin from a living fossil, the cephalopod Nautilus pompilius: protein structure, gene organization, and evolution.

    PubMed

    Bergmann, Sandra; Lieb, Bernhard; Ruth, Peter; Markl, Jürgen

    2006-03-01

    By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M(r) = approximately 3.5 million), assembled from 10 identical copies of an approximately 350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of approximately 50 kDa each (FUs a-g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5' UTR of 58 bp, a 3' UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M(r) = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin "OdH" from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 +/- 24 million years ago, in close agreement with fossil records from the early Devonian. PMID:16501879

  20. Further characterization of Escherichia coli alanyl-tRNA synthetase.

    PubMed

    Sood, S M; Slattery, C W; Filley, S J; Wu, M X; Hill, K A

    1996-04-15

    Selected physical and thermodynamic parameters for Escherichia coli alanyl-tRNA synthetase (AlaRS) have been determined primarily to assess the quaternary structure of this enzyme. The extinction coefficient (epsilon) at 280 nm was determined experimentally to be 0.71 ml mg-1 cm-1, and the partial specific volume (nu) was calculated from the amino acid composition to be 0.73 ml g-1. From viscosity experiments the intrinsic viscosity (eta) of AlaRS was extrapolated to be 3.4 ml g-1 and the degree of hydration (delta 1) estimated to be 0.67 gH2O g(-1)(AlaRS). Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D20,W) of 3.89 x 10(-7) cm2 s-1. In 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM 2-mercaptoethanol and at a protein concentration of 4.2 mg ml-1 the sedimentation coefficient (S20,W) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination of S20,W and D20,W under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. The S20,W was virtually independent of temperature in the range of 10-37 degrees C, while an Arrhenius plot of aminoacylation activity was biphasic. The isoelectric point was determined experimentally to be 4.9. Sedimentation equilibrium data were best fit to a decamer association complex in which dimeric AlaRS is the predominant species at 25 degrees C. PMID:8645007

  1. The structure of Medicago truncatula δ1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

    SciTech Connect

    Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; Dauter, Zbigniew

    2015-10-30

    The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.

  2. A dark energy camera search for missing supergiants in the LMC after the advanced LIGO gravitational-wave event GW150914

    DOE PAGES

    Annis, J.

    2016-05-27

    The collapse of a stellar core is expected to produce gravitational waves (GWs), neutrinos, and in most cases a luminous supernova. Sometimes, however, the optical event could be significantly less luminous than a supernova and a direct collapse to a black hole, where the star just disappears, is possible. The GW event GW150914 was detected by the LIGO Virgo Collaboration via a burst analysis that gave localization contours enclosing the Large Magellanic Cloud (LMC). Shortly thereafter, we used DECam to observe 102 deg2 of the localization area, including 38 deg2 on the LMC for a missing supergiant search. We constructmore » a complete catalog of LMC luminous red supergiants, the best candidates to undergo invisible core collapse, and collected catalogs of other candidates: less luminous red supergiants, yellow supergiants, blue supergiants, luminous blue variable stars, and Wolf–Rayet stars. Of the objects in the imaging region, all are recovered in the images. The timescale for stellar disappearance is set by the free-fall time, which is a function of the stellar radius. Our observations at 4 and 13 days after the event result in a search sensitive to objects of up to about 200 solar radii. We conclude that it is unlikely that GW150914 was caused by the core collapse of a relatively compact supergiant in the LMC, consistent with the LIGO Collaboration analyses of the gravitational waveform as best interpreted as a high mass binary black hole merger. Lastly, we discuss how to generalize this search for future very nearby core-collapse candidates.« less

  3. Structure of the Threonine-Rich Extensin from Zea mays1

    PubMed Central

    Kieliszewski, Marcia J.; Leykam, Joseph F.; Lamport, Derek T. A.

    1990-01-01

    Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete post-translational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the `P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains alternate with high regularity. The THRGP is not in the polyproline-II conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HF-solvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot. Images Figure 3 Figure 4 PMID:16667276

  4. Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

    PubMed

    Dragićević, Milan; Todorović, Slađana; Bogdanović, Milica; Filipović, Biljana; Mišić, Danijela; Simonović, Ana

    2014-06-01

    Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

  5. The Resolved Stellar Halo and Dwarf Satellite Population of NGC 3109

    NASA Astrophysics Data System (ADS)

    Hargis, Jonathan R.; Crnojevic, Denija; Sand, David J.; Willman, Beth; Spekkens, Kristine; Grillmair, Carl J.; Strader, Jay

    2016-01-01

    The stellar halo and halo substructure of dwarf galaxies provides an important window into both LCDM cosmology and galaxy formation theory on the smallest scales. We are undertaking a deep, wide-field imaging survey of nearby, isolated sub-Milky Way mass galaxies in order to (1) map the substructure, spatial extent, and metallicity of their stellar halos in resolved stars, and (2) search for faint dwarf satellite companions (i.e., the ``dwarfs of dwarfs"). These studies will allow us to explore the role of in-situ versus accretion processes in forming stellar halos in dwarfs, as well as constrain the faint end of the satellite galaxy luminosity function. This work presents a preliminary analysis of the pilot galaxy in our survey: NGC 3109 (Mv = -15 mag), a nearby (d = 1.3 Mpc) dwarf irregular, approximately 1/6th the stellar mass of the SMC. We imaged ~40 sq. deg around NGC 3109 (projected radius of ~100 kpc) using CTIO 4m/DECam to depths ~2 mag below the TRGB. We disovered a new gas-rich dwarf satellite of NGC 3109, dubbed Antlia B (Mv = -9.7 mag), similar to the recently-discovered Leo P. We also discovered five candidate dwarf satellites, with sizes (~100 pc) and luminosities (Mv ~ -6 mag) consistent with being ultra-faint dwarfs at the distance of NGC 3109. Lastly, we present stellar halo maps of resolved RGB stars on both large and small scales. We discuss the various substructures found in these maps and the future directions of our survey. This work was supported by NSF AST-1151462.

  6. The Photometric Calibration of the Dark Energy Survey (DES): Results from the Summer 2013 Re-processing of the DES Science Verification Data

    NASA Astrophysics Data System (ADS)

    Tucker, Douglas L.; Allam, S. S.; Annis, J. T.; Armstrong, R.; Bauer, A.; Bernstein, G.; Burke, D.; Fix, M.; Foust, W.; Gruendl, R. A.; Head, H.; Kuehn, K.; Kuhlmann, S.; Li, T.; Lin, H.; Rykoff, E. S.; Smith, J.; Wester, W.; Wyatt, S.; Yanny, B.; Energy Survey, Dark

    2014-01-01

    The Dark Energy Survey (DES) -- a five-year 5000 sq deg grizY survey of the Southern sky to probe the parameters of dark energy -- recently began operations using the new 3 sq deg DECam imager on the Blanco 4m telescope at the Cerro Tololo Interamerican Observatory. In order to achieve its science goals, the DES has tight requirements on both its relative and absolute photometric calibrations. The 5-year requirements are (1) an internal (relative) photometric calibration of 2% rms (2) an absolute color calibration of 0.5%, and (3) an absolute flux calibration of 0.5% (in i-band relative to BD+17 4708). In preparation for DES operations, the instrument+telescope underwent a period of Science Verification between November 2012 and February 2013. These Science Verification (SV) data were quickly processed to determine whether the image data were being produced with sufficient quality and efficiency to meet DES science goals. These data were also useful for initial science, and they were re-processed and re-calibrated during Summer 2013. The photometric goals for Summer 2013 re-processing of the DES SV were intentionally more relaxed than the requirements for the final 5-year survey: (1) an all-sky internal (relative) calibration goal of 3%, (2) an absolute color goal of 3%, and (3) an absolute flux goal of 3%. Here, we describe the results from the photometric calibration of the Summer 2013 re-processing of the DES SV data, the lessons learned, and plans for the future.

  7. A single autoimmune T cell receptor recognizes more than a million different peptides.

    PubMed

    Wooldridge, Linda; Ekeruche-Makinde, Julia; van den Berg, Hugo A; Skowera, Anna; Miles, John J; Tan, Mai Ping; Dolton, Garry; Clement, Mathew; Llewellyn-Lacey, Sian; Price, David A; Peakman, Mark; Sewell, Andrew K

    2012-01-01

    The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <10(8) human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>10(15) distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8(+) T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type "index" preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >10(8) peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8(+) T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease. PMID:22102287

  8. Structural and functional analysis of native peroxiredoxin 2 in human red blood cells.

    PubMed

    Ogasawara, Yuki; Ohminato, Takuya; Nakamura, Yusuke; Ishii, Kazuyuki

    2012-07-01

    Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H(2)O(2) results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin-thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin-thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160 kDa) oligomers and that decamers or higher order molecular weight oligomers (260-460 kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities. PMID:22537912

  9. A Detailed Study of Contamination in Deep Rapid Searches for Gravitational Wave Optical Counterparts

    NASA Astrophysics Data System (ADS)

    Cowperthwaite, Philip; Berger, Edo; Chornock, Ryan; Fong, Wen-fai

    2015-01-01

    The first direct detection of gravitational waves (GW) by the Advanced LIGO/VIRGO (aLIGO/VIRGO) collaboration is expected to occur within the next few years. In order to maximize the science gains from such a detection it is essential that we identify an electromagnetic counterpart. One of the most promising counterparts is the so-called 'kilonovae,' a fast-evolving (t ~ days) and faint (z ~ 24 AB mag) optical transient powered by the radioactive decay of r-process elements generated in the merger. However, the poor initial localization of aLIGO/VIRGO (~ 100 sq. deg.) demand the use of wide-field telescopes. Furthermore, the cadences and depths used by current and future wide-field optical surveys (e.g. PTF/ZTF, PANStarrs, LSST) are sub-optimal for kilonovae detection. We present our attempts to tackle these issues by investigating the theoretical and practical issues associated with optical follow-up of an aLIGO/VIRGO GW event. This includes a systematic study of the potential contaminant population and their impact on kilonovae detectability in simulated observations. We show that kilonovae can remain separated from contaminants by the virtue of their red colors (i - z > 0.5) and short timescales. This theoretical analysis will be tested against observations obtained by the DECam wide-field imager on the CTIO Blanco 4m telescope. These data attempt to simulate the wide area coverage (~ 70 sq. deg.) and rapid cadence (two visits per night in i,z) necessary for targeted GW follow-up and will provide an excellent test bed for understanding the practical issues associated with this endeavor. This work is supported in part by the NSF GRFP grant DGE1144152.

  10. Cosmology with superluminous supernovae

    NASA Astrophysics Data System (ADS)

    Scovacricchi, D.; Nichol, R. C.; Bacon, D.; Sullivan, M.; Prajs, S.

    2016-02-01

    We predict cosmological constraints for forthcoming surveys using superluminous supernovae (SLSNe) as standardizable candles. Due to their high peak luminosity, these events can be observed to high redshift (z ˜ 3), opening up new possibilities to probe the Universe in the deceleration epoch. We describe our methodology for creating mock Hubble diagrams for the Dark Energy Survey (DES), the `Search Using DECam for Superluminous Supernovae' (SUDSS) and a sample of SLSNe possible from the Large Synoptic Survey Telescope (LSST), exploring a range of standardization values for SLSNe. We include uncertainties due to gravitational lensing and marginalize over possible uncertainties in the magnitude scale of the observations (e.g. uncertain absolute peak magnitude, calibration errors). We find that the addition of only ≃100 SLSNe from SUDSS to 3800 Type Ia Supernovae (SNe Ia) from DES can improve the constraints on w and Ωm by at least 20 per cent (assuming a flat wCDM universe). Moreover, the combination of DES SNe Ia and 10 000 LSST-like SLSNe can measure Ωm and w to 2 and 4 per cent, respectively. The real power of SLSNe becomes evident when we consider possible temporal variations in w(a), giving possible uncertainties of only 2, 5 and 14 per cent on Ωm, w0 and wa, respectively, from the combination of DES SNe Ia, LSST-like SLSNe and Planck. These errors are competitive with predicted Euclid constraints, indicating a future role for SLSNe for probing the high-redshift Universe.

  11. Supramolecular design of biocompatible nanocontainers based on amphiphilic derivatives of a natural compound isosteviol.

    PubMed

    Gabdrakhmanov, Dinar R; Voronin, Mikhail A; Zakharova, Lucia Ya; Konovalov, Alexander I; Khaybullin, Ravil N; Strobykina, Irina Yu; Kataev, Vladimir E; Faizullin, Dzhigangir A; Gogoleva, Natalia E; Konnova, Tatiana A; Salnikov, Vadim V; Zuev, Yuriy F

    2013-10-21

    Two diterpenoid surfactants with ammonium head groups and bromide (S1) or tosylate (S2) counterions have been synthesized. Exploration of these biomimetic species made it possible to demonstrate that even minor structural changes beyond their chemical nature may dramatically affect their solution behavior. While their aggregation thresholds differ inconsiderably, morphological behavior and affinity to lipid bilayer are strongly dependent on the counterion nature. Compound S2 demonstrates properties of typical surfactants and forms small micelle-like aggregates above critical micelle concentration. For surfactant S1, two critical concentrations and two types of aggregates occur. Structural transitions have been observed between small micelles and aggregates with higher aggregation numbers and hydrodynamic diameter of ca. 150 nm. Unlike S2, surfactant S1 is shown to integrate with liposomes based on dipalmitoylphosphatidylcholine, resulting in a decrease of the temperature of the main phase transition. Both surfactants demonstrate an effective complexation capacity toward oligonucleotide (ONu), which is supported by recharging the surfactant-ONu complexes and the ethidium bromide exclusion at a low N/P ratio. Meanwhile, a very weak complexation of plasmid DNA with the surfactants has been revealed in the gel electrophoresis experiment. The DNA transfer to bacterial cells mediated by the surfactant S1 is shown to depend on the protocol used. In the case of the electroporation, the inhibition of the cell transformation occurs in the presence of the surfactant, while upon the chemical treatment no surfactant effect has been observed. The variability in the morphology, the biocompatibility, the nanoscale dimension and the high binding capacity toward the DNA decamer make it possible to nominate the designed surfactants as promising carriers for biosubstrates or as a helper surfactant for the mixed liposome-surfactant nanocontainers.

  12. Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales

    SciTech Connect

    Klinke,S.; Zylberman, V.; Bonomi, H.; Haase, I.; Guimaraes, B.; Braden, B.; Bacher, A.; Fischer, M.; Goldbaum, F.

    2007-01-01

    6, 7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of ?-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs from Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.

  13. DES14X3taz: A type I superluminous supernova showing a luminous, rapidly cooling initial pre-peak bump

    DOE PAGES

    Smith, M.

    2016-02-03

    Here, we present DES14X3taz, a new hydrogen-poor superluminous supernova (SLSN-I) discovered by the Dark Energy Survey (DES) supernova program, with additional photometric data provided by the Survey Using DECam for Superluminous Supernovae. Spectra obtained using Optical System for Imaging and low-Intermediate-Resolution Integrated Spectroscopy on the Gran Telescopio CANARIAS show DES14X3taz is an SLSN-I at z = 0.608. Multi-color photometry reveals a double-peaked light curve: a blue and relatively bright initial peak that fades rapidly prior to the slower rise of the main light curve. Our multi-color photometry allows us, for the first time, to show that the initial peak cools from 22,000more » to 8000 K over 15 rest-frame days, and is faster and brighter than any published core-collapse supernova, reaching 30% of the bolometric luminosity of the main peak. No physical (56)Ni-powered model can fit this initial peak. We show that a shock-cooling model followed by a magnetar driving the second phase of the light curve can adequately explain the entire light curve of DES14X3taz. Models involving the shock-cooling of extended circumstellar material at a distance of ≃400 R⊙ are preferred over the cooling of shock-heated surface layers of a stellar envelope. We compare DES14X3taz to the few double-peaked SLSN-I events in the literature. Although the rise times and characteristics of these initial peaks differ, there exists the tantalizing possibility that they can be explained by one physical interpretation.« less

  14. QuatIdent: a web server for identifying protein quaternary structural attribute by fusing functional domain and sequential evolution information.

    PubMed

    Shen, Hong-Bin; Chou, Kuo-Chen

    2009-03-01

    Many proteins exist in vivo as oligomers with various different quaternary structural attributes rather than as single individual chains. They are the structural bases of various marvelous biological functions such as cooperative effects, allosteric mechanism, and ion-channel gating. Therefore, with the avalanche of protein sequences generated in the postgenomic era, it is very important for both basic research and drug discovery to identify their quaternary structural attributes in a timely manner. In view of this, a powerful ensemble identifier, called QuatIdent, is developed by fusing the functional domain and sequential evolution information. QuatIdent is a 2-layer predictor. The 1st layer is for identifying a query protein as belonging to which one of the following 10 main quaternary structural attributes: (1) monomer, (2) dimer, (3) trimer, (4) tetramer, (5) pentamer, (6) hexamer, (7) heptamer, (8) octamer, (9) decamer, and (10) dodecamer. If the result thus obtained turns out to be anything but monomer, the process will be automatically continued to further identify it as belonging to a homo-oligomer or hetero-oligomer. The overall success rate by QuatIdent for the 1st layer identification was 71.1% and that for the 2nd layer ranged from 84 to 96%. These rates were derived by the jackknife cross-validation tests on the stringent benchmark data sets where none of proteins has > or =60% pairwise sequence identity to any other in a same subset. QuatIdent is freely accessible to the public as a web server via the site at http://www.csbio.sjtu.edu.cn/bioinf/Quaternary/ , by which one can get the desired 2-level results for a query protein sequence in around 25 seconds. The longer the sequence is, the more time that is needed.

  15. Full-depth Coadds of the WISE and NEOWISE-Reactivation Data

    NASA Astrophysics Data System (ADS)

    Meisner, Aaron M.; Lang, Dustin; Schlegel, David J.

    2016-01-01

    Thanks to the Wide-field Infrared Survey Explorer (WISE) mission's NEOWISE-Reactivation program, an entire year of post-reactivation W1 and W2 exposures has been made publicly available in 2015. This data set consists of ˜2.5 million new exposures in each band, effectively doubling the amount of WISE imaging available at 3.4μm and 4.6μm relative to the AllWISE release. However, no full sky coadds have yet been published which incorporate both pre and post-reactivation WISE imaging. We are creating a custom, full-sky set of coadds combining all W1 and W2 exposures from both the AllWISE and NEOWISE-Reactivation mission phases, employing the unWISE image coaddition framework (Lang 2014), which preserves the native WISE angular resolution and is optimized for forced photometry. We anticipate that our resulting unWISE data products will have a broad range of applications, from studies of quasar variability to the identification of faint moving objects in the solar neighborhood. Using our full-depth coadds, we plan to perform W1 and W2 forced photometry at the positions of ˜400 million SDSS sources, extending to WISE depths 0.4 magnitudes fainter than previously possible. Our full-depth unWISE coadds will represent an important input for DESI target selection, and preliminary versions are already in use within the DECam Legacy Survey (DECaLS) reduction pipeline.

  16. An Exocyclic Methylene Group Acts As a Bioisostere of the 2′-Oxygen Atom in LNA

    SciTech Connect

    Seth, Punit P.; Allerson, Charles R.; Berdeja, Andres; Siwkowski, Andrew; Pallan, Pradeep S.; Gaus, Hans; Prakash, Thazha P.; Watt, Andrew T.; Egli, Martin; Swayze, Eric E.

    2010-12-07

    We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2{prime}-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2{prime}-position and a propynyl group at the C-4{prime} position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2{prime}-deoxyribose residue of the 5{prime}-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2{prime}-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.

  17. A single autoimmune T cell receptor recognizes more than a million different peptides.

    PubMed

    Wooldridge, Linda; Ekeruche-Makinde, Julia; van den Berg, Hugo A; Skowera, Anna; Miles, John J; Tan, Mai Ping; Dolton, Garry; Clement, Mathew; Llewellyn-Lacey, Sian; Price, David A; Peakman, Mark; Sewell, Andrew K

    2012-01-01

    The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <10(8) human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>10(15) distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8(+) T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type "index" preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >10(8) peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8(+) T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.

  18. DES14X3taz: A Type I Superluminous Supernova Showing a Luminous, Rapidly Cooling Initial Pre-peak Bump

    NASA Astrophysics Data System (ADS)

    Smith, M.; Sullivan, M.; D'Andrea, C. B.; Castander, F. J.; Casas, R.; Prajs, S.; Papadopoulos, A.; Nichol, R. C.; Karpenka, N. V.; Bernard, S. R.; Brown, P.; Cartier, R.; Cooke, J.; Curtin, C.; Davis, T. M.; Finley, D. A.; Foley, R. J.; Gal-Yam, A.; Goldstein, D. A.; González-Gaitán, S.; Gupta, R. R.; Howell, D. A.; Inserra, C.; Kessler, R.; Lidman, C.; Marriner, J.; Nugent, P.; Pritchard, T. A.; Sako, M.; Smartt, S.; Smith, R. C.; Spinka, H.; Thomas, R. C.; Wolf, R. C.; Zenteno, A.; Abbott, T. M. C.; Benoit-Lévy, A.; Bertin, E.; Brooks, D.; Buckley-Geer, E.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Crocce, M.; Cunha, C. E.; da Costa, L. N.; Desai, S.; Diehl, H. T.; Doel, P.; Estrada, J.; Evrard, A. E.; Flaugher, B.; Fosalba, P.; Frieman, J.; Gerdes, D. W.; Gruen, D.; Gruendl, R. A.; James, D. J.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Li, T. S.; Marshall, J. L.; Martini, P.; Miller, C. J.; Miquel, R.; Nord, B.; Ogando, R.; Plazas, A. A.; Reil, K.; Romer, A. K.; Roodman, A.; Rykoff, E. S.; Sanchez, E.; Scarpine, V.; Schubnell, M.; Sevilla-Noarbe, I.; Soares-Santos, M.; Sobreira, F.; Suchyta, E.; Swanson, M. E. C.; Tarle, G.; Walker, A. R.; Wester, W.; DES Collaboration

    2016-02-01

    We present DES14X3taz, a new hydrogen-poor superluminous supernova (SLSN-I) discovered by the Dark Energy Survey (DES) supernova program, with additional photometric data provided by the Survey Using DECam for Superluminous Supernovae. Spectra obtained using Optical System for Imaging and low-Intermediate-Resolution Integrated Spectroscopy on the Gran Telescopio CANARIAS show DES14X3taz is an SLSN-I at z = 0.608. Multi-color photometry reveals a double-peaked light curve: a blue and relatively bright initial peak that fades rapidly prior to the slower rise of the main light curve. Our multi-color photometry allows us, for the first time, to show that the initial peak cools from 22,000 to 8000 K over 15 rest-frame days, and is faster and brighter than any published core-collapse supernova, reaching 30% of the bolometric luminosity of the main peak. No physical 56Ni-powered model can fit this initial peak. We show that a shock-cooling model followed by a magnetar driving the second phase of the light curve can adequately explain the entire light curve of DES14X3taz. Models involving the shock-cooling of extended circumstellar material at a distance of ≃400 {\\text{}}{R}⊙ are preferred over the cooling of shock-heated surface layers of a stellar envelope. We compare DES14X3taz to the few double-peaked SLSN-I events in the literature. Although the rise times and characteristics of these initial peaks differ, there exists the tantalizing possibility that they can be explained by one physical interpretation.

  19. The Dark Energy Survey: more than dark energy - an overview

    NASA Astrophysics Data System (ADS)

    Dark Energy Survey Collaboration; Abbott, T.; Abdalla, F. B.; Aleksić, J.; Allam, S.; Amara, A.; Bacon, D.; Balbinot, E.; Banerji, M.; Bechtol, K.; Benoit-Lévy, A.; Bernstein, G. M.; Bertin, E.; Blazek, J.; Bonnett, C.; Bridle, S.; Brooks, D.; Brunner, R. J.; Buckley-Geer, E.; Burke, D. L.; Caminha, G. B.; Capozzi, D.; Carlsen, J.; Carnero-Rosell, A.; Carollo, M.; Carrasco-Kind, M.; Carretero, J.; Castander, F. J.; Clerkin, L.; Collett, T.; Conselice, C.; Crocce, M.; Cunha, C. E.; D'Andrea, C. B.; da Costa, L. N.; Davis, T. M.; Desai, S.; Diehl, H. T.; Dietrich, J. P.; Dodelson, S.; Doel, P.; Drlica-Wagner, A.; Estrada, J.; Etherington, J.; Evrard, A. E.; Fabbri, J.; Finley, D. A.; Flaugher, B.; Foley, R. J.; Fosalba, P.; Frieman, J.; García-Bellido, J.; Gaztanaga, E.; Gerdes, D. W.; Giannantonio, T.; Goldstein, D. A.; Gruen, D.; Gruendl, R. A.; Guarnieri, P.; Gutierrez, G.; Hartley, W.; Honscheid, K.; Jain, B.; James, D. J.; Jeltema, T.; Jouvel, S.; Kessler, R.; King, A.; Kirk, D.; Kron, R.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Li, T. S.; Lima, M.; Lin, H.; Maia, M. A. G.; Makler, M.; Manera, M.; Maraston, C.; Marshall, J. L.; Martini, P.; McMahon, R. G.; Melchior, P.; Merson, A.; Miller, C. J.; Miquel, R.; Mohr, J. J.; Morice-Atkinson, X.; Naidoo, K.; Neilsen, E.; Nichol, R. C.; Nord, B.; Ogando, R.; Ostrovski, F.; Palmese, A.; Papadopoulos, A.; Peiris, H. V.; Peoples, J.; Percival, W. J.; Plazas, A. A.; Reed, S. L.; Refregier, A.; Romer, A. K.; Roodman, A.; Ross, A.; Rozo, E.; Rykoff, E. S.; Sadeh, I.; Sako, M.; Sánchez, C.; Sanchez, E.; Santiago, B.; Scarpine, V.; Schubnell, M.; Sevilla-Noarbe, I.; Sheldon, E.; Smith, M.; Smith, R. C.; Soares-Santos, M.; Sobreira, F.; Soumagnac, M.; Suchyta, E.; Sullivan, M.; Swanson, M.; Tarle, G.; Thaler, J.; Thomas, D.; Thomas, R. C.; Tucker, D.; Vieira, J. D.; Vikram, V.; Walker, A. R.; Wechsler, R. H.; Weller, J.; Wester, W.; Whiteway, L.; Wilcox, H.; Yanny, B.; Zhang, Y.; Zuntz, J.

    2016-08-01

    This overview paper describes the legacy prospect and discovery potential of the Dark Energy Survey (DES) beyond cosmological studies, illustrating it with examples from the DES early data. DES is using a wide-field camera (DECam) on the 4 m Blanco Telescope in Chile to image 5000 sq deg of the sky in five filters (grizY). By its completion, the survey is expected to have generated a catalogue of 300 million galaxies with photometric redshifts and 100 million stars. In addition, a time-domain survey search over 27 sq deg is expected to yield a sample of thousands of Type Ia supernovae and other transients. The main goals of DES are to characterize dark energy and dark matter, and to test alternative models of gravity; these goals will be pursued by studying large-scale structure, cluster counts, weak gravitational lensing and Type Ia supernovae. However, DES also provides a rich data set which allows us to study many other aspects of astrophysics. In this paper, we focus on additional science with DES, emphasizing areas where the survey makes a difference with respect to other current surveys. The paper illustrates, using early data (from `Science Verification', and from the first, second and third seasons of observations), what DES can tell us about the Solar system, the Milky Way, galaxy evolution, quasars and other topics. In addition, we show that if the cosmological model is assumed to be Λ+cold dark matter, then important astrophysics can be deduced from the primary DES probes. Highlights from DES early data include the discovery of 34 trans-Neptunian objects, 17 dwarf satellites of the Milky Way, one published z > 6 quasar (and more confirmed) and two published superluminous supernovae (and more confirmed).

  20. A Dark Energy Camera Search for Missing Supergiants in the LMC after the Advanced LIGO Gravitational-wave Event GW150914

    NASA Astrophysics Data System (ADS)

    Annis, J.; Soares-Santos, M.; Berger, E.; Brout, D.; Chen, H.; Chornock, R.; Cowperthwaite, P. S.; Diehl, H. T.; Doctor, Z.; Drlica-Wagner, A.; Drout, M. R.; Farr, B.; Finley, D. A.; Flaugher, B.; Foley, R. J.; Frieman, J.; Gruendl, R. A.; Herner, K.; Holz, D.; Kessler, R.; Lin, H.; Marriner, J.; Neilsen, E.; Rest, A.; Sako, M.; Smith, M.; Smith, N.; Sobreira, F.; Walker, A. R.; Yanny, B.; Abbott, T. M. C.; Abdalla, F. B.; Allam, S.; Benoit-Lévy, A.; Bernstein, R. A.; Bertin, E.; Buckley-Geer, E.; Burke, D. L.; Capozzi, D.; Carnero Rosell, A.; Carrasco Kind, M.; Carretero, J.; Castander, F. J.; Cenko, S. B.; Crocce, M.; Cunha, C. E.; D’Andrea, C. B.; da Costa, L. N.; Desai, S.; Dietrich, J. P.; Eifler, T. F.; Evrard, A. E.; Fernandez, E.; Fischer, J.; Fong, W.; Fosalba, P.; Fox, D. B.; Fryer, C. L.; Garcia-Bellido, J.; Gaztanaga, E.; Gerdes, D. W.; Goldstein, D. A.; Gruen, D.; Gutierrez, G.; Honscheid, K.; James, D. J.; Karliner, I.; Kasen, D.; Kent, S.; Kuehn, K.; Kuropatkin, N.; Lahav, O.; Li, T. S.; Lima, M.; Maia, M. A. G.; Martini, P.; Metzger, B. D.; Miller, C. J.; Miquel, R.; Mohr, J. J.; Nichol, R. C.; Nord, B.; Ogando, R.; Peoples, J.; Petravic, D.; Plazas, A. A.; Quataert, E.; Romer, A. K.; Roodman, A.; Rykoff, E. S.; Sanchez, E.; Santiago, B.; Scarpine, V.; Schindler, R.; Schubnell, M.; Sevilla-Noarbe, I.; Sheldon, E.; Smith, R. C.; Stebbins, A.; Swanson, M. E. C.; Tarle, G.; Thaler, J.; Thomas, R. C.; Tucker, D. L.; Vikram, V.; Wechsler, R. H.; Weller, J.; Wester, W.; The DES Collaboration

    2016-06-01

    The collapse of a stellar core is expected to produce gravitational waves (GWs), neutrinos, and in most cases a luminous supernova. Sometimes, however, the optical event could be significantly less luminous than a supernova and a direct collapse to a black hole, where the star just disappears, is possible. The GW event GW150914 was detected by the LIGO Virgo Collaboration via a burst analysis that gave localization contours enclosing the Large Magellanic Cloud (LMC). Shortly thereafter, we used DECam to observe 102 deg2 of the localization area, including 38 deg2 on the LMC for a missing supergiant search. We construct a complete catalog of LMC luminous red supergiants, the best candidates to undergo invisible core collapse, and collected catalogs of other candidates: less luminous red supergiants, yellow supergiants, blue supergiants, luminous blue variable stars, and Wolf–Rayet stars. Of the objects in the imaging region, all are recovered in the images. The timescale for stellar disappearance is set by the free-fall time, which is a function of the stellar radius. Our observations at 4 and 13 days after the event result in a search sensitive to objects of up to about 200 solar radii. We conclude that it is unlikely that GW150914 was caused by the core collapse of a relatively compact supergiant in the LMC, consistent with the LIGO Collaboration analyses of the gravitational waveform as best interpreted as a high mass binary black hole merger. We discuss how to generalize this search for future very nearby core-collapse candidates.

  1. Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro

    SciTech Connect

    Zhao Guohui; Liu Jun; Liu Xiang; Chen Min; Zhang Houcheng Wang, Peng George

    2007-11-23

    GDP-perosamine synthetase (Per, E.C. not yet classified) is important to the synthesis of Escherichia coli O157:H7 O-antigen. The mutant in per gene can disrupt the synthesis of O157 O-antigen. In this study, GDP-perosamine synthetase was cloned from E. coli O157:H7 and over-expressed in E. coli BL21 (DE3). The recombinant His-tagged Per fusion protein was a decamer with molecular weight of 431 kDa. The optimal pH value of this recombinant protein was 7.5. The divalent ions had no significant effect on Per-catalyzed reaction. The K{sub m} and K{sub cat}/K{sub m} for GDP-4-keto-6-deoxy-D-mannose were 0.09 mM and 2.1 x 10{sup 5} M{sup -1} S{sup -1}, and those for L-glutamate were 2 mM and 0.52 x 10{sup 5} M{sup -1}S{sup -1}, respectively. Per was used to synthesize GDP-perosamine from GDP-mannose together with recombinant GDP-mannose dehydratase (GMD, E.C. 4.2.1.47). The purified GDP-perosamine was identified by MS and NMR. In summary, this work provided a feasible approach for the synthesis of GDP-perosamine which can lead to the study of LPS biosynthesis of pathogenic E. coli O157:H7.

  2. Evolution from primordial oligomeric repeats to modern coding sequences.

    PubMed

    Ohno, S

    1987-01-01

    It seems as though nature was most innovative at the very beginning of life on this Earth a few billion years ago. For example, the functional competence of most, if not all, of the sugar-metabolizing enzymes was clearly established before the division of eukaryotes from prokaryotes eons ago, each critical active-site amino acid sequence being conserved ever since by bacteria as well as by mammals. I contend that this initial innovativeness was due to the first set of coding sequences being repeats of base oligomers, thus encoding polypeptide chains of various periodicities; such periodical polypeptide chains can easily acquire alpha-helical and beta-sheet-forming segments. In fact, the entire length of sugar-metabolizing enzymes is comprised of alternating alpha-helical and beta-sheet-forming segments. In the prebiotic (therefore nonenzymatic) replication of nucleic acids, what was in short supply was long templates, for there apparently was no inherent obstacle in copying of long templates, if such existed, in the presence of Zn2+. I submit that in this prebiotic condition, only those nucleotide oligomers that were internal doubles were automatically assured of progressive elongation to become long templates. For example, a decamer that was a pentameric repeat and its complementary sequence may pair unequally to initiate the next round of replication: first unit pairing with second, and a paired segment serving as a primer. As a consequence of this unequal pairing, decameric templates managed to become pentadecameric templates only after one round of replication, and this elongation process had no inherent limit.

  3. Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker--an endangered medicinal herb.

    PubMed

    Lattoo, S K; Bamotra, S; Sapru Dhar, R; Khan, S; Dhar, A K

    2006-06-01

    An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4 x 10(-6) M Kn and 2 x 10(-6) MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1 x 10(-5) M Kinetin (Kn) and 5 x 10(-6) M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm +/- 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5 x 10(-6) M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb. PMID:16477407

  4. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    NASA Technical Reports Server (NTRS)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  5. The Dark Energy Survey: More than dark energy - An overview

    DOE PAGES

    Abbott, T.

    2016-03-21

    This overview article describes the legacy prospect and discovery potential of the Dark Energy Survey (DES) beyond cosmological studies, illustrating it with examples from the DES early data. DES is using a wide-field camera (DECam) on the 4m Blanco Telescope in Chile to image 5000 sq deg of the sky in five filters (grizY). By its completion the survey is expected to have generated a catalogue of 300 million galaxies with photometric redshifts and 100 million stars. In addition, a time-domain survey search over 27 sq deg is expected to yield a sample of thousands of Type Ia supernovae andmore » other transients. The main goals of DES are to characterise dark energy and dark matter, and to test alternative models of gravity; these goals will be pursued by studying large scale structure, cluster counts, weak gravitational lensing and Type Ia supernovae. However, DES also provides a rich data set which allows us to study many other aspects of astrophysics. In this paper we focus on additional science with DES, emphasizing areas where the survey makes a difference with respect to other current surveys. The paper illustrates, using early data (from `Science Verification', and from the first, second and third seasons of observations), what DES can tell us about the solar system, the Milky Way, galaxy evolution, quasars, and other topics. In addition, we show that if the cosmological model is assumed to be Lambda+ Cold Dark Matter (LCDM) then important astrophysics can be deduced from the primary DES probes. Lastly, highlights from DES early data include the discovery of 34 Trans Neptunian Objects, 17 dwarf satellites of the Milky Way, one published z > 6 quasar (and more confirmed) and two published superluminous supernovae (and more confirmed).« less

  6. Effect of Currently Approved Carriers and Adjuvants on the Pre-Clinical Efficacy of a Conjugate Vaccine against Oxycodone in Mice and Rats

    PubMed Central

    Pravetoni, Marco; Vervacke, Jeffrey S.; Distefano, Mark D.; Tucker, Ashli M.; Laudenbach, Megan; Pentel, Paul R.

    2014-01-01

    Vaccination against the highly abused prescription opioid oxycodone has shown pre-clinical efficacy for blocking oxycodone effects. The current study further evaluated a candidate vaccine composed of oxycodone derivatized at the C6 position (6OXY) conjugated to the native keyhole limpet hemocyanin (nKLH) carrier protein. To provide an oxycodone vaccine formulation suitable for human studies, we studied the effect of alternative carriers and adjuvants on the generation of oxycodone-specific serum antibody and B cell responses, and the effect of immunization on oxycodone distribution and oxycodone-induced antinociception in mice and rats. 6OXY conjugated to tetanus toxoid (TT) or a GMP grade KLH dimer (dKLH) was as effective as 6OXY conjugated to the nKLH decamer in mice and rats, while the 6OXY hapten conjugated to a TT-derived peptide was not effective in preventing oxycodone-induced antinociception in mice. Immunization with 6OXY-TT s.c. absorbed on alum adjuvant provided similar protection to 6OXY-TT administered i.p. with Freund’s adjuvant in rats. The toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) adjuvant, alone or in combination with alum, offered no advantage over alum alone for generating oxycodone-specific serum antibodies or 6OXY-specific antibody secreting B cells in mice vaccinated with 6OXY-nKLH or 6OXY-TT. The immunogenicity of oxycodone vaccines may be modulated by TLR4 signaling since responses to 6OXY-nKLH in alum were decreased in TLR4-deficient mice. These data suggest that TT, nKLH and dKLH carriers provide consistent 6OXY conjugate vaccine immunogenicity across species, strains and via different routes of administration, while adjuvant formulations may need to be tailored to individual immunogens or patient populations. PMID:24797666

  7. Cloning and functional characterization of a typical 2-Cys peroxiredoxin from southern bluefin tuna (Thunnus maccoyii).

    PubMed

    Sutton, Drew L; Loo, Grace H; Menz, R Ian; Schuller, Kathryn A

    2010-06-01

    Peroxiredoxins (Prxs, EC: 1.11.1.15) are cysteine-dependent peroxidases proposed to function as antioxidant enzymes and also in H2O2-mediated cell signaling. They have been well characterized in yeast, mammals, protists and bacteria but not yet in fish. Here we describe the cloning and functional characterization of a Prx 2 cDNA from southern bluefin tuna (SBT, Thunnus maccoyii), an important aquaculture species in South Australia. The SBT Prx sequence was closely related (76-92% identical) to Prx 1 and 2 sequences from other fish and mammals and phylogenetic analyses showed that it was most likely a Prx 2. The deduced amino acid sequence contained the peroxidatic and resolving Cys residues characteristic of typical 2-Cys Prx proteins from all kingdoms of life. It also contained the GGLG motif associated with the sensitivity of eukaryotic typical 2-Cys Prx proteins to overoxidation and consequent inactivation by H2O2. When the SBT Prx 2 was expressed in E. coli, it showed thioredoxin (Trx)-dependent peroxidase activity with H2O2, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH). The SBT Prx displayed Michaelis-Menten kinetics with Trx but sigmoidal kinetics with H2O2 and CuOOH. The K(m)(Trx) was 12 microM and the S(0.5) values for H2O2 and CuOOH were 29 and 25 microM, respectively. At mM concentrations of H2O2, SBT Prx progressively lost its peroxidase activity as has been observed for other eukaryotic typical 2-Cys Prx proteins. The native SBT Prx enzyme existed as a mixture of dimers, tetramers, decamers and a higher order aggregate.

  8. Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides: Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2'-O-Ribonucleic Acid Modifications

    SciTech Connect

    Egli, Martin; Minasov, George; Tereshko, Valentina; Pallan, Pradeep S.; Teplova, Marianna; Inamati, Gopal B.; Lesnik, Elena A.; Owens, Steve R.; Ross, Bruce S.; Prakash, Thazha P.; Manoharan, Muthiah

    2010-03-05

    The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the

  9. Aβ(39–42) Modulates Aβ Oligomerization but Not Fibril Formation

    PubMed Central

    Gessel, Megan Murray; Wu, Chun; Li, Huiyuan; Bitan, Gal; Shea, Joan-Emma; Bowers, Michael T.

    2012-01-01

    Recently, certain C-terminal fragments (CTFs) of Aβ42 have been shown to be effective inhibitors of Aβ42 toxicity. Here, we examine the interactions between the shortest CTF in the original series, Aβ(39–42) and full-length Aβ. Mass spectrometry results indicate that Aβ(39–42) binds directly to Aβ monomers and to the n=2,4, and 6 oligomers. The Aβ42:Aβ(39–42) complex is further probed using in molecular dynamics simulations. Although the CTF was expected to bind to the hydrophobic C-terminus of Aβ42, the simulations show that Aβ(39–42) binds at several locations on Aβ42, including the C-terminus, other hydrophobic regions, and preferentially in the N-terminus. Ion mobility-mass spectrometry (IM-MS) and electron microscopy experiments indicate that Aβ(39–42) disrupts the early assembly of full-length Aβ. Specifically, the ion-mobility results show that Aβ(39–42) prevents the formation of large decamer/dodecamer Aβ42 species and, moreover, can remove these structures from solution. At the same time, thioflavin T fluorescence and electron microscopy results show that the CTF does not inhibit fibril formation, lending strong support to the hypothesis that oligomers and not amyloid fibrils are the Aβ form responsible for toxicity. The results emphasize the role of small, soluble assemblies in Aβ-induced toxicity and suggest that Aβ(39–42) inhibits Aβ-induced toxicity by a unique mechanism, modulating early assembly into non-toxic heterooligomers, without preventing fibril formation. PMID:22129303

  10. Aβ(39-42) modulates Aβ oligomerization but not fibril formation.

    PubMed

    Gessel, Megan Murray; Wu, Chun; Li, Huiyuan; Bitan, Gal; Shea, Joan-Emma; Bowers, Michael T

    2012-01-10

    Recently, certain C-terminal fragments (CTFs) of Aβ42 have been shown to be effective inhibitors of Aβ42 toxicity. Here, we examine the interactions between the shortest CTF in the original series, Aβ(39-42), and full-length Aβ. Mass spectrometry results indicate that Aβ(39-42) binds directly to Aβ monomers and to the n = 2, 4, and 6 oligomers. The Aβ42:Aβ(39-42) complex is further probed using molecular dynamics simulations. Although the CTF was expected to bind to the hydrophobic C-terminus of Aβ42, the simulations show that Aβ(39-42) binds at several locations on Aβ42, including the C-terminus, other hydrophobic regions, and preferentially in the N-terminus. Ion mobility-mass spectrometry (IM-MS) and electron microscopy experiments indicate that Aβ(39-42) disrupts the early assembly of full-length Aβ. Specifically, the ion-mobility results show that Aβ(39-42) prevents the formation of large decamer/dodecamer Aβ42 species and, moreover, can remove these structures from solution. At the same time, thioflavin T fluorescence and electron microscopy results show that the CTF does not inhibit fibril formation, lending strong support to the hypothesis that oligomers and not amyloid fibrils are the Aβ form responsible for toxicity. The results emphasize the role of small, soluble assemblies in Aβ-induced toxicity and suggest that Aβ(39-42) inhibits Aβ-induced toxicity by a unique mechanism, modulating early assembly into nontoxic hetero-oligomers, without preventing fibril formation.

  11. Searching for the Most UV-Luminous Galaxies in the Distant Universe

    NASA Astrophysics Data System (ADS)

    Stevans, Matthew L.; Finkelstein, Steven L.; Wold, Isak; Gebhardt, Karl; Jogee, Shardha; Papovich, Casey J.; Ciardullo, Robin; Gronwall, Caryl; Acquaviva, Viviana; Casey, Caitlin; HETDEX Team

    2016-01-01

    How galaxies grow and evolve over cosmic time is one of the largest unanswered questions in astronomy. With 50% of the stellar mass in today's galaxies having formed before z~1 (Dickinson et al. 2003) and the cosmic star formation density peaking between 1 < z < 3 (Madau & Dickinson 2014), the epoch at z>3 is particularly important for understanding the rise of the Hubble sequence at later times. We present multi-wavelength photometry from the the unprecedentedly large 28 deg2 Hobby Eberly Telescope Dark Energy Experiment (HETDEX) / Spitzer-HETDEX Exploratory Large Area (SHELA) survey in a study of 800,000 galaxies at redshifts of 1.9 to 3.5 including significant numbers of the most massive and most rare galaxies, unseen in pencil-beam deep field surveys. We use ugriz photometry from the Dark Energy Camera (DECam) and fit galaxy spectral energy distributions (SEDs) with stellar population spectral templates to select LBGs at z~3-4 and measure the rest-frame ultraviolet (UV) luminosity function. Our survey's large area and moderate depth provide a unique view of the bright-end (MAB<-22). Contamination by stellar sources or active galactic nuclei (AGNs) can be ruled out by the inclusion of multi-wavelength data. Probing a volume of 0.5 Gpc3 at 1.9 < z < 3.5, similar to that of the Sloan Digital Sky Survey (SDSS) at z < 0.5, we provide the most definitive constraints for numerical models of cosmic galaxy evolution, expanding our knowledge of galaxy growth during this critical era in cosmic history.

  12. Simulation study of noncovalent hybridization of carbon nanotubes by single-stranded DNA in water.

    PubMed

    Martin, Willis; Zhu, Wusheng; Krilov, Goran

    2008-12-18

    Recent discovery that single-stranded DNA (ssDNA) binds to carbon nanotubes with high affinity to form soluble hybrids has received great attention as a promising approach to solving the long-standing problem of nanotube solubilization and separation. The mechanism of this process, including the nature of the DNA-nanotube interactions and the molecular structure of the hybrids is still not well understood. Here, we use all-atom replica-exchange molecular dynamics simulations to study the association of several ssDNA decamers with single-walled carbon nanotubes of different chirality in an aqueous environment. The oligonucleotides are found to readily adsorb onto the nanotube surface, after which they undergo a slow structural rearrangement. Cluster analysis of bound DNA conformations as well as population distribution maps computed as a function of several local and global order parameters show that the hybrids exhibit a complex morphology with DNA strands assuming a number of distinct backbone geometries, which depend on both DNA sequence and nanotube diameter. In contrast, the nucleotide bases are found to align parallel to the nanotube surface with a high degree of orientational order. While the binding appears to be primarily driven by energetically favorable pi-stacking of DNA bases onto the nanotube surface, equilibrium distribution of hybrid conformations is modulated by a complex interplay of forces, including the DNA conformational strain and solvent interactions. As a result, the hybrid free-energy landscapes are found to be rugged, with multiple low-lying minima separated by high barriers, several of which are significantly populated at room temperature. Qualitative differences are observed in free energy profiles of purine- and pyrimidine-based oligonucleotide sequences and are attributed to the difference in self-stacking propensity of the bases.

  13. Solution structure of the octamer motif in immunoglobulin genes via restrained molecular dynamics calculations.

    PubMed

    Weisz, K; Shafer, R H; Egan, W; James, T L

    1994-01-11

    The solution structure of the DNA decamer d(CATTTGCATC)-d(GATGCAAATG), comprising the octamer motif of immunoglobulin genes, is determined by restrained molecular dynamics (rMD) simulations. The restraint data set includes interproton distances and torsion angles for the deoxyribose sugar ring which were previously obtained by a complete relaxation matrix analysis of the two-dimensional nuclear Overhauser enhancement (2D NOE) intensities and by the quantitative simulation of cross-peaks in double-quantum-filtered correlated (2QF-COSY) spectra. The influence of torsion angles and the number of experimental distance restraints on the structural refinement has been systematically examined. Omitting part of the experimental NOE-derived distances results in reduced restraint violations and lower R factors but impairs structural convergence in the rMD refinement. Eight separate restrained molecular dynamics simulations were carried out for 20 ps each, starting from either energy-minimized A- or B-DNA. Mutual atomic root-mean-square (rms) differences among the refined structures are well below 1 A and comparable to the rms fluctuations of the atoms about their average position, indicating convergence to essentially identical structures. The average refined structure was subjected to an additional 100 ps of rMD simulations and analyzed in terms of average torsion angles and helical parameters. The B-type duplex exhibits clear sequence-dependent variations in its geometry with a narrow minor groove at the T3.A3 tract and a large positive roll at the subsequent TG.CA step. This is accompanied by a noticeable bend of the global helix axis into the major groove. There is also evidence of significant flexibility of the sugar-phosphate backbone with rapid interconversion among different conformers.

  14. Variability of Optical Counterparts to X-ray Selected Sources in the Galactic Bulge Survey

    NASA Astrophysics Data System (ADS)

    Johnson, Christopher; Hynes, Robert I.; Jonker, Peter; Torres, Manuel; Maccarone, Thomas J.; Britt, Christopher; Steeghs, Danny; Galactic Bulge Survey Collaboration

    2016-01-01

    The Galactic Bulge Survey (GBS) is a wide-field, multi-wavelength survey of new X-ray sources in the Galactic Bulge detected with the Chandra X-ray Observatory. The goals of the GBS are to test binary population models by uncovering quiescent Low-Mass X-Ray Binaries (LMXB), and to identify suitable systems for follow-up mass determination using multi-wavelength observations. This follow-up is essential to better determine black hole and neutron star mass distributions. We present preliminary results from the southernmost portion of the GBS positioned 1.5-2.0 degrees below the Galactic Center which contains 424 unique X-ray sources. The optical photometry presented here were acquired using the DECam imager and the previous Mosaic-II imager on the 4m Blanco telescope at Cerro-Tololo Inter-American Observatory (CTIO). We combine photometry with optical spectroscopy from several different telescopes to help characterize the detected X-ray sources. To accomplish this goal, we analyze the light curve morphology and the spectroscopic features of the optical counterparts to classify these binary systems. I will describe the technique for determining the correct optical counterpart within the error circle using image subtraction and report on the statistics of the sample. I will then summarize the candidate LMXBs we have identified so far and highlight other interesting sources. This work was supported by the National Science Foundation under Grant No. AST-0908789 and by NASA through Chandra Award Number AR3-14002X issued by the Chandra X-ray Observatory Center, which is operated by the Smithsonian Astrophysical Observatory for and on behalf of the National Aeronautics Space Administration under contract NAS8-03060. We also acknowledge support from a Graduate Student Research Award administered by the Louisiana Space Grant Consortium (LaSPACE).

  15. Structures of MART-126/27-35Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

    SciTech Connect

    Borbulevych, Oleg Y; Insaidoo, Francis K; Baxter, Tiffany K; Powell, Jr., Daniel J.; Johnson, Laura A; Restifo, Nicholas P; Baker, Brian M

    2008-09-17

    Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26-35 decamer, although only the 27-35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26-35 and 27-35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1{sub 26/27-35}-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27-35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.

  16. Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker--an endangered medicinal herb.

    PubMed

    Lattoo, S K; Bamotra, S; Sapru Dhar, R; Khan, S; Dhar, A K

    2006-06-01

    An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4 x 10(-6) M Kn and 2 x 10(-6) MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1 x 10(-5) M Kinetin (Kn) and 5 x 10(-6) M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm +/- 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5 x 10(-6) M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb.

  17. Structure of a Protozoan Virus from the Human Genitourinary Parasite Trichomonas vaginalis

    PubMed Central

    Parent, Kristin N.; Takagi, Yuko; Cardone, Giovanni; Olson, Norman H.; Ericsson, Maria; Yang, May; Lee, Yujin; Asara, John M.; Fichorova, Raina N.; Baker, Timothy S.; Nibert, Max L.

    2013-01-01

    ABSTRACT The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves −2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date. PMID:23549915

  18. A major Sm epitope anchored to sequential oligopeptide carriers is a suitable antigenic substrate to detect anti-Sm antibodies.

    PubMed

    Petrovas, C J; Vlachoyiannopoulos, P G; Tzioufas, A G; Alexopoulos, C; Tsikaris, V; Sakarellos-Daitsiotis, M; Sakarellos, C; Moutsopoulos, H M

    1998-11-01

    A sensitive, highly reproducible, solid-phase enzyme immunoassay (ELISA), was developed in order to investigate whether the synthetic heptapeptide PPGMRPP-a major epitope of the Sm autoantigen-anchored in five copies to a sequential oligopeptide carrier (SOC), [(PPGMRPP)5-SOC5] is a suitable antigenic substrate to identify anti-Sm/antibodies. Sera with different autoantibody specificities [45 anti-Sm, 40 anti-U1RNP, 40 anti-Ro (SSA)/La(SSB) positive, 21 Antinuclear antibody positive, but negative for antibodies to extractable nuclear antigens (ANA + /ENA - ) and 75 normal human sera, ANA negative] and 75 sera from patients with rheumatoid arthritis (RA) were tested for anti-(PPGMRPP)5-(SOC)5 reactivity in order to evaluate the specificity and sensitivity of the method to detect anti-Sm antibodies. RNA immunoprecipitation assays for the detection of anti-Sm and anti-U1RNP antibodies and counter immunoelectrophoresis (CIE) for the detection of anti-Ro(SSA) and anti-La(SSB) antibodies were used as reference techniques. The sensitivity of the method was 98% and the specificity was 68% for the determination of anti-Sm antibodies, while for the determination of anti-Sm and/or anti-U1RNP reactivity (antibodies to snRNPs) the corresponding values were 82% and 86%, respectively. In a comparison of the above assay with an ELISA, using Sm/U1RNP purified complex as immobilized antigen it was shown that the sensitivity of the anti-Sm/U1RNP ELISA in detecting anti-snRNPs was 74%; in addition sera with anti-Sm antibodies gave higher binding in the anti-(PPGMRPP)5-(SOC)5 ELISA compared with anti-Sm/U1RNP ELISA. Intra- and inter-assay precision was measured on four sera with reactivities extending into a wide range of absorbance values showed that the intra-assay coefficient of variation (CV%) ranged from 2.7 to 6 and the inter-assay CV% ranged from 9 to 14.5. These results indicate that the PPGMRPP peptide anchored to a pentameric SOC as a carrier is a suitable antigen for

  19. Genome-wide identification and characterization of WRKY gene family in Salix suchowensis.

    PubMed

    Bi, Changwei; Xu, Yiqing; Ye, Qiaolin; Yin, Tongming; Ye, Ning

    2016-01-01

    WRKY proteins are the zinc finger transcription factors that were first identified in plants. They can specifically interact with the W-box, which can be found in the promoter region of a large number of plant target genes, to regulate the expressions of downstream target genes. They also participate in diverse physiological and growing processes in plants. Prior to this study, a plenty of WRKY genes have been identified and characterized in herbaceous species, but there is no large-scale study of WRKY genes in willow. With the whole genome sequencing of Salix suchowensis, we have the opportunity to conduct the genome-wide research for willow WRKY gene family. In this study, we identified 85 WRKY genes in the willow genome and renamed them from SsWRKY1 to SsWRKY85 on the basis of their specific distributions on chromosomes. Due to their diverse structural features, the 85 willow WRKY genes could be further classified into three main groups (group I-III), with five subgroups (IIa-IIe) in group II. With the multiple sequence alignment and the manual search, we found three variations of the WRKYGQK heptapeptide: WRKYGRK, WKKYGQK and WRKYGKK, and four variations of the normal zinc finger motif, which might execute some new biological functions. In addition, the SsWRKY genes from the same subgroup share the similar exon-intron structures and conserved motif domains. Further studies of SsWRKY genes revealed that segmental duplication events (SDs) played a more prominent role in the expansion of SsWRKY genes. Distinct expression profiles of SsWRKY genes with RNA sequencing data revealed that diverse expression patterns among five tissues, including tender roots, young leaves, vegetative buds, non-lignified stems and barks. With the analyses of WRKY gene family in willow, it is not only beneficial to complete the functional and annotation information of WRKY genes family in woody plants, but also provide important references to investigate the expansion and evolution of

  20. An osteopontin-NADPH oxidase signaling cascade promotes pro-matrix metalloproteinase 9 activation in aortic mesenchymal cells.

    PubMed

    Lai, Chung-Fang; Seshadri, Venkat; Huang, Kane; Shao, Jian-Su; Cai, Jun; Vattikuti, Radhika; Schumacher, Arwyn; Loewy, Arleen P; Denhardt, David T; Rittling, Susan R; Towler, Dwight A

    2006-06-23

    Osteopontin (OPN) is a cytokine upregulated in diabetic vascular disease. To better understand its role in vascular remodeling, we assessed how OPN controls metalloproteinase (MMP) activation in aortic adventitial myofibroblasts (AMFs) and A7r5 vascular smooth muscle cells (VSMCs). By zymography, OPN and tumor necrosis factor (TNF)-alpha preferentially upregulate pro-matrix metalloproteinase 9 (pro-MMP9) activity. TNF-alpha upregulated pro-MMP9 in AMFs isolated from wild-type (OPN(+/+)) mice, but pro-MMP9 induction was abrogated in AMFs from OPN(-/-) mice. OPN treatment of VSMCs enhanced pro-MMP9 activity, and TNF-alpha induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F(2) alpha-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-alpha, 8-IsoP preferentially activated pro-MMP9. Superoxide, 8-IsoP, and NADPH oxidase 2 (Nox2) subunits were reduced in OPN(-/-) AMFs. Treatment of A7r5 VSMCs with OPN upregulated NADPH oxidase subunit accumulation. OPN structure/function studies mapped these activities to the SVVYGLR heptapeptide motif in the thrombin-liberated human OPN N-terminal domain (SLAYGLR in mouse OPN). Treatment of aortic VSMCs with SVVYGLR upregulated pro-MMP9 activity and restored TNF-alpha activation of pro-MMP9 in OPN(-/-) AMFs. Injection of OPN-deficient OPN(+/-) mice with SVVYGLR peptide upregulated pro-MMP9 activity, 8-IsoP levels, and Nox2 protein levels in aorta and increased panmural superoxide production (dihydroethidium staining). At equivalent hyperglycemia and dyslipidemia, 8-IsoP levels and aortic pro-MMP9 were reduced with complete OPN deficiency in a model of diet-induced diabetes, achieved by comparing OPN(-/-)/LDLR(-/-) versus OPN(+/-)/LDLR(-/-) siblings. Thus, OPN provides a paracrine signal that augments vascular pro-MMP9 activity, mediated in part via superoxide generation and oxylipid

  1. Where Does the Electron Go? Stable and Metastable Peptide Cation Radicals Formed by Electron Transfer

    NASA Astrophysics Data System (ADS)

    Pepin, Robert; Layton, Erik D.; Liu, Yang; Afonso, Carlos; Tureček, František

    2016-10-01

    Electron transfer to doubly and triply charged heptapeptide ions containing polar residues Arg, Lys, and Asp in combination with nonpolar Gly, Ala, and Pro or Leu generates stable and metastable charge-reduced ions, (M + 2H)+●, in addition to standard electron-transfer dissociation (ETD) fragment ions. The metastable (M + 2H)+● ions spontaneously dissociate upon resonant ejection from the linear ion trap, giving irregularly shaped peaks with offset m/z values. The fractions of stable and metastable (M + 2H)+● ions and their mass shifts depend on the presence of Pro-4 and Leu-4 residues in the peptides, with the Pro-4 sequences giving larger fractions of the stable ions while showing smaller mass shifts for the metastables. Conversion of the Asp and C-terminal carboxyl groups to methyl esters further lowers the charge-reduced ion stability. Collisional activation and photodissociation at 355 nm of mass-selected (M + 2H)+● results in different dissociations that give sequence specific MS3 spectra. With a single exception of charge-reduced (LKGLADR + 2H)+●, the MS3 spectra do not produce ETD sequence fragments of the c and z type. Hence, these (M + 2H)+● ions are covalent radicals, not ion-molecule complexes, undergoing dramatically different dissociations in the ground and excited electronic states. The increased stability of the Pro-4 containing (M + 2H)+● ions is attributed to radicals formed by opening of the Pro ring and undergoing further stabilization by hydrogen atom migrations. UV-VIS photodissociation action spectroscopy and time-dependent density functional theory calculations are used in a case in point study of the stable (LKGPADR + 2H)+● ion produced by ETD. In contrast to singly-reduced peptide ions, doubly reduced (M + 3H)+ ions are stable only when formed from the Pro-4 precursors and show all characteristics of even electron ions regarding no photon absorption at 355 nm or ion-molecule reactions, and exhibiting proton driven collision

  2. The presence of microcystins and other cyanobacterial bioactive peptides in aquatic fauna collected from Greek freshwaters.

    PubMed

    Gkelis, S; Lanaras, T; Sivonen, K

    2006-06-10

    Toxic bloom-forming cyanobacteria can cause animal death and adversely affect human health. Blooms may contain microcystins (MCs), cyanobacterial heptapeptide hepatotoxins and other peptides such as anabaenopeptins and anabaenopeptilides. MCs have been shown to occur in various aquatic organisms including mussels, water snails, crustaceans and fish. Muscle and viscera samples from eight species of fish (Acipenser gueldenstaedtii, Carassius auratus, Carassius gibelio, Cyprinus carpio, Perca fluviatilis, Rutilus rubilio, Silurus aristotelis and Silurus glanis), a frog (Rana eperotica), a mussel (Anodonta sp.) and a water snail (Viviparus contectus) were analyzed by high-performance liquid chromatography (HPLC), protein phosphatase 1 (PP1) inhibition assay (PP1IA) and ELISA. MC(s) was detected in all fish, frog, mussel and water snail samples tested by PP1IA and ELISA, including the frog R. eperotica and the freshwater snail V. contectus, in which the occurrence of MCs was not previously known. MC concentration ranged from 20 to 1500 ng g(-1)dw and from 25 to 5400 ng g(-1)dw in muscle and visceral tissue of fishes and frogs, respectively. In mussel and water snail tissue MC concentration ranged from 1650 to 3495 ng g(-1)dw. HPLC analysis revealed peaks having the same UV spectrum as anabaenopeptin- or anabaenopeptilide-like compounds, not previously known to occur in aquatic fauna tissue. The concentrations of the compounds detected ranged from 1.5 to 230 microg g(-1)dw. Comparison of the PP1IA and ELISA showed that values obtained with PP1IA where higher than those obtained with ELISA. Anabaenopeptins and/or anabaenopeptilides occurring in faunal tissue may account for the higher PP1IA values as we found that PP1 activity was inhibited by the purified anabaenopeptins A (45-60% inhibition) and B (5-75% inhibition). Purified anabaenopeptilides 90A and 90B exhibited weaker PP1 inhibition activity (5-35 and 5-23% inhibition, respectively). This is the first report of MC

  3. Genome-wide identification and characterization of WRKY gene family in Salix suchowensis

    PubMed Central

    Ye, Qiaolin; Yin, Tongming

    2016-01-01

    WRKY proteins are the zinc finger transcription factors that were first identified in plants. They can specifically interact with the W-box, which can be found in the promoter region of a large number of plant target genes, to regulate the expressions of downstream target genes. They also participate in diverse physiological and growing processes in plants. Prior to this study, a plenty of WRKY genes have been identified and characterized in herbaceous species, but there is no large-scale study of WRKY genes in willow. With the whole genome sequencing of Salix suchowensis, we have the opportunity to conduct the genome-wide research for willow WRKY gene family. In this study, we identified 85 WRKY genes in the willow genome and renamed them from SsWRKY1 to SsWRKY85 on the basis of their specific distributions on chromosomes. Due to their diverse structural features, the 85 willow WRKY genes could be further classified into three main groups (group I–III), with five subgroups (IIa–IIe) in group II. With the multiple sequence alignment and the manual search, we found three variations of the WRKYGQK heptapeptide: WRKYGRK, WKKYGQK and WRKYGKK, and four variations of the normal zinc finger motif, which might execute some new biological functions. In addition, the SsWRKY genes from the same subgroup share the similar exon–intron structures and conserved motif domains. Further studies of SsWRKY genes revealed that segmental duplication events (SDs) played a more prominent role in the expansion of SsWRKY genes. Distinct expression profiles of SsWRKY genes with RNA sequencing data revealed that diverse expression patterns among five tissues, including tender roots, young leaves, vegetative buds, non-lignified stems and barks. With the analyses of WRKY gene family in willow, it is not only beneficial to complete the functional and annotation information of WRKY genes family in woody plants, but also provide important references to investigate the expansion and evolution

  4. Testing protozoacidal activity of ligand-lytic peptides against termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into termite hindgut).

    PubMed

    Husseneder, Claudia; Sethi, Amit; Foil, Lane; Delatte, Jennifer

    2010-01-01

    We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004). In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic

  5. Environmental conditions associating microcystins production to Microcystis aeruginosa in a reservoir of Thailand.

    PubMed

    Wang, Xiaofeng; Parkpian, Preeda; Fujimoto, Naoshi; Ruchirawat, Khunying Mathuros; DeLaune, R D; Jugsujinda, A

    2002-08-01

    Three heptapeptide toxins, microcystin-RR, microcystin-RY and microcystin-LR, which can cause health problems in animals and humans were monitored in Bang Phra Reservoir, Thailand using reversed-phase high performance liquid chromatography. The concentrations of the three toxins in the reservoir varied greatly depending on location and time water samples were collected. Water quality parameters such as light intensity, temperature, pH, dissolved oxygen, suspended solid, chemical oxygen demand, dissolved organic carbon, total nitrogen, total phosphorus, ammonia, nitrate, phosphate, total dissolved nitrogen, t