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Sample records for aequorea victoria gfp

  1. Development of a photoactivatable fluorescent protein from Aequorea victoria GFP

    NASA Astrophysics Data System (ADS)

    Patterson, George H.; Lippincott-Schwartz, Jennifer

    2004-06-01

    Photoactivation, the rapid conversion of photoactivatable molecules to a fluorescent state by intense irradiation, can be used to mark and monitor selected molecules within cells1. We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) based on a mutation at position 203 that upon intense irradiation with 413 nm light exhibits a stable 60-100 fluorescence increase under 488 nm excitation. The photoactivated form of this mutant named photoactivatable GFP (PA-GFP), is stable under a number of conditions. PA-GFP can be used to analyze protein dynamics in living cells, offering enormous potential for addressing outstanding questions in protein trafficking and turnover, organelle dynamics, and cell lineage patterns.

  2. Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

    PubMed

    Gorokhovatsky, Andrey Yu; Marchenkov, Victor V; Rudenko, Natalia V; Ivashina, Tanya V; Ksenzenko, Vladimir N; Burkhardt, Nils; Semisotnov, Gennady V; Vinokurov, Leonid M; Alakhov, Yuli B

    2004-07-30

    The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

  3. [Expression of green fluorescent protein (GFP) gene of Aequorea victoria [correction of Aequoria victoria] in Lactobacillus plantarum bacterium ].

    PubMed

    Shkoporov, A N; Khokhlova, E V; Efimov, B A; Kafarskaia, L I

    2008-01-01

    Results of development of shuttle expressing plasmid vector Escherichia coli-Lactobacillus which allowed high level expression of heterologous genes in lactobacilli are represented. Vector pTRKH2 which is able to replicate in E. coli and in wide range of Gram-positive bacteria was used as the base. In order to provide high level of cloned gene expression constitutive-active synthetic promoter, site of initiation of translation, and terminator of transcription were introduced in the vector. Functional activity of this vector was confirmed using green fluorescent protein (GFP) gene from Aequoria victoria. Transformation of model strain by gfp gene-carrying plasmid resulted in appearance of typical fluorescent phenotype.

  4. Bacterial plasmid conjugation on semi-solid surfaces monitored with the green fluorescent protein (GFP) from Aequorea victoria as a marker.

    PubMed

    Christensen, B B; Sternberg, C; Molin, S

    1996-01-01

    Horizontal transfer of the TOL plasmid was examined in Pseudomonas putida (Pp) KT2442 micro-colonies on semi-solid agar surfaces. Horizontal gene transfer is usually studied in large populations where all information is based on average estimates of the transfer events in the entire population. We have used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a plasmid marker, in combination with single-cell observations. This provided hitherto unknown details on the distribution of cells active in conjugation. In the present study, donor cells containing the gfp gene expressed from the bacteriophage T7 phi 10 promoter on the TOL plasmid, and recipient cells expressing the corresponding phage RNA polymerase allowed us to monitor the occurrence of ex-conjugants as green fluorescent cells upon illumination with blue light (470-490 nm). Further, the recipients were labeled with the luxAB genes to distinguish micro-colonies of donor cells from recipient cells. We conclude that conjugal plasmid transfer in Pp KT2442 cells on semi-solid surfaces occurs mainly during a short period of time after the initial contact of donors and recipients, indicating that spread of the TOL plasmid is limited in static, but viable cultures.

  5. Deletion mapping of the Aequorea victoria green fluorescent protein.

    PubMed

    Dopf, J; Horiagon, T M

    1996-01-01

    Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2-232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300-500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.

  6. Primary structure of the Aequorea victoria green-fluorescent protein.

    PubMed

    Prasher, D C; Eckenrode, V K; Ward, W W; Prendergast, F G; Cormier, M J

    1992-02-15

    Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.

  7. Crystal structure of the Aequorea victoria green fluorescent protein.

    PubMed

    Ormö, M; Cubitt, A B; Kallio, K; Gross, L A; Tsien, R Y; Remington, S J

    1996-09-06

    The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

  8. Crystal Structure of the Aequorea victoria Green Fluorescent Protein

    NASA Astrophysics Data System (ADS)

    Ormo, Mats; Cubitt, Andrew B.; Kallio, Karen; Gross, Larry A.; Tsien, Roger Y.; Remington, S. James

    1996-09-01

    The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded β barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

  9. Aequorea victoria bioluminescence moves into an exciting new era.

    PubMed

    Kendall, J M; Badminton, M N

    1998-05-01

    Bioluminescence has revolutionized research into many cellular and molecular-biological processes, ranging from intracellular signalling to gene transcription. This article focuses on the chemistry and biotechnological exploitation of the two proteins involved in bioluminescence of the jellyfish Aequorea victoria--aequorin and green fluorescent protein. Engineered recombinant aequorin has led to a novel technological approach to monitoring calcium signals in organelles and subcellular domains. A new generation of intracellular calcium indicators has been produced in which engineered variants of green fluorescent protein are used to probe their ionic environment using intramolecular fluorescence-resonance-energy transfer.

  10. The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore.

    PubMed

    Mishin, Alexander S; Subach, Fedor V; Yampolsky, Ilia V; King, William; Lukyanov, Konstantin A; Verkhusha, Vladislav V

    2008-04-22

    Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags.

  11. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence.

    PubMed

    Li, X; Zhang, G; Ngo, N; Zhao, X; Kain, S R; Huang, C C

    1997-11-07

    The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a widely used marker for gene expression and protein localization studies. Dissection of the structure of the protein would be expected to shed light on its potential applications to other fields such as the detection of protease activity. Using deletion analysis, we have defined the minimal domain in GFP required for fluorescence to amino acids 7-229. This domain starts at the middle of the first small alpha helix at the N terminus of GFP and ends immediately following the last beta sheet. Studies of the amino acids at both termini of the minimal domain revealed that positions 6 and 7 at the N terminus are Glu-specific. Change of the Glu residues to other amino acids results in reduction of GFP fluorescence. Position 229 at the C terminus of GFP, however, is nonspecific: the Ile can be replaced with other amino acids with no measurable loss of fluorescence. A total of only 15 terminal amino acids can be deleted from GFP without disrupting fluorescence, consistent with findings of a previous study of GFP crystal structure (Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., Remington, S. J. (1996) Science 273, 1392-1395 and Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nat. Biotechnol. 14, 1246-1251) that a tightly packed structure exists in the protein. We also generated internal deletions within the loop regions of GFP according to its crystal structure and found that all such deletions eliminated GFP fluorescence.

  12. Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis.

    PubMed

    Deo, S K; Daunert, S

    2001-02-01

    Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.

  13. Functional expression of the Aequorea victoria green fluorescent protein in insect cells using the baculovirus expression system.

    PubMed

    Reiländer, H; Haase, W; Maul, G

    1996-02-06

    A DNA fragment encoding the green fluorescent protein (GFP) was isolated via PCR from a jellyfish Aequorea victoria cDNA, cloned and sequenced. Subsequently, a recombinant baculovirus bearing the coding region of the GFP under the transcriptional control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin gene promoter was constructed and isolated. High-level expression of GFP could be easily monitored in Spodoptera frugiperda (Sf9) insect cells after infection with recombinant baculovirus, due to the intrinsic fluorescence (lambda(max) = 508 nm) of the recombinant protein after excitation with blue light (lambda(max) = 400 nm). The functional recombinant GFP displayed an apparent molecular mass of approximately 43 kDa and the fluorescence emission spectrum of the recombinant protein was virtually identical to that of the native green fluorescent protein.

  14. The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos.

    PubMed

    Amsterdam, A; Lin, S; Hopkins, N

    1995-09-01

    The green fluorescent protein (GFP) from the cnidarian Aequorea victoria is capable of producing fluorescence without an exogenously added substrate. Here we demonstrate that a cDNA for GFP driven by a Xenopus elongation factor 1 alpha enhancer-promoter can confer fluorescence upon live zebrafish embryos, either as an injected plasmid or as a transgene after passage through the germline. When injected into zebrafish embryos at the one-cell stage, this construct starts to express detectable GFP after about 4 hr of development at 28 degrees C, about 1 hr after the midblastula transition. Fluorescence can be observed in cells of many tissue types in the embryo for at least 3 weeks after injection. We used three different expression constructs, each employing a modified ef1 alpha enhancer-promoter, to generate 12 transgenic lines. Eight of the 12 lines, including 5 of 5 derived from one construct with an intron, express detectable fluorescence in the F1 and, where tested, in the F2 generation. Most expressing lines showed very similar expression patterns. Generally, fluorescence is not seen in the transgenic embryos before 20 hr postfertilization, at which point it appears uniformly throughout the embryo. Fluorescence is most visible between 24-36 hr, and it becomes less visible after this, except that in many lines strong fluorescence remains visible in the eye for at least 5 days. A single inherited copy of the transgene is sufficient to produce detectable fluorescence in hemizygous F1 and F2 embryos.

  15. Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code.

    PubMed

    Budisa, Nediljko; Pal, Prajna Paramita; Alefelder, Stefan; Birle, Petra; Krywcun, Tatjana; Rubini, Marina; Wenger, Waltraud; Bae, Jae Hyun; Steiner, Thomas

    2004-02-01

    The expanded genetic code in combination with site-directed mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange ('atomic mutation') and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphur-aromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.

  16. Sound scattering by the gelatinous zooplankters Aequorea victoria and Pleurobrachia bachei

    NASA Astrophysics Data System (ADS)

    Monger, B. C.; Chinniah-Chandy, S.; Meir, E.; Billings, S.; Greene, C. H.; Wiebe, P. H.

    Target strengths were determined for individual Aequorea victoria and Pleurobrachia bachei using separate 200, 420 and 1,000 kHz transducers in an enclosure deployed off the dock of the Friday Harbor Laboratories in northern Puget Sound, Washington, USA. The target strength for Aequorea, when averaged over all animal sizes and all three trnasducer frequencies, was -72.6 dB. The target strength for Pleurobrachia was -75.9 dB when similarly averaged. Average reduced target strengths for Aequorea and Pleurobrachia were -43.3 and -33.1 dB, respectively. Differences in the sound scattering contrasts for Pleurobrachia and Aequorea are proposed to explain the larger reduced target strength of Pleurobrachia, relative to that of Aequorea. A new sound scattering model was developed to predict the backscattering cross-section of Aequorea. The model takes into account expected variations in target strength due to changes in shape of the jellyfish as it propels itself through the water. The scattering model was tuned specifically for Aequorea in the present study, but has a general formulation that is widely applicable to any medusa-shaped gelatinous zooplankter.

  17. Detection of Aequorea victoria green fluorescent protein by capillary electrophoresis laser induced fluorescence detection.

    PubMed

    Craig, D B; Wong, J C; Dovichi, N J

    1997-01-01

    Aequorea victoria green fluorescent protein was assayed by capillary electrophoresis using post-capillary laser-induced fluorescence detection in a sheath flow cuvette. The limit of detection was 3.0 x 10(-12) M protein in an injection volume of 17 nL, corresponding to a mass of 3100 molecules.

  18. Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination.

    PubMed

    Pal, Prajna P; Bae, Jae Hyun; Azim, M Kamran; Hess, Petra; Friedrich, Rainer; Huber, Robert; Moroder, Luis; Budisa, Nediljko

    2005-03-15

    Global replacements of tyrosine by 2- and 3-fluorotyrosine in "enhanced green" and "enhanced yellow" mutants of Aequorea victoria green fluorescent proteins (avGFPs) provided protein variants with novel biophysical properties. While crystallographic and modeled structures of these proteins are indistinguishable from those of their native counterparts (i.e., they are perfectly isomorphous), there are considerable differences in their spectroscopic properties. The fluorine being an integral part of the avGFP chromophore induces changes in the titration curves, variations in the intensity of the absorbance and fluorescence, and spectral shifts in the emission maxima. Furthermore, targeted fluorination in close proximity to the fluorinated chromophore yielded additional variants with considerably enhanced spectral changes. These unique spectral properties are intrinsic features of the fluorinated avGFPs, in the context of the rigid chromophore-microenvironment interactions. The availability of the isomorpohous crystal structures of fluorinated avGFPs allowed mapping of novel, unusual interaction distances created by the presence of fluorine atoms. In addition, fluorine atoms in the ortho position of the chromophore tyrosyl moiety exhibit a single conformation, while in the meta position two conformer states were observed in the crystalline state. Such global replacements in chromophores of avGFPs and similar proteins result in "atomic mutations" (i.e., H --> F replacements) in the structures, offering unprecedented opportunities to understand and manipulate the relationships between protein structure and spectroscopic properties.

  19. Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria.

    PubMed

    Hannick, L I; Prasher, D C; Schultz, L W; Deschamps, J R; Ward, K B

    1993-01-01

    Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) A. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis.

  20. Control of swimming in the hydrozoan jellyfish Aequorea victoria: subumbrellar organization and local inhibition.

    PubMed

    Satterlie, Richard A

    2008-11-01

    The subumbrella of the hydrozoan jellyfish Aequorea victoria (previously classified as Aequorea aequorea) is divided by numerous radial canals and attached gonads, so the subumbrellar musculature is partitioned into subumbrellar segments. The ectoderm of each segment includes two types of muscle: smooth muscle with a radial orientation, used for local (feeding and righting) and widespread (protective) radial responses, and striated muscle with a circular orientation which produces swim contractions. Two subumbrellar nerve nets were found, one of which stained with a commercial antibody produced against the bioactive peptide FMRFamide. Circular muscle cells produce a single, long-duration action potential with each swim, triggered by a single junctional potential. In addition, the circular cells are electrically coupled so full contractions require both electrotonic depolarization from adjacent cells and synaptic input from a subumbrellar nerve net. The radial cells, which form a layer superficial to the circular cells, are also activated by a subumbrellar nerve net, and produce short-duration action potentials. The radial muscle cells are electrically coupled to one another. No coupling exists between the two muscle layers. Spread of excitation between adjacent segments is decremental, and nerve net-activated junctional potentials disappear during local inhibition of swimming (such as with a radial response). Variable swim contractions are controlled by a combination of synaptic input from the motor network of the inner nerve ring, synaptic input from a subumbrellar nerve net, and electrotonic depolarization from adjacent, active muscle cells.

  1. Flow structures and fluid transport for the hydromedusae Sarsia tubulosa and Aequorea victoria.

    PubMed

    Lipinski, Doug; Mohseni, Kamran

    2009-08-01

    The flow structures produced by the hydromedusae Sarsia tubulosa and Aequorea victoria are examined using direct numerical simulation and Lagrangian coherent structures (LCS). Body motion of each hydromedusa is digitized and input to a CFD program. Sarsia tubulosa uses a jetting type of propulsion, emitting a single, strong, fast-moving vortex ring during each swimming cycle while a secondary vortex of opposite rotation remains trapped within the subumbrellar region. The ejected vortex is highly energetic and moves away from the hydromedusa very rapidly. Conversely, A. victoria, a paddling type hydromedusa, is found to draw fluid from the upper bell surface and eject this fluid in pairs of counter-rotating, slow-moving vortices near the bell margins. Unlike S. tubulosa, both vortices are ejected during the swimming cycle of A. victoria and linger in the tentacle region. In fact, we find that A. victoria and S. tubulosa swim with Strouhal numbers of 1.1 and 0.1, respectively. This means that vortices produced by A. victoria remain in the tentacle region roughly 10 times as long as those produced by S. tubulosa, which presents an excellent feeding opportunity during swimming for A. victoria. Finally, we examine the pressure on the interior bell surface of both hydromedusae and the velocity profile in the wake. We find that S. tubulosa produces very uniform pressure on the interior of the bell as well as a very uniform jet velocity across the velar opening. This type of swimming can be well approximated by a slug model, but A. victoria creates more complicated pressure and velocity profiles. We are also able to estimate the power output of S. tubulosa and find good agreement with other hydromedusan power outputs. All results are based on numerical simulations of the swimming jellyfish.

  2. Single amino acid replacement makes Aequorea victoria fluorescent proteins reversibly photoswitchable.

    PubMed

    Bizzarri, Ranieri; Serresi, Michela; Cardarelli, Francesco; Abbruzzetti, Stefania; Campanini, Barbara; Viappiani, Cristiano; Beltram, Fabio

    2010-01-13

    Reversibly photoswitchable (i.e., photochromic) fluorescent proteins open the way to a number of advanced bioimaging techniques applicable to living-cell studies such as sequential photolabeling of distinct cellular regions, innovative FRET schemes, or nanoscopy. Owing to the relevance of fluorescent proteins from Aequorea victoria (AFPs) for cell biology, a photochromic "toolbox" constituted by several AFPs is highly desirable. Here we introduce four new photochromic AFPs whose reversible photoswitching occurs between the native bright and a dark state at low illumination power, on account of a very efficient cis-trans photoisomerization. Most remarkably, the optical bistability of these AFPs derives from the single E222Q mutation in the primary sequence. Apparently, the E222Q substitution can restore the intrinsic photochromic behavior of the isolated chromophore. The significance of these mutants for high-resolution in vivo cell imaging is shown by means of photochromic FRET experiments.

  3. Identifying and modeling motion primitives for the hydromedusae Sarsia tubulosa and Aequorea victoria.

    PubMed

    Sledge, Isaac; Krieg, Michael; Lipinski, Doug; Mohseni, Kamran

    2015-10-23

    The movements of organisms can be thought of as aggregations of motion primitives: motion segments containing one or more significant actions. Here, we present a means to identify and characterize motion primitives from recorded movement data. We address these problems by assuming that the motion sequences can be characterized as a series of dynamical-system-based pattern generators. By adopting a nonparametric, Bayesian formalism for learning and simplifying these pattern generators, we arrive at a purely data-driven model to automatically identify breakpoints in the movement sequences. We apply this model to swimming sequences from two hydromedusa. The first hydromedusa is the prolate Sarsia tubulosa, for which we obtain five motion primitives that correspond to bell cavity pressurization, jet formation, jetting, cavity fluid refill, and coasting. The second hydromedusa is the oblate Aequorea victoria, for which we obtain five motion primitives that correspond to bell compression, vortex separation, cavity fluid refill, vortex formation, and coasting. Our experimental results indicate that the breakpoints between primitives are correlated with transitions in the bell geometry, vortex formation and shedding, and changes in derived dynamical quantities. These dynamics quantities include terms like pressure, power, drag, and thrust. Such findings suggest that dynamics information is inherently present in the observed motions.

  4. Detection of protein-protein interactions using Aequorea victoria bioluminescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Vinokurov, Leonid M.; Gorokhovatsky, Andrey Y.; Rudenko, Natalia V.; Marchenkov, Victor V.; Skosyrev, Vitaly S.; Arzhanov, Maxim A.; Zakharov, Mikhail V.; Burkhardt, Nils; Semisotnov, Gennady V.; Alakhov, Yuli B.

    2003-07-01

    Bioluminescence resonance energy transfer (BRET) is a naturally occurring phenomenon taking place in some marine coelenterates. Emission of light in these organisms involves the energy transfer between chromophores of donor luciferase and acceptor fluorescent protein. Due to the strict dependence of BRET efficiency on the inter-chromophore distance, the phenomenon has been applied to study protein-protein interactions by fusing interacting partners with either donor or acceptor proteins. Here we describe a BRET-based homogeneous protein-protein interaction assay exploiting novel donor-acceptor pair formed by photoproteins of jellyfish Aequorea victoria bioluminescent system, aequorin and green fluorescent protein enhanced variant (EGFP). Two known interacting proteins, streptavidin (SAV) and biotin carboxyl carrier protein (BCCP) were fused, respectively, with aequorin and EGFP. The fusions were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-Aequorin and BCCP-EGFP was followed by BRET between aequorin (donor) and EGFP (acceptor) resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-Aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

  5. Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins.

    PubMed

    Joosen, L; Hink, M A; Gadella, T W J; Goedhart, J

    2014-12-01

    Fluorescence lifetime imaging microscopy can be used to study protein-protein interactions by Förster Resonance Energy Transfer or to perform lifetime-based multiplexing. Fixation of samples with cells producing fluorescent fusion proteins is commonly used for preservation of samples and for staining with membrane impermeable reagents such as antibodies. However, the effect of fixation methods and mounting media on fluorescence lifetime is poorly documented so far. Here, we demonstrate that fixation by formaldehyde or methanol itself does not affect the lifetime of fluorescent proteins produced in cells but that several widely used mounting media decrease the fluorescence lifetime by up to 20%. It is shown that fixed cells producing Aequorea victoria derived fluorescent proteins mounted in Tris buffer have fluorescence lifetimes indistinguishable from values measured in living cells. Tris buffer also allows accurate Förster Resonance Energy Transfer quantification in fixed cells, as shown with an mTurquoise2-SYFP2 fusion protein. Moreover, identical lifetime contrasts are measured in living and fixed cells mounted in Tris buffer after introducing a single plasmid expressing two lifetime variants of cyan fluorescent proteins, each targeted to different locations in the cell. Our findings will aid the preparation of fixed cells producing fluorescent proteins for reliable measurement of fluorescence lifetimes for Förster Resonance Energy Transfer determination, lifetime based multiplexing and for instrument calibration for standardization purposes.

  6. Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria.

    PubMed

    Freitag, N E; Jacobs, K E

    1999-04-01

    The ActA protein of Listeria monocytogenes is an essential virulence factor and is required for intracellular bacterial motility and cell-to-cell spread. plcB, cotranscribed with actA, encodes a broad-specificity phospholipase C that contributes to lysis of host cell vacuoles and cell-to-cell spread. Construction of a transcriptional fusion between actA-plcB and the green fluorescent protein gene of Aequorea victoria has facilitated the detailed examination of patterns of actA/plcB expression within infected tissue culture cells. actA/plcB expression began approximately 30 min postinfection and was dependent upon entry of L. monocytogenes into the host cytosol. L. monocytogenes Deltahly mutants, which are unable to escape from host cell vacuoles, did not express actA/plcB at detectable levels within infected tissue culture cells; however, complementation of the hly defect allowed entry of the bacteria into the host cytoplasm and subsequent actA/plcB expression. These results emphasize the ability of L. monocytogenes to sense the different host cell compartment environments encountered during the course of infection and to regulate virulence gene expression in response.

  7. Expression of Aequorea green fluorescent protein in plant cells.

    PubMed

    Hu, W; Cheng, C L

    1995-08-07

    The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

  8. The numerical comparison of flow patterns and propulsive performances for the hydromedusae Sarsia tubulosa and Aequorea victoria.

    PubMed

    Sahin, Mehmet; Mohseni, Kamran; Colin, Sean P

    2009-08-01

    The thrust-generating mechanism of a prolate hydromedusa Sarsia tubulosa and an oblate hydromedusa Aequorea victoria was investigated by solving the incompressible Navier-Stokes equations in the swirl-free cylindrical coordinates. The calculations clearly show the vortex dynamics related to the thrust-generating mechanism, which is very important for understanding the underlying propulsion mechanism. The calculations for the prolate jetting hydromedusa S. tubulosa indicate the formation of a single starting vortex ring for each pulse cycle with a relatively high vortex formation number. However, the calculations for the oblate jet-paddling hydromedusa A. victoria indicate shedding of the opposite-signed vortex rings very close to each other and the formation of large induced velocities along the line of interaction as the vortices move away from the hydromedusa in the wake. In addition to this jet propulsion mechanism, the hydromedusa's bell margin acts like a paddle and the highly flexible bell margin deforms in such a way that the low pressure leeward side of the bell margin has a projected area in the direction of motion. This thrust is particularly important during refilling of the subumbrella cavity where the stopping vortex causes significant pressure drag. The swimming performances based on our numerical simulations, such as swimming velocity, thrust, power requirement and efficiency, were computed and support the idea that jet propulsion is very effective for rapid body movement but is energetically costly and less efficient compared with the jet-paddling propulsion mechanism.

  9. Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators.

    PubMed

    Kotera, Ippei; Iwasaki, Takuya; Imamura, Hiromi; Noji, Hiroyuki; Nagai, Takeharu

    2010-02-19

    Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.

  10. Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K.

    PubMed

    von Stetten, David; Noirclerc-Savoye, Marjolaine; Goedhart, Joachim; Gadella, Theodorus W J; Royant, Antoine

    2012-08-01

    The green fluorescent protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan fluorescent protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations.

  11. An arbitrary Lagrangian-Eulerian formulation for the numerical simulation of flow patterns generated by the hydromedusa Aequorea victoria

    NASA Astrophysics Data System (ADS)

    Sahin, Mehmet; Mohseni, Kamran

    2009-07-01

    A new geometrically conservative arbitrary Lagrangian-Eulerian (ALE) formulation is presented for the moving boundary problems in the swirl-free cylindrical coordinates. The governing equations are multiplied with the radial distance and integrated over arbitrary moving Lagrangian-Eulerian quadrilateral elements. Therefore, the continuity and the geometric conservation equations take very simple form similar to those of the Cartesian coordinates. The continuity equation is satisfied exactly within each element and a special attention is given to satisfy the geometric conservation law (GCL) at the discrete level. The equation of motion of a deforming body is solved in addition to the Navier-Stokes equations in a fully-coupled form. The mesh deformation is achieved by solving the linear elasticity equation at each time level while avoiding remeshing in order to enhance numerical robustness. The resulting algebraic linear systems are solved using an ILU(k) preconditioned GMRES method provided by the PETSc library. The present ALE method is validated for the steady and oscillatory flow around a sphere in a cylindrical tube and applied to the investigation of the flow patterns around a free-swimming hydromedusa Aequorea victoria (crystal jellyfish). The calculations for the hydromedusa indicate the shed of the opposite signed vortex rings very close to each other and the formation of large induced velocities along the line of interaction while the ring vortices moving away from the hydromedusa. In addition, the propulsion efficiency of the free-swimming hydromedusa is computed and its value is compared with values from the literature for several other species.

  12. Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis

    PubMed Central

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2009-01-01

    Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 Å resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 Å, α = 90.96, β = 103.41, γ = 101.79°. The Matthews coefficient (V M = 2.10 Å3 Da−1) indicated that the crystal contained two mAG molecules per asymmetric unit. PMID:20054132

  13. Crystallization and preliminary X-ray analysis of a monomeric mutant of Azami-Green (mAG), an Aequorea victoria green fluorescent protein-like green-emitting fluorescent protein from the stony coral Galaxea fascicularis.

    PubMed

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2009-12-01

    Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first monomeric green-emitting fluorescent protein that is not a derivative of Aequorea victoria green fluorescent protein (avGFP). mAG and avGFP are 27% identical in amino-acid sequence. Diffraction-quality crystals of recombinant mAG were obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The mAG crystal diffracted X-rays to 2.20 A resolution on beamline AR-NW12A at the Photon Factory (Tsukuba, Japan). The crystal belonged to space group P1, with unit-cell parameters a = 41.78, b = 51.72, c = 52.89 A, alpha = 90.96, beta = 103.41, gamma = 101.79 degrees. The Matthews coefficient (V(M) = 2.10 A(3) Da(-1)) indicated that the crystal contained two mAG molecules per asymmetric unit.

  14. Aequorea green fluorescent protein analysis by flow cytometry.

    PubMed

    Ropp, J D; Donahue, C J; Wolfgang-Kimball, D; Hooley, J J; Chin, J Y; Hoffman, R A; Cuthbertson, R A; Bauer, K D

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263: 802-805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T-GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm.

  15. Aequorea green fluorescent protein analysis by flow cytometry

    SciTech Connect

    Ropp, J.D.; Cuthbertson, R.A.; Donahue, C.J.; Wolfgang-Kimball, D.

    1995-12-01

    The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.

  16. GFP-like proteins stably accumulate in lysosomes.

    PubMed

    Katayama, Hiroyuki; Yamamoto, Akitsugu; Mizushima, Noboru; Yoshimori, Tamotsu; Miyawaki, Atsushi

    2008-01-01

    Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its GFP variants (Aequorea GFPs), and more recently the novel GFP-like proteins from Anthozoa have greatly advanced our technologies for fluorescently labeling cells, organelles, and proteins. It has been shown, however, that some GFP-like proteins have a tendency to oligomerize and aggregate. Transfection of GFP-like proteins into cultured mammalian cells results in bright punctate structures, which are thought to be cytosolic protein aggregates. In this study, we demonstrate that these structures are not cytosolic aggregates but lysosomes that have accumulated the GFP-like proteins. Our biochemical and immunocytochemical experiments have revealed that certain GFP-like proteins expressed in the cytosol enter lysosomes possibly by an autophagy-related mechanism, but retain their fluorescence because of resistance not only to acidity but also to lysosomal proteases.

  17. Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells.

    PubMed

    Reichel, C; Mathur, J; Eckes, P; Langenkemper, K; Koncz, C; Schell, J; Reiss, B; Maas, C

    1996-06-11

    The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.

  18. Bioluminescence of Aequorea macrodactyla, a common jellyfish species in the East China Sea.

    PubMed

    Xia, Ning-Shao; Luo, Wen-Xin; Zhang, Jun; Xie, Xiao-Yan; Yang, Hai-Jie; Li, Shao-Wei; Chen, Ming; Ng, Mun-Hon

    2002-03-01

    Studies of the bioluminescent mechanisms of jellyfish have been mainly confined to one species, Aequorea victoria. We describe the luminescent system of another species, Aequorea macrodactyla, which is commonly found in the warmer waters on the coastal region of East China Sea. The luminescent system of this species consists of a green fluorescent protein (GFP) and one or more aequorins. The GFP gene is 1042 bp. It encompasses a coding sequence of 717 bp organized as 3 exons, and it is predicted to specify a 27-kDa peptide, which shares 80% amino acid sequence identity with the GFP of A. victoria. The entire coding sequence was cloned into the pTO-T7 expression vector and expressed in Escherichia coli. Compared with GFP of A. victoria, the purified expressed protein exhibited an excitation peak at a higher wavelength of 476 nm and an emission peak at a lower wavelength of 496 nm, with a higher quantum yield of 1.0. The other photoprotein, aequorin, is encoded in a single open reading frame of 585 bp specifying a 23-kDa apoprotein. The gene was cloned in to the same expression vector and expressed in E. coli. The activity of the photoprotein was reconstituted by incubating the expressed apoprotein with coelenterazine f. In the presence of Ca(2+) the reconstituted aequorin exhibits an emission peak at 470 nm. The kinetics of regeneration and the photoactivities of the reconstituted aequorins of the 2 species of jellyfish are similar. Nevertheless, Aequorea macrodactyla is expected to appear brighter and more "blue" than Aequorea victorea because of the differences in the photoactivity of their GFPs.

  19. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.

    PubMed

    Cody, C W; Prasher, D C; Westler, W M; Prendergast, F G; Ward, W W

    1993-02-09

    The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.

  20. A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells

    NASA Astrophysics Data System (ADS)

    Patterson, George H.; Lippincott-Schwartz, Jennifer

    2002-09-01

    We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.

  1. A photoactivatable GFP for selective photolabeling of proteins and cells.

    PubMed

    Patterson, George H; Lippincott-Schwartz, Jennifer

    2002-09-13

    We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.

  2. [Construction and characterization of TetR and GFP fusion protein].

    PubMed

    Zuo, Yan; Yang, Ke-Qian

    2005-01-01

    Tetracycline repressor gene (tetR) from E. coli transposon Tn10 was fused in frame with green fluorescent protein gene (gfp) from jellyfish Aequorea Victoria on an E. coli expression vector and the fusion protein (TR::GFP) was purified. The binding of TR::GFP with tetracycline (tc) was demonstrated by nitrocellulose filter binding assay. TR::GFP also maintained the fluorescence property of GFP. Most significantly, fluorescence emission intensity of TR::GFP increased by 2-fold in the presence of tc, from 1.132 to 2.214, while those of GFP and TetR showed little change under similar conditions. The results indicated TR::GFP possesses characteristics of a tetracycline biosensor.

  3. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor.

    PubMed

    Arosio, Daniele; Ricci, Fernanda; Marchetti, Laura; Gualdani, Roberta; Albertazzi, Lorenzo; Beltram, Fabio

    2010-07-01

    Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.

  4. Laser microbeam abalation of GFP-labeled nuclear organelles in a living cell.

    NASA Astrophysics Data System (ADS)

    LaMorte, Vickie J.; Krasieva, Tatiana B.; Evans, Ronald M.; Berns, Michael W.; Tromberg, Bruce J.

    1997-05-01

    Cancer, development, cellular growth and differentiation are governed by gene expression. Recent molecular and cellular advances to visualize and perturb the pathways of transcriptional regulation, nascent RNA processing, and protein trafficking at the single cell level have been developed. More recently, applications utilizing the green fluorescent marker (GFP) from Aequorea victoria have facilitated visualization of these molecular events in a living cell. Specifically, we will describe a novel approach to perturb cellular processes by labeling discrete cellular components of interest with GFP and subsequently altering/ablating them with a laser microbeam.

  5. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants.

    PubMed

    Davis, S J; Vierstra, R D

    1998-03-01

    Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is 'brighter' because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.

  6. Convenient and sensitive evaluation of a superoxide anion-generating reagent methyl viologen by Escherichia coli harboring a soxS'::gfp reporter plasmid.

    PubMed

    Shibuya, Akihito; Tsukagoshi, Norihiko; Ohtsu, Iwao; Dokyu, Noriyuki; Aono, Rikizo

    2004-12-01

    We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria. E. coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen. The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 microM to 10 microM.

  7. Producing a Mammalian GFP Expression Vector Containing Neomycin Resistance Gene.

    PubMed

    Izadi, Manizheh; Abiri, Maryam; Keramatipour, Mohammad

    2009-04-01

    The green fluorescent protein (GFP) was originally isolated from the Jellyfish Aequorea Victoria that fluoresces green when exposed to blue light. GFP protein is composed of 238 amino acids with the molecular mass of 26.9 kD. The GFP gene is frequently used in cellular and molecular biology as a reporter gene. To date, many bacterial, yeast, fungal, plants, fly and mammalian cells, including human, have been created which express GFP. Martin Chalfie, Osamu Shimomura, and Roger Tsien were awarded the 2008 noble prize in chemistry for their discovery and development of GFP. In many studies on mammalian cells, GFP gene is introduced into cells using vector-based systems or a recombinant virus to track the location of a target protein or to study the expression level of the gene of interest, but in these studies there is no selection marker to normalize transfection. According to the importance of neomycin gene as a selection marker in mammalian cells, we aimed to produce a GFP expression vector that contains neomycin gene. GFP gene was separated from pEGFP-N1 vector and was inserted in the back-bone of pCDNA3.1/His/LacZ vector that contained the neomycin gene. The resulted vector contained GFP beside neomycin gene.

  8. Prolongation of GFP-expressed skin graft after intrathymic injection of GFP positive splenocytes in adult rat

    NASA Astrophysics Data System (ADS)

    Hakamata, Yoji; Igarashi, Yuka; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

    GFP is a fluorescent product of the jellyfish Aequorea victoria and has been used for a variety of biological experiments as a reporter molecule. While GFP possesses advantages for the non-invasive imaging of viable cells, GFP-positive cells are still considered potential xeno-antigens. It is difficult to observe the precise fate of transplanted cells/organs in recipients without immunological control. The aim of this study was to determine whether intrathymic injection of GFP to recipients and the depletion of peripheral lymphocytes could lead to donor-specific unresponsiveness to GFP-expressed cell. LEW rats were administered intraperitoneally with 0.2 ml of anti-rat lymphocyte serum (ALS) 1 day prior to intrathymic injection of donor splenocytes or adeno-GFP vector. Donor cells and vector were non-invasively inoculated into the thymus under high frequency ultrasound imaging using an echo-guide. All animals subsequently received a 7 days GFP-expressed skin graft from the same genetic background GFP LEW transgenic rat. Skin graft survival was greater in rats injected with donor splenocytes (23.6+/-9.1) compared with adeno-GFP (13.0+/-3.7) or untreated control rats (9.5+/-1.0). Intrathymic injection of donor antigen into adult rats can induce donor-specific unresponsiveness. Donor cells can be observed for a long-term in recipients with normal immunity using this strategy.

  9. Photoactivation in green to red converting EosFP and other fluorescent proteins from the GFP family

    NASA Astrophysics Data System (ADS)

    Wiedenmann, Jörg; Nienhaus, G. Ulrich

    2006-02-01

    The green fluorescent protein (GFP) from the hydromedusa Aequorea victoria and its derivatives have become indispensable imaging devices in cell biology. In previous years, a wide variety of GFP-like proteins were discovered in non-bioluminescent anthozoa. Some of them displayed exciting new properties, including photoactivated changes of the fluorescence emission intensity and wavelength. Photoactivatable proteins offer a high potential as tools for regional optical marking in live cells and tissues. This review aims to give an overview of photoactivatable marker proteins, focusing on the molecular basis of light-induced green to red photoconversion in EosFP.

  10. Chemical nature of the light emitter of the Aequorea green fluorescent protein.

    PubMed

    Niwa, H; Inouye, S; Hirano, T; Matsuno, T; Kojima, S; Kubota, M; Ohashi, M; Tsuji, F I

    1996-11-26

    The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

  11. Using GFP--ligand fusions to measure receptor-mediated endocytosis in living cells.

    PubMed

    Medina-Kauwe, Lali K; Chen, Xinhua

    2002-01-01

    Recombinant DNA technology has enabled the production of many types of chimeric proteins containing heterologous functional domains that have served a variety of useful capacities for cell biology research. Among proteins gaining wide use as a fusion partner is Aequorea victoria green fluorescent protein (GFP). GFP has been employed by numerous groups as a reporter gene for cell transfection and as an autofluorescent tag by recombinant fusion to foreign sequences. Here we describe the use of GFP as a tag for ligands, and provide examples of how purified recombinant GFP-ligand fusion proteins may be used to detect ligand-receptor interactions, including receptor-mediated endocytosis. Both its utility and limitations are discussed.

  12. The use of GFP to localize Rho GTPases in living cells.

    PubMed

    Michaelson, David; Philips, Mark

    2006-01-01

    The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP-Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP-Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems.

  13. Cell-based assays in practice: cell markers from autofluorescent proteins of the GFP-family.

    PubMed

    Wolff, Michael; Kredel, Simone; Wiedenmann, Jörg; Nienhaus, G Ulrich; Heilker, Ralf

    2008-09-01

    The more recently discovered anthozoan fluorescent proteins (FPs) and the classic Aequorea victoria Green Fluorescent Protein (avGFP) as well as their derivatives have become versatile tools as live cell markers in fluorescence microscopy. In this review, we show the use of these FPs in drug discovery assays. Assay examples are given for the application of FPs in multiplexed imaging, as photosensitizers, as fluorescent timers, as pulse-chase labels and for robotically integrated compound testing. The development of fast microscopic imaging devices has enabled the application of automated fluorescence microscopy combined with image analysis to pharmaceutical high throughput drug discovery assays, generally referred to as High Content Screening (HCS).

  14. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    NASA Technical Reports Server (NTRS)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  15. Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells.

    PubMed

    von Arnim, Albrecht

    2007-02-01

    INTRODUCTIONRecombinant tags (i.e., reporter proteins) offer an excellent alternative to antibodies for determining the subcellular localization of proteins. The most user-friendly tags are the ß-glucuronidase (GUS) reporter enzyme from Escherichia coli and fluorescent proteins derived primarily from the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. GUS is useful primarily as a tag to address nuclear localization, whereas GFP is more versatile. Moreover, GFP is detectable directly in living cells, whereas GUS is only detected indirectly by staining of fixed tissue. This may lead to artifacts or it may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active, and preliminary targeting data can be obtained.

  16. Visualization of GFP-expressing tumors and metastasis in vivo.

    PubMed

    Hoffman, R M

    2001-05-01

    We have developed mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged in fresh tissue, in situ, as well as externally. To achieve this capability, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of human and rodent cancer cell lines that were selected in vitro to stably express GFP in vivo after transplantation to metastatic rodent models. Techniques were also developed for transduction of tumors by GFP in vivo. With this fluorescent tool, we detected and visualized for the first time tumors and metastasis in fresh viable tissue or in situ in host organs down to the single-cell level. GFP tumors on the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone, and other organs can also be visualized externally, transcutaneously by quantitative whole-body fluorescence optical imaging. Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs can be imaged and quantified for rapid antitumor, antimetastatic, and antiangiogenesis drug screening. The GFP-transfected tumor cells enabled a fundamental advance in the visualization of tumor growth and metastasis in real time in vivo.

  17. Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation.

    PubMed

    Morschhäuser, J; Michel, S; Hacker, J

    1998-02-01

    Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicans ACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the noncanonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans.

  18. Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

    PubMed

    Liu, J; Wang, Y; Szalay, A A; Escher, A

    2000-01-01

    We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells.

  19. Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry.

    PubMed

    Barondeau, David P; Kassmann, Carey J; Tainer, John A; Getzoff, Elizabeth D

    2005-02-15

    The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.

  20. Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis.

    PubMed

    Barondeau, David P; Tainer, John A; Getzoff, Elizabeth D

    2006-03-15

    The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1/2 = 39 min-1) upon exposure to air. Isolation of this enolate intermediate will now allow specific probing of the rate-limiting oxidation step for fluorophore biosynthesis in GFP and its red fluorescent protein homologues. Such targeted characterizations may lead to the design of faster maturing proteins with enhanced applications in biotechnology and cell biology. Moreover, our results reveal how the GFP protein environment mimics enzyme systems, by stabilizing an otherwise high energy enolate intermediate to achieve its post-translational modification.

  1. Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506.

    PubMed

    Lowder, M; Unge, A; Maraha, N; Jansson, J K; Swiggett, J; Oliver, J D

    2000-08-01

    The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5 degrees C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30 degrees C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30 degrees C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50 degrees C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because

  2. Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain.

    PubMed

    Sarrocco, Sabrina; Mikkelsen, Lisbeth; Vergara, Mariarosaria; Jensen, Dan Funck; Lübeck, Mette; Vannacci, Giovanni

    2006-02-01

    The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.

  3. Stability of green fluorescent protein using luminescence spectroscopy: is GFP applicable to field analysis of contaminants?

    PubMed

    Smith, C B; Anderson, J E; Fischer, R L; Webb, S R

    2002-01-01

    Green fluorescent protein (GFP) was first isolated in the early 1970s for experimental use from coelenterates or the Pacific jellyfish. Aequorea victoria (Morin and Hastings, 1971). GFP has since become a favored biomarker in the photophysical analysis of molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (Creemers et al., 2000). Here we report using Bacillus subtilis expressing GFP to evaluate the influence of different environmental pH conditions on GFP fluorescence. Emission acquisitions were configured to excite at 471 nm and detect at an emission from 490 to 650 nm at 1-nm increments. Fluorescence intensity was significantly better at pH 7 (4.2 x 105 cps; P-value < 0.01) than at acid or alkaline conditions. GFP is a good biomarker for environments near netural conditions: however, GFP may be unsuitable where soils or waters are below or above pH 7 because of loss in fluorescence intensity. Alternative fluorescent markers and delivery systems must be examined in different environments to optimize responses from bioreporter molecules.

  4. Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

    PubMed

    Kahana, J A; Silver, P A

    2001-05-01

    Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.

  5. Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

    PubMed

    Kahana, J A; Silver, P A

    2001-05-01

    Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has heretofore been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.

  6. Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway.

    PubMed

    Di Sansebastiano, G P; Paris, N; Marc-Martin, S; Neuhaus, J M

    1998-08-01

    The green fluorescent protein (GFP) from Aequorea victoria can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C-terminal endoplasmic reticulum (ER)-retention motif of mGFP5-ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18-24 h) depending on the cell type. Most chloroplast-rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast-poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non-acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.

  7. Evaluation of GFP tag as a screening reporter in directed evolution of a hyperthermophilic beta-glucosidase.

    PubMed

    Lima, André O S; Davis, Diane F; Swiatek, Gavin; McCarthy, James K; Yernool, Dinesh; Pizzirani-Kleiner, Aline A; Eveleigh, Douglas E

    2009-06-01

    By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP's fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

  8. Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region.

    PubMed

    Schneider, Marc; Barozzi, Sara; Testa, Ilaria; Faretta, Mario; Diaspro, Alberto

    2005-08-01

    This report covers the two-photon activation and excitation properties of the PA-GFP, a photoactivatable variant of the Aequorea victoria green fluorescent protein in the spectral region from 720 to 920 nm. It is known from this special form of the molecule that it has an increased level of fluorescence emission when excited at 488 nm after irradiation at lambda approximately 413 nm, under single-photon excitation conditions. Here, we show that upon two-photon irradiation, PA-GFP yields activation in the spectral region from 720 to 840 nm. After photoactivation, the excitation spectrum shifts maintaining the very same emission spectrum of the single-photon case for the native and photoactivated protein. Additionally, when comparing the conventional photoactivation at lambda = 405 nm with a two-photon one, a sharper and better controllable three-dimensional volume of activation is obtained.

  9. The role of green fluorescent protein (GFP) in transgenic plants to reduce gene silencing phenomena.

    PubMed

    El-Shemy, Hany A; Khalafalla, Mutasim M; Ishimoto, Masao

    2009-01-01

    The green fluorescent protein (GFP) of jellyfish (Aequorea victoria) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. However, sGFP(S65T) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(S65T) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(S65T ) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system.

  10. Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP).

    PubMed

    Wiehler, Jens; Jung, Gregor; Seebacher, Christian; Zumbusch, Andreas; Steipe, Boris

    2003-11-07

    The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.

  11. Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures.

    PubMed

    Su, Wei Wen; Liu, Bo; Lu, Wei-Bin; Xu, Ning-Shou; Du, Guo-Cheng; Tan, Jing-Lu

    2005-07-20

    The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein

  12. Structural basis for red-shifted emission of a GFP-like protein from the marine copepod Chiridius poppei.

    PubMed

    Suto, Kyoko; Masuda, Hiromi; Takenaka, Yasuhiro; Tsuji, Frederick I; Mizuno, Hiroshi

    2009-06-01

    The fluorescence excitation and emission maxima of a GFP-like protein from the marine copepod Chiridius poppei (CpYGFP) show a significant red shift (lambda(ex) = 509 nm, lambda(em) = 517 nm) compared with those of GFP from Aequorea victoria (avGFP) and other GFP-like proteins from marine copepods. We performed crystallographic and biochemical studies to understand why this shift occurs in CpYGFP. The structure of CpYGFP showed that the imidazole side chain of His52 is involved in stacking on the phenol moiety of the chromophore. We investigated the potential role of His52 in causing the red-shifted spectral properties by performing mutational analyses of H52T, H52D and H52F. The emission wavelengths of H52T and H52D were blue-shifted and that of H52F was red-shifted relative to the wild type. Comparison of its structure of another copepod GFP (ppluGFP2) having an emission maximum at 502 nm showed that the imidazole ring of His54 (corresponding to His52 in CpYGFP) is flipped out of the stacking position with the chromophore. These findings suggest that pi-pi stacking interaction between His52 and the phenol moiety of the chromophore is the likely cause of the red-shift in light emission.

  13. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  14. The construction of bifunctional fusion proteins consisting of MutS and GFP.

    PubMed

    Stanisławska-Sachadyn, Anna; Sachadyn, Paweł; Ihle, Karolina; Sydorczuk, Cezary; Wiejacha, Katarzyna; Kur, Józef

    2006-01-24

    MutS as a mismatch binding protein is a promising tool for SNP detection. Green fluorescent protein (GFP) is known as an excellent reporter domain. We constructed chimeric proteins consisting of MutS from Thermus thermophilus and GFPuv from Aequorea victoria by cloning the GFPuv gene into the plasmid vectors carrying the mutS gene. The GFPuv domain fused to the N-terminus of MutS (histag-GFP-MutS) exhibited the same level of green fluorescence as free GFPuv. To obtain the fluorescing histag-GFP-MutS protein the expression at 30 degrees C was required, while free GFPuv fluoresces when expressed both at 30 and 37 degrees C. The chimeric protein where the GFPuv domain was fused to the C-terminus of MutS exhibited much weaker green fluorescence (20-25% compared with those of histag-GFP-MutS or free GFPuv). The insertion of (ProGly)5 peptide linker between the MutS and GFP domains resulted in no significant improvement in GFP fluorescence. No shifts in the excitation and emission spectra have been observed for the GFP domain in the fusion proteins. The fusion proteins with GFP at the N- and C-terminus of MutS recognised DNA mismatches similarly like T. thermophilus MutS. The fluorescent proteins recognising DNA mismatches could be useful for SNP scanning or intracellular DNA analysis. The fusion proteins around 125 kDa were efficiently expressed in E. coli and purified in milligram amounts using metal chellate affinity chromatography.

  15. Identification of GFP-like proteins in nonbioluminescent, azooxanthellate anthozoa opens new perspectives for bioprospecting.

    PubMed

    Wiedenmann, Jörg; Ivanchenko, Sergey; Oswald, Franz; Nienhaus, G Ulrich

    2004-01-01

    We screened nonbioluminescent, azooxanthellate cnidaria as potential sources for advanced marker proteins and succeeded in cloning a tetrameric green fluorescent protein (GFP) from the tentacles of Cerianthus membranaceus. The fluorescence of this protein (cmFP512) is characterized by excitation maximum at 503 nm, emission maximum at 512 nm, extinction coefficient of 58,800 M-1 cm-1, quantum yield of 0.66, and fluorescence lifetime of 2.4 ns. The chromophore is formed from the tripeptide Gln-Tyr-Gly. The amino acid sequence of this protein shares 17.8% identical residues with GFP from Aequorea victoria. Weak interactions between the subunits of the tetramer make cmFP512 a promising lead structure for the generation of monomeric variants of fluorescent proteins. Both red fluorescent proteins and nonfluorescent proteins of the GFP family were also purified from tissue homogenates of Adamsia palliata and Calliactis parasitica. The results presented here indicate that a photoprotective function of GFP-like proteins is unlikely in the examined anthozoa species.

  16. Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio).

    PubMed

    Mattingly, C J; McLachlan, J A; Toscano, W A

    2001-08-01

    We developed an inducible in vivo reporter system to examine expression of the aryl hydrocarbon receptor (AhR) during development in zebrafish (Danio rerio). AhR is a ligand-activated transcription factor that mediates the toxic actions of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Induction of cytochrome P4501A1 (CYP1A1) is an early biomarker of AhR activation. A 1905 base pair region of the human CYP1A1 promoter/enhancer region was regulated by AhR in zebrafish liver cells after exposure to TCDD (10 nM) in a transient transfection assay. This regulatory region was fused to the cDNA sequence encoding green fluorescent protein (GFP) of jellyfish (Aequorea victoria). Transgenic zebrafish were generated to express this AhR-regulated GFP construct. Injected fish exposed to TCDD exhibited induction of GFP in the eye, nose, and vertebrae of zebrafish embryos (48 and 72 hr after fertilization) compared to vehicle controls (DMSO), which did not express GFP. To investigate whether AhR-regulated GFP expression correlated with sites of TCDD toxicity, we exposed wild-type zebrafish to DMSO or TCDD and examined them for morphologic abnormalities. By 5 days after fertilization, TCDD-exposed fish exhibited gross dysmorphogenesis in cranio-facial and vertebral development.

  17. Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle.

    PubMed

    Wallace, Lindsay M; Moreo, Andrew; Clark, K Reed; Harper, Scott Q

    2013-04-16

    Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP

  18. Spectroscopic and structural study of proton and halide ion cooperative binding to gfp.

    PubMed

    Arosio, Daniele; Garau, Gianpiero; Ricci, Fernanda; Marchetti, Laura; Bizzarri, Ranieri; Nifosì, Riccardo; Beltram, Fabio

    2007-07-01

    This study reports the influence of halogens on fluorescence properties of the Aequorea victoria Green Fluorescent Protein variant S65T/T203Y (E(2)GFP). Halide binding forms a specific nonfluorescent complex generating a substantial drop of the fluorescence via static quenching. Spectroscopic analysis under different solution conditions reveals high halogen affinity, which is strongly dependent on the pH. This evidences the presence in E(2)GFP of interacting binding sites for halide ions and for protons. Thermodynamic link and cooperative interaction are assessed demonstrating that binding of one halide ion is associated with the binding of one proton in a cooperative fashion with the formation, in the pH range 4.5-10, of a single fully protonated E(2)GFP.halogen complex. To resolve the structural determinants of E(2)GFP sensitivity to halogens, high-resolution crystallographic structures were obtained for the halide-free and I(-), Br(-), and Cl(-) bound E(2)GFP. Remarkably the first high-resolution (1.4 A) crystallographic structure of a chloride-bound GFP is reported. The chloride ion occupies a specific and unique binding pocket in direct contact (3.4 A) with the chromophore imidazolidinone aromatic ring. Unanticipated flexibility, strongly modulated by halide ion interactions, is observed in the region surrounding the chromophore. Furthermore molecular dynamics simulations identified E222 residue (along with the chromophore Y66 residue) being in the protonated state when E(2)GFP.halogen complex is formed. The impact of these results on high-sensitivity biosensor design will be discussed.

  19. T2P-GFP: two-photon photoactivation of PA-GFP in the 720-840 nm spectral region.

    NASA Astrophysics Data System (ADS)

    Testa, Ilaria; Schneider, Marc; Barozzi, Sara; Vicidomini, Giuseppe; Parazzoli, Dario; Faretta, Mario; Diaspro, Alberto

    2006-02-01

    We report about a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm1. The aim in this work was to evaluate the use of two-photon interactions for activation of the molecules2. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The latter objects were used to investigate the ability of different wavelengths to activate the paGFP due to the anticipated more homogeneous density distribution. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation with a single photon at 413 nm and two-photons demonstrates clearly the advantages using non linear processes: much smaller volume in the cell can be activated unlike to a whole cell activation in single photon excitation regime.

  20. Application of GFP technique for cytoskeleton visualization onboard the International Space Station

    NASA Astrophysics Data System (ADS)

    Kordyum, E. L.; Shevchenko, G. V.; Yemets, A. I.; Nyporko, A. I.; Blume, Ya. B.

    2005-03-01

    Cytoskeleton recently attracted wide attention of cell and molecular biologists due to its crucial role in gravity sensing and trunsduction. Most of cytoskeletal research is conducted by the means of immunohistochemical reactions, different modifications of which are beneficial for the ground-based experiments. But for the performance onboard the space vehicles, they represent quite complicated technique which requires time and special skills for astronauts. In addition, immunocytochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for the better understanding of cytoskeletal function. In this connection, we propose a new approach for cytoskeletal visualization onboard the ISS, namely, application of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. The creation of chimerical protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of the cytoskeleton functional status in microgravity.

  1. 3D localized photoactivation of pa-GFP in living cells using two-photon interactions.

    PubMed

    Diaspro, Alberto; Testa, Ilaria; Faretta, Mario; Magrassi, Raffaella; Barozzi, Sara; Parazzoli, Dario; Vicidomini, Giuseppe

    2006-01-01

    We report about two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm. The aim in this work was to evaluate the use of two-photon interactions for confining the molecular switching of pa-GFP in the bright state. Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation and two-photon mode demonstrates clearly the better three- dimensional (3D) confinement and the possibility of selection of cell volumes of interest. This enables molecular trafficking studies at high signal to noise ratio.

  2. Application of GFP technique for cytoskeleton visualization onboard the International Space Station.

    PubMed

    Kordyum, E L; Shevchenko, G V; Yemets, A I; Nyporko, A I; Blume, Ya B

    2005-03-01

    Cytoskeleton recently attracted wide attention of cell and molecular biologists due to its crucial role in gravity sensing and trunsduction. Most of cytoskeletal research is conducted by the means of immunohistochemical reactions, different modifications of which are beneficial for the ground-based experiments. But for the performance onboard the space vehicles, they represent quite complicated technique which requires time and special skills for astronauts. In addition, immunocytochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for the better understanding of cytoskeletal function. In this connection, we propose a new approach for cytoskeletal visualization onboard the ISS, namely, application of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. The creation of chimerical protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of the cytoskeleton functional status in microgravity.

  3. The color of mice: in the light of GFP-variant reporters.

    PubMed

    Hadjantonakis, A K; Nagy, A

    2001-01-01

    The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ, the bacterial beta-galactosidase gene has been the marker of choice for most studies in the mouse. However, over the past 3 years another valuable reporter has emerged, and its attractiveness is reflected by an explosion in its use in mice. Green fluorescent protein (GFP), a novel autofluorescent genetic reporter derived from the bioluminescent jellyfish Aequorea victoria, currently represents a unique alternative to other gene-based reporters in that its visualization is non-invasive and so can be monitored in real-time in vitro or in vivo. It has the added advantage that it can be quantified by, for example, flow cytometry, confocal microscopy, and fluorometric assays. Several mutants of the original wild-type GFP gene that improve thermostability and fluorescence have been engineered. Enhanced GFP is one such variant, which has gained popularity for use in transgenic or targeted mice. Moreover, various GFP spectral variants have also been developed, and two of these novel color variants, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), can also be used in mice. Since the spectral profiles of the ECFP and EYFP color variants are distinct and non-overlapping, these two reporters can be co-visualized, and are therefore ideal for in vivo double-labeling or fluorescent energy transfer analyses. The use of GFP and its color variants as reporters provides an unprecedented level of sophistication and represents the next step in mouse genome engineering technology by opening up the possibility

  4. Structure and reactivity of the chromophore of a GFP-like chromoprotein from Condylactis gigantea.

    PubMed

    Pakhomov, Alexey A; Pletneva, Nadezhda V; Balashova, Tamara A; Martynov, Vladimir I

    2006-06-13

    Here we present the study of the chromophore structure of the purple chromoprotein from Condylactis gigantea. Tandem mass spectrometry and 1H and 13C NMR of the chromopeptide reveal that the protein contains a chromophore with a chemical structure identical to that of the red fluorescent protein from Discosoma sp. A single A63G substitution demonstrates that the nature of the first amino acid of the XYG chromophore-forming sequence is dispensable for the chromoprotein red shift development. It has been recently proposed that post-translational reactions at the acylimine, a chemical group that accounts for the red fluorescence, might be an additional source of spectral diversity of proteins homologous to the Aequorea victoria green fluorescent protein (GFP). We have examined the reactivity of the chromophore acylimine group within the C. gigantea purple chromoprotein. Like other proteins with the acylimine-modified chromophore, the purple chromoprotein suffers a hypsochromic spectral shift to the GFP-like absorbance (386 nm) upon mild denaturation. NMR analysis of the chromopeptide suggests this hypsochromic spectral shift is due to H2O addition across the C=N bond of the acylimine. However, unlike the red fluorescent protein from Discosoma sp., denatured under harsh conditions, the wild-type chromoprotein exhibits only slight fragmentation, which is induced by complete hydrolysis of the acylimine. A model suggesting the influence of the amino acid X side chain on protein fragmentation is presented.

  5. Expression of GFP in tumor cells and fluorescent examination by confocal microscope

    NASA Astrophysics Data System (ADS)

    Jin, Ying; Xing, Da; Xu, Chaoyang

    2002-04-01

    The green fluorescent protein (GFP), from the bioluminescent jellyfish Aequorea victoria, yields a bright green fluorescence when expressed in either eukaryotic or prokaryotic cells and illuminated by blue or UV light. The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the plasmid EGFP encoding GFP was used to transfect SWO cells (a cancer cell line of nerve gelatinous tissue) mediated by liposome: (1) The plasmid EGFP-C1, purchased from Clontech Co., propagated in suitable E. coli strain (JM 109), was extracted by Concert High Purity Plasmid Miniprep (Gibco). (2) SWO was cultured in RPMI 1640 (10% FCS and 25 mM HEPES), 37 degree(s)C, 5% CO2. Cancer cells were transfected in 6-cm tissue culture dishes by Lipofectin Reagent (Gibco) for 6-12 hr using 2 ug DNA. (3) Then, infected cells were collected in medium containing 800 ug/ml G418, and the resistant clones were harvested and subcloned in fresh culture medium maintaining 800 ug/ml G418. (4) The cells were examined by using Nikon fluorescent microscope (E600) and Bio-Rad confocal microscope (MRC 600). (5) Next step, the cancer cells, stably expressing GFP after in vivo transduction, were implanted by surgical orthotopic implantation (SOI) in nude mice. Tracking of these cancer cells will become more sensitive and rapid than the traditional procedure of histopathological examination or immunohistochemistry. This method demonstrates external, noninvasive, whole-body, real-time fluorescence optical imaging of internally growing tumors and metastases in transplanted animals.

  6. pH-dependent fluorescence of a heterologously expressed Aequorea green fluorescent protein mutant: in situ spectral characteristics and applicability to intracellular pH estimation.

    PubMed

    Robey, R B; Ruiz, O; Santos, A V; Ma, J; Kear, F; Wang, L J; Li, C J; Bernardo, A A; Arruda, J A

    1998-07-14

    The green fluorescent protein of Aequorea victoria (GFP) is a natural peptide chromophore without substrate or cofactor requirements for fluorescence. In vitro, a recombinant F64L/S65T GFP mutant (GFPmut1) exhibited pH sensitive fluorescence within the physiologic range. When heterologously expressed in BS-C-1 cells or rabbit proximal tubule cells, uniform cytosolic and nuclear fluorescence was observed. Cytosolic fluorescence constituted over 80% of the total. Excitation scanning of transfected cells revealed two GFPmut1-specific regions that were pH-sensitive over the physiologic range, and each region exhibited a unique pH "bias" in fluorescence emission. Excitation at or near the expected maximum of 488 nm (region II) uniformly resulted in fluorescence that was preferentially altered at acidic pH. In contrast, a novel "wild-type" excitation peak at 400 nm (region I) resulted in alkaline-biased fluorescence similar to that described for the wild-type chromophore in vitro, suggesting that wild-type spectral features disrupted in vitro by mutagenesis may be recovered in intact cells. Calibration of intracellular pH (pHi) with in situ fluorescence following excitation in either region revealed a semilogarithmic relationship between fluorescence intensity and pH within the physiologic range. We therefore measured pHi changes attributable to altered Na/HCO3 cotransport (NBC) activity both in GFPmut1-expressing cells and in paired untransfected cells loaded with BCECF. Basal NBC activity was the same in each group, as was the stimulation of activity by 10% CO2, thus validating the utility of GFPmut1 as a fluorescent probe for pHi and establishing a novel, useful, and practical application for GFPmut1 in monitoring pHi in real time.

  7. A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

    PubMed

    Kelloniemi, Jani; Mäkinen, Kristiina; Valkonen, Jari P T

    2006-05-01

    Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

  8. Reporter system for the detection of in vivo gene conversion: changing colors from blue to green using GFP variants.

    PubMed

    Sommer, Jeffrey R; Alderson, Jon; Laible, Goetz; Petters, Robert M

    2006-06-01

    We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr(66)-His(66)). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reporter cells using a variety of methodologies and strategies.Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.

  9. Reporter genes lucFF, luxCDABE, gfp, and dsred have different characteristics in whole-cell bacterial sensors.

    PubMed

    Hakkila, Kaisa; Maksimow, Mikael; Karp, Matti; Virta, Marko

    2002-02-15

    The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.

  10. The role of localized cell surface-associated glycoproteins during fertilization in the hydrozoan Aequorea.

    PubMed

    Freeman, G

    1996-10-10

    Hydrozoan eggs can be fertilized only at the site of polar body formation and first acquire this ability during second polar body formation. The eggs of Aequorea victoria form a discrete Triticum lectin binding moiety in the jelly coat near the first polar body as it is being given off and also a discrete concanavalin A binding moiety associated with the egg surface where the second polar body forms, which disappears immediately after fertilization. The germinal vesicle has an eccentric position in full grown Aequorea oocytes. The region of oocyte surface closest to the germinal vesicle is the site where the polar bodies normally form. When oocytes are centrifuged during oocyte maturation, the meiotic apparatus sometimes shifts to a different position with reference to the egg surface and polar bodies are given off at this new site. There is a corresponding shift in the position of the Triticum and concanavalin A lectin binding moieties, indicating that their formation is associated with local events occurring at the site of polar body formation. Treatment of Aequorea eggs with Triticum or concanavalin A causes a marked reduction in the ability of these eggs to be fertilized, suggesting that sugar-containing moieties, to which the lectins bind, play a role in fertilization. Removal of sugars on these moieties with mannosidase or N-acetylglucosaminidase, or the cleavage of the protein the sugars are attached to with trypsin, results in eggs that do not bind Triticum or concanavalin A and also show a marked reduction in the ability to be fertilized. These experiments suggest that the lectin binding moieties are glycoproteins.

  11. X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals.

    PubMed

    Perozzo, M A; Ward, K B; Thompson, R B; Ward, W W

    1988-06-05

    The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P6122 or P6522 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallel-epipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, beta = 108 degrees, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Time-resolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.

  12. Application of GFP-Technique for Cytoskeleton Visualization Onboard

    NASA Astrophysics Data System (ADS)

    Kordyum, E. L.; Shevchenko, G. V.; Yemets, A. I.; Nyporko, A. I.; Blume, Ya. B.

    2002-01-01

    gravity perception and transduction. Elucidation of cytoskeleton involvement in the above processes will be a great contribution into the fundamental biology, namely, in signaling mechanisms. One of the useful models for investigation of cell gravisensing are higher plants where the sites of gravity perception and reaction are spatially separated. From this point of view the localization of cytoskeletal elements at the different stages of plant development with the special emphasis on cytoskeleton dynamics during development of gravisensitive regions are of special interests. It should be noted that most of plant cytoskeletal researches are conducted by the means of immunohistochemical reactions with the application of monoclonal antibodies specific to specific cytoskeletal proteins. Different modifications of these methods are beneficial for the ground-based experiments, but for the performance onboard the latter represents quite routine and complicated technique, which requires time and special skills for astronauts. Besides, immunohistochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for better understanding of cytoskeleton function. In this connection we propose new approach for cytoskeleton visualization onboard in particular, use of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. Recently appeared this method helped to obtain significant data on the cytoskeleton dynamics in living cells, including plant cells. The creation of chimaeric protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of cytoskeleton functional status in microgravity. To realize this idea at present state of art it would be possible to produce a respective chimaeric plant tubulin gene

  13. Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy.

    PubMed

    Fang, Chong; Frontiera, Renee R; Tran, Rosalie; Mathies, Richard A

    2009-11-12

    Tracing the transient atomic motions that lie at the heart of chemical reactions requires high-resolution multidimensional structural information on the timescale of molecular vibrations, which commonly range from 10 fs to 1 ps. For simple chemical systems, it has been possible to map out in considerable detail the reactive potential-energy surfaces describing atomic motions and resultant reaction dynamics, but such studies remain challenging for complex chemical and biological transformations. A case in point is the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, which is a widely used gene expression marker owing to its efficient bioluminescence. This feature is known to arise from excited-state proton transfer (ESPT), yet the atomistic details of the process are still not fully understood. Here we show that femtosecond stimulated Raman spectroscopy provides sufficiently detailed and time-resolved vibrational spectra of the electronically excited chromophore of GFP to reveal skeletal motions involved in the proton transfer that produces the fluorescent form of the protein. In particular, we observe that the frequencies and intensities of two marker bands, the C-O and C = N stretching modes at opposite ends of the conjugated chromophore, oscillate out of phase with a period of 280 fs; we attribute these oscillations to impulsively excited low-frequency phenoxyl-ring motions, which optimize the geometry of the chromophore for ESPT. Our findings illustrate that femtosecond simulated Raman spectroscopy is a powerful approach to revealing the real-time nuclear dynamics that make up a multidimensional polyatomic reaction coordinate.

  14. Imaging long distance propagating calcium signals in intact plant leaves with the BRET-based GFP-aequorin reporter.

    PubMed

    Xiong, Tou Cheu; Ronzier, Elsa; Sanchez, Frédéric; Corratgé-Faillie, Claire; Mazars, Christian; Thibaud, Jean-Baptiste

    2014-01-01

    Calcium (Ca(2+)) is a second messenger involved in many plant signaling processes. Biotic and abiotic stimuli induce Ca(2+) signals within plant cells, which, when decoded, enable these cells to adapt in response to environmental stresses. Multiple examples of Ca(2+) signals from plants containing the fluorescent yellow cameleon sensor (YC) have contributed to the definition of the Ca(2+) signature in some cell types such as root hairs, pollen tubes and guard cells. YC is, however, of limited use in highly autofluorescent plant tissues, in particular mesophyll cells. Alternatively, the bioluminescent reporter aequorin enables Ca(2+) imaging in the whole plant, including mesophyll cells, but this requires specific devices capable of detecting the low amounts of emitted light. Another type of Ca(2+) sensor, referred to as GFP-aequorin (G5A), has been engineered as a chimeric protein, which combines the two photoactive proteins from the jellyfish Aequorea victoria, the green fluorescent protein (GFP) and the bioluminescent protein aequorin. The Ca(2+)-dependent light-emitting property of G5A is based on a bioluminescence resonance energy transfer (BRET) between aequorin and GFP. G5A has been used for over 10 years for enhanced in vivo detection of Ca(2+) signals in animal tissues. Here, we apply G5A in Arabidopsis and show that G5A greatly improves the imaging of Ca(2+) dynamics in intact plants. We describe a simple method to image Ca(2+) signals in autofluorescent leaves of plants with a cooled charge-coupled device (cooled CCD) camera. We present data demonstrating how plants expressing the G5A probe can be powerful tools for imaging of Ca(2+) signals. It is shown that Ca(2+) signals propagating over long distances can be visualized in intact plant leaves and are visible mainly in the veins.

  15. Thermal effect on Aequorea green fluorescent protein anionic and neutral chromophore forms fluorescence.

    PubMed

    dos Santos, Andrea Martins

    2012-01-01

    The emission behaviour of Aequorea green fluorescent protein (A-GFP) chromophore, in both neutral (N) and anionic (A) form, was studied in the temperature range from 20 °C to 75 °C and at pH = 7. Excitation wavelengths of 399 nm and 476 nm were applied to probe the N and A forms environment, respectively. Both forms exhibit distinct fluorescence patterns at high temperature values. The emission quenching rate, following a temperature increase, is higher for the chromophore N form as a result of the hydrogen bond network weakening. The chromophore anionic form emission maximum is red shifted, upon temperature increase, due to a charge transfer process occurring after A form excitation.

  16. A zebrafish histone variant H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development.

    PubMed

    Pauls, S; Geldmacher-Voss, B; Campos-Ortega, J A

    2001-12-01

    Abstract. We have generated transgenic zebrafish lines expressing a fusion of a histone variant, H2A.F/Z, to the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. Here, we describe the molecular cloning, partial characterisation and expression of the zebrafish H2A.F/Z histone gene, as well as the construction of the transgene and its transformation into the zebrafish germ line. No abnormality can be detected in transgenic fish expressing the H2A.F/Z:GFP fusion protein. The nuclear localisation of the fusion protein correlates with the start of zygotic transcription, in that it is present in the unfertilised egg and in the cytoplasm of cells after the first cleavages, being found in some nuclei after the seventh or eighth cleavage, whereas all nuclei from the 1,000-cell stage on, i.e. after midblastula transition, contain protein. In addition to these data, we present a few examples of the many possible applications of this transgenic line for developmental studies in vivo. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00427-001-0196-x

  17. Characterization of the groESL operon in Listeria monocytogenes: utilization of two reporter systems (gfp and hly) for evaluating in vivo expression.

    PubMed

    Gahan, C G; O'Mahony, J; Hill, C

    2001-06-01

    The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is an important factor governing virulence. We have sequenced the operon encoding the major heat shock proteins GroES and GroEL in the gram-positive food-borne pathogen Listeria monocytogenes. The operon has a conserved orientation in the order groES groEL. Upstream of groES and in the opposite orientation is a gene encoding a homologue of the Bacillus subtilis protein YdiL, while downstream of groEL is a gene encoding a putative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to the UV-optimized Aequorea victoria green fluorescent protein (GFP(UV)) to analyze expression of groESL under various environmental stress conditions, including heat shock, ethanol stress, and acid shock, and during infection of J774 mouse macrophage cells. Strains harboring GFP(UV) transcriptional fusions to the promoter region of groESL demonstrated a significant increase in fluorescence following heat shock that was detected by both fluorimetry and fluorescence microscopy. Using both RT-PCR and GFP technology we detected expression of groESL following internalization by J774 cells. Increased intracellular expression of dnaK was also determined using RT-PCR. We have recently described a system which utilizes L. monocytogenes hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C. G. M. Gahan and C. Hill, Mol. Microbiol. 36:498-507, 2000). In this study a strain was constructed in which hemolysin expression was placed under the control of the groESL promoter. In this strain hemolysin expression during infection also confirms transcription from the groESL promoter during J774 and murine infection, albeit at lower levels than the known virulence factor plcA.

  18. Victoria Cowling.

    PubMed

    Cowling, Victoria; Bobrowska, Anna

    2015-08-15

    Victoria Cowling received her BA from the University of Cambridge. She completed her PhD with Julian Downward and Gerard Evan at Cancer Research UK in London, and then moved to the US for a postdoctoral position at Princeton University and Dartmouth College with Michael Cole. Since 2008, she has been running a lab at Dundee University. She is an MRC Senior Research Fellow, a Lister Institute Research Fellow and an EMBO Young Investigator and, in 2015, she was the first recipient of the Women in Cell Biology medal, awarded by the British Society for Cell Biology.

  19. Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells.

    PubMed

    Kofoed, Eric M; Guerbadot, Martin; Schaufele, Fred

    2008-01-01

    Forster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ERalpha) labeled with "wild-type" CFP and YFP is compared with that of ERalpha labeled with "monomeric" A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced ERalpha-ERalpha interaction is similar for ERalpha labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERalpha-ERalpha interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERalpha-ERalpha interaction but, when brought close together via ERalpha-ERalpha interaction, FP dimerization modestly improves FRET measurement.

  20. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein.

    PubMed

    Brejc, K; Sixma, T K; Kitts, P A; Kain, S R; Tsien, R Y; Ormö, M; Remington, S J

    1997-03-18

    The 2.1-A resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.

  1. Structural Basis for Dual Excitation and Photoisomerization of the Aequorea victoria Green Fluorescent Protein

    NASA Astrophysics Data System (ADS)

    Brejc, Katjusa; Sixma, Titia K.; Kitts, Paul A.; Kain, Steven R.; Tsien, Roger Y.; Ormo, Mats; Remington, S. James

    1997-03-01

    The 2.1- angstrom resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 --> Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.

  2. Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

    PubMed

    Gorokhovatsky, Andrey Yu; Rudenko, Natalia V; Marchenkov, Victor V; Skosyrev, Vitaly S; Arzhanov, Maxim A; Burkhardt, Nils; Zakharov, Mikhail V; Semisotnov, Gennady V; Vinokurov, Leonid M; Alakhov, Yuli B

    2003-02-01

    Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

  3. Synthetic Biomimetic Fluorophores for Micro/Nanosensor

    DTIC Science & Technology

    2006-11-01

    as a companion protein to aequorin, the famous chemiluminescent protein from the brightly luminescent Aequorea jellyfish , with glowing points...peaked near 470 nm, which was close to one of the excitation peaks of GFP. Aequorin, isolated from the jellyfish Aequorea victoria, is a complex of...partially homologous to green fluorescent protein (GFP) (Matz et al. 1999, Dove et al. 2000), first found in the luminescent jellyfish Aequorea and used

  4. Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

    PubMed

    Tsuji, Frederick I

    2010-08-01

    The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment.

  5. Room temperature spectrally resolved single-molecule spectroscopy reveals new spectral forms and photophysical versatility of aequorea green fluorescent protein variants.

    PubMed

    Blum, Christian; Meixner, Alfred J; Subramaniam, Vinod

    2004-12-01

    It is known from ensemble spectroscopy at cryogenic temperatures that variants of the Aequorea green fluorescent protein (GFP) occur in interconvertible spectroscopically distinct forms which are obscured in ensemble room temperature spectroscopy. By analyzing the fluorescence of the GFP variants EYFP and EGFP by spectrally resolved single-molecule spectroscopy we were able to observe spectroscopically different forms of the proteins and to dynamically monitor transitions between these forms at room temperature. In addition to the predominant EYFP B-form we have observed the blue-shifted I-form thus far only seen at cryogenic temperatures and have followed transitions between these forms. Further we have identified for EYFP and for EGFP three more, so far unknown, forms with red-shifted fluorescence. Transitions between the predominant forms and the red-shifted forms show a dark time which indicates the existence of a nonfluorescent intermediate. The spectral position of the newly-identified red-shifted forms and their formation via a nonfluorescent intermediate hint that these states may account for the possible photoactivation observed in bulk experiments. The comparison of the single-protein spectra of the red-shifted EYFP and EGFP forms with single-molecule fluorescence spectra of DsRed suggest that these new forms possibly originate from an extended chromophoric pi-system analogous to the DsRed chromophore.

  6. Proton nuclear magnetic resonance and fluorescence spectroscopic studies of segmental mobility in aequorin and a green fluorescent protein from Aequorea forskalea

    SciTech Connect

    Nageswara Rao, B.D.; Kemple, M.D.; Prendergast, F.G.

    1980-10-01

    Aequorin is a protein of low molecular weight (20,000) isolated from the jellyfish Aequorea forskalea which emits blue light upon the binding of Ca/sup 2 +/ ions. This bioluminescence requires neither exogenous oxygen nor any other cofactors. The light emission occurs from an excited state of a chromophore (an imidazolopyrazinone) which is tightly and noncovalently bound to the protein. Apparently the binding of Ca/sup 2 +/ by the protein induces changes in the protein conformation which allow oxygen, already bound or otherwise held by the protein, to react with and therein oxidize the chromophore. The resulting discharged protein remains intact, with the Ca/sup 2 +/ and the chromophore still bound, but is incapable of further luminescence. The fluorescence spectrum of this discharged protein and the bioluminescence spectrum of the original charged aequorin are identical. A green fluorescent protein (GFP) of approx. 30,000 mol wt isolated from the same organism, functions in vivo as an acceptor of energy from aequorin and subsequently emits green light. We are applying proton nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy to examine structural details of, and fluctuations associated with the luminescent reaction of aequorin and the in vivo energy transfer from aequorin to the GFP.

  7. Big Spherules near 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This frame from the microscopic imager on NASA's Mars Exploration Rover Opportunity shows spherules up to about 5 millimeters (one-fifth of an inch) in diameter. The camera took this image during the 924th Martian day, or sol, of Opportunity's Mars-surface mission (Aug. 30, 2006), when the rover was about 200 meters (650 feet) north of 'Victoria Crater.'

    Opportunity discovered spherules like these, nicknamed 'blueberries,' at its landing site in 'Eagle Crater,' and investigations determined them to be iron-rich concretions that formed inside deposits soaked with groundwater. However, such concretions were much smaller or absent at the ground surface along much of the rover's trek of more than 5 kilometers (3 miles) southward to Victoria. The big ones showed up again when Opportunity got to the ring, or annulus, of material excavated and thrown outward by the impact that created Victoria Crater. Researchers hypothesize that some layer beneath the surface in Victoria's vicinity was once soaked with water long enough to form the concretions, that the crater-forming impact dispersed some material from that layer, and that Opportunity might encounter that layer in place if the rover drives down into the crater.

  8. Marketing ACE in Victoria.

    ERIC Educational Resources Information Center

    2001

    This publication presents options raised through various forums for marketing adult and community education (ACE) in Victoria, Australia, and suggested strategies. After an introduction (chapter 1), chapters 2 and 3 provide a broad view of the current situation for marketing ACE. Chapter 2 discusses general issues in the current position--ACE…

  9. Stretched View Showing 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Stretched View Showing 'Victoria'

    This pair of images from the panoramic camera on NASA's Mars Exploration Rover Opportunity served as initial confirmation that the two-year-old rover is within sight of 'Victoria Crater,' which it has been approaching for more than a year. Engineers on the rover team were unsure whether Opportunity would make it as far as Victoria, but scientists hoped for the chance to study such a large crater with their roving geologist. Victoria Crater is 800 meters (nearly half a mile) in diameter, about six times wider than 'Endurance Crater,' where Opportunity spent several months in 2004 examining rock layers affected by ancient water.

    When scientists using orbital data calculated that they should be able to detect Victoria's rim in rover images, they scrutinized frames taken in the direction of the crater by the panoramic camera. To positively characterize the subtle horizon profile of the crater and some of the features leading up to it, researchers created a vertically-stretched image (top) from a mosaic of regular frames from the panoramic camera (bottom), taken on Opportunity's 804th Martian day (April 29, 2006).

    The stretched image makes mild nearby dunes look like more threatening peaks, but that is only a result of the exaggerated vertical dimension. This vertical stretch technique was first applied to Viking Lander 2 panoramas by Philip Stooke, of the University of Western Ontario, Canada, to help locate the lander with respect to orbiter images. Vertically stretching the image allows features to be more readily identified by the Mars Exploration Rover science team.

    The bright white dot near the horizon to the right of center (barely visible without labeling or zoom-in) is thought to be a light-toned outcrop on the far wall of the crater, suggesting that the rover can see over the low rim of Victoria. In figure 1, the northeast and southeast rims are labeled

  10. The Role of Protein Kinase-C in Breast Cancer Invasion and Metastasis

    DTIC Science & Technology

    1997-09-01

    the jellyfish Aequorea victoria . A modified form of this protein has been generated that has altered excitation and emission spectra so that it...relatively new reporter molecule that is being increasingly used in a variety of studies is the green fluorescent protein (GFP) from the jellyfish Aequorea ... victoria . A modified form of this protein has been generated that has altered excitation and emission spectra so that it works well with detection

  11. [Fluorescent proteins: physical-chemical properties and application in cell biology].

    PubMed

    Stepanenko, O V; Verkhusha, V V; Kuznetsova, I M; Turoverov, K K

    2007-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. At present novel naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by stability, value of quantum yield, absorption and fluorescence spectra position and photochemical properties. GFP-like proteins are the fast growing family. This review is an attempt to characterize the main groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

  12. GFP as potential cellular viscosimeter

    NASA Astrophysics Data System (ADS)

    Visser, Antonie J. W. G.; Westphal, Adrie H.; Skakun, Victor V.; Borst, Jan Willem

    2016-09-01

    The molecular dimensions of proteins such as green fluorescent protein (GFP) are large as compared to the ones of solvents like water or glycerol. The microscopic viscosity, which determines the resistance to diffusion of, e.g. GFP, is then the same as that determined from the resistance of the solvent to flow, which is known as macroscopic viscosity. GFP in water/glycerol mixtures senses this macroscopic viscosity, because the translational and rotational diffusion coefficients are proportional to the reciprocal value of the viscosity as predicted by the Stokes-Einstein equations. To test this hypothesis, we have performed time-resolved fluorescence anisotropy (reporting on rotational diffusion) and fluorescence correlation spectroscopy (reporting on translational diffusion) experiments of GFP in water/glycerol mixtures. When the solvent also contains macromolecules of similar or larger dimensions as GFP, the microscopic and macroscopic viscosities can be markedly different and the Stokes-Einstein relations must be adapted. It was established from previous dynamic fluorescence spectroscopy observations of diffusing proteins with dextran polysaccharides as co-solvents (Lavalette et al 2006 Eur. Biophys. J. 35 517-22), that rotation and translation sense a different microscopic viscosity, in which the one arising from rotation is always less than that from translation. A microscopic viscosity parameter is defined that depends on scaling factors between GFP and its immediate environment. The direct consequence is discussed for two reported diffusion coefficients of GFP in living cells.

  13. Generation of AcGFP fusion with single-chain Fv selected from a phage display library constructed from mice hyperimmunized against 5-methyl 2'-deoxycytidine.

    PubMed

    Ohshima, Motohiro; Inoue, Kazuyuki; Hayashi, Hideki; Tsuji, Daiki; Mizugaki, Michinao; Itoh, Kunihiko

    2010-11-01

    DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

  14. [The epilepsy of Guadalupe Victoria].

    PubMed

    Soto-Pérez-de-Celis, Enrique

    2008-01-01

    Guadalupe Victoria, the first President of Mexico, passed away in 1843 uictim of an ailment that, at the time, was diagnosed as epilepsy. The clinical data and the pathologic findings, however, suggest the possibility that Victoria had an underlying disease that was responsible for the seizures that affected him. In this article I propose that Guadalupe Victoria had in fact Chagas Disease, and that he was infected with this parasitic malady while he lived in the tropical jungles of Veracruz, in eastern Mexico. Even though there aren't many published works regarding seizures secondary to chronic Chagas Disease, there are good descriptions of epileptic syndromes in patients with this infection. At the same time, the cardiac findings in Victoria's autopsy support the idea that he had some kind of cardiac pathology; in this case Chagasic dilated cardiomyopathy, which ultimately led to his death.

  15. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  16. 33 CFR 165.837 - Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, Texas.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, Texas. 165.837 Section 165.837 Navigation and Navigable Waters... Guard District § 165.837 Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria,...

  17. Early dentistry in Victoria, Australia.

    PubMed

    Atkinson, Henry

    2015-01-01

    At the time of settlement and in the years leading up to the establishment in 1884 of the Odontological Society of Victoria, dentistry was an unregulated activity practised simultaneously by those that had received the best apprenticeship training and those that had no training what-so-ever. Under the influence of dentists such as John Iliffe however, this situation was soon to change. In 1887 the first Dental Act was passed making it a legal requirement for anyone practicing dentistry to be registered. In 1890, the Melbourne Dental Hospital opened its doors to its first patients, and in 1897, the Australian College of Dentistry, later to become a school within the University of Melbourne, began teaching a dental course. Combined, these three moments in history lead to the eradication of the unscrupulous practitioner and laid the path for the development and professionalization of dentistry in the state of Victoria.

  18. Overview of Approach to 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Figure 1

    This image from the Mars Orbiter Camera aboard NASA's Mars Global Surveyor spacecraft shows an overview of 'Victoria Crater' and a portion of the area NASA's Mars Exploration Rover Opportunity has covered to reach the enormous depression.

    Images such as this one from the Mars Orbiter Camera on NASA's Mars Global Surveyor are helping scientists and engineers decide the best path for NASA's Mars Exploration Rover Opportunity as it approaches 'Victoria Crater.'

    In figure 1, a blue dot indicates 'Cape Verde' and a red dot 'Cabo Frio.' These two points mark the extent of the crater visible from the rover's position on its 945th Martian day, or sol (Sept. 20, 2006), a location it had reached two sols earlier and from which much of this monster depression was still out of sight. The green annotations indicate 'Duck Bay,' a location expected to allow a view to the other side of the crater. A dune, or ripple, is to the left of the crater, right in front of the green dot location. This is where the team initially talked about sending Opportunity for the rover's first view down into the crater. After further consideration, the team opted for a drive to the right of that ripple (south of the green dot) near the rim.

    The yellow lines that surround and intersect Victoria Crater are used to measure the crater and the distance to the far 'bays.' North is up. Victoria Crater is about 800 meters (half a mile) in diameter.

  19. Degradation of Victoria Crater, Mars

    NASA Technical Reports Server (NTRS)

    Wilson, Sharon A.; Grant, John A.; Cohen, Barbara A.; Golombek, Mathew P.; Geissler, Paul E.; Sullivan, Robert J.; Kirk, Randolph L.; Parker, Timothy J.

    2008-01-01

    The $\\sim$750 m diameter and $\\sim$75 m deep Victoria crater in Meridiani Planum, Mars, presents evidence for significant degradation including a low, serrated, raised rim characterized by alternating alcoves and promontories, a surrounding low relief annulus, and a floor partially covered by dunes. The amount and processes of degradation responsible for the modified appearance of Victoria crater were evaluated using images obtained in situ by the Mars Exploration Rover Opportunity in concert with a digital elevation model created using orbital HiRISE images. Opportunity traversed along the north and northwest rim and annulus, but sufficiently characterized features visible in the DEM to enable detailed measurements of rim relief, ejecta thickness, and wall slopes around the entire degraded, primary impact structure. Victoria retains a 5 m raised rim consisting of 1-2 m of uplifted rocks overlain by 3 m of ejecta at the rim crest. The rim is $\\sim$120 to 220 m wide and is surrounded by a dark annulus reaching an average of 590 m beyond the raised rim. Comparison between observed morphology and that expected for pristine craters 500 to 750 m across indicate the original, pristine crater was close to 600 m in diameter. Hence, the crater has been erosionally widened by approximately 150 m and infilled by about 50 m of sediments. Eolian processes are responsible for modification at Victoria, but lesser contributions from mass wasting or other processes cannot be ruled out. Erosion by prevailing winds is most significant along the exposed rim and upper walls and accounts for $\\sim$50 m widening across a WNW-ESE diameter. The volume of material eroded from the crater walls and rim is $\\sim$20% less than the volume of sediments partially filling the crater, indicating eolian infilling from sources outside the crater over time. The annulus formed when $\\sim$1 m deflation of the ejecta created a lag of more resistant hematite spherules that trapped darker, regional

  20. Degradation of Victoria Crater, Mars

    NASA Astrophysics Data System (ADS)

    Wilson, S. A.; Grant, J. A.; Cohen, B. A.; Golombek, M. P.; Geissler, P. E.; Sullivan, R. J.; Kirk, R. L.; Parker, T. J.

    2008-12-01

    The approximately 750 m diameter and 75 m deep Victoria crater in Meridiani Planum, Mars, presents evidence for significant degradation including a low, serrated, raised rim characterized by alternating alcoves and promontories, a surrounding low relief annulus, and a floor partially covered by dunes. The amount and processes of degradation responsible for the modified appearance of Victoria crater were evaluated using images obtained in situ by the Mars Exploration Rover Opportunity in concert with a digital elevation model (DEM) created using orbital HiRISE images. Opportunity traversed along the north and northwest rim and annulus and sufficiently characterized features visible in the DEM, thereby enabling detailed measurements of rim relief, ejecta thickness, and wall slopes around the entire degraded, primary impact structure. Victoria retains a 5 m raised rim consisting of 1 to 2 m of uplifted rocks overlain by 3 m of ejecta at the rim crest. The rim is 120 to 220 m wide and is surrounded by a dark annulus reaching an average of 590 m beyond the raised rim. Comparison between observed morphology and that expected for pristine craters 500 to 750 m across indicate the original, pristine crater was close to 600 m in diameter. Hence, the crater has been erosionally widened by approximately 150 m and infilled by about 50 m of sediments. Eolian processes are responsible for modification at Victoria, but lesser contributions from mass wasting or other processes cannot be ruled out. Erosion by prevailing winds is most significant along the exposed rim and upper walls and accounts for roughly 50 m widening across a WNW to ESE diameter. The volume of material eroded from the crater walls and rim is about 20 percent less than the volume of sediments partially filling the crater, indicating eolian infilling from sources outside the crater over time. The annulus formed when less than 1 m deflation of the ejecta created a lag of more resistant hematite spherules that

  1. vVICTORIA Console Development: Design and Fabrication of VICTORIA Console Emulations

    DTIC Science & Technology

    2011-07-01

    Canada vVICTORIA Console Development Design and Fabrication of VICTORIA Console Emulations Contract Project Manager: Keith Bowden, 902 832 7862...intentionally left blank. vVICTORIA Console Development Design and Fabrication of VICTORIA Console Emulations Keith Bowden Earl Gosse Prepared...integration, optimum equipment configurations and human factors. Résumé …..... Le présent rapport porte sur le développement et la fabrication de

  2. 75 FR 13433 - Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, TX

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-22

    ... SECURITY Coast Guard 33 CFR Part 165 RIN 1625-AA00 Safety Zone; Invista Inc Facility Docks, Victoria Barge Canal, Victoria, TX AGENCY: Coast Guard, DHS. ACTION: Interim final rule with request for comments. SUMMARY: The Coast Guard is establishing a safety zone for a partial blockage of the Victoria Barge...

  3. 'Victoria Crater' from 'Duck Bay'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    This view is presented as a cylindrical projection with geometric seam correction.

  4. YGFP: a spectral variant of GFP.

    PubMed

    Hansen, Flemming G; Atlung, Tove

    2011-06-01

    We describe YGFP, a slow bleaching green fluorescent protein (GFP) with unique spectral properties. YGFP is derived from an Escherichia coli codon-optimized synthetic gfp mutant 2 derivative. In addition to the GFP-mut 2 changes, it also carries S202F and T203I substitutions. YGFP can be used as a substitute for yellow fluorescent protein (YFP) in experiments in which two or more fluorescent proteins are fused to different cellular protein components, expanding the ability to study multiple labeled proteins in a cell at once.

  5. Degradation of Victoria crater, Mars

    NASA Astrophysics Data System (ADS)

    Grant, John A.; Wilson, Sharon A.; Cohen, Barbara A.; Golombek, Matthew P.; Geissler, Paul E.; Sullivan, Robert J.; Kirk, Randolph L.; Parker, Timothy J.

    2008-11-01

    The ~750 m diameter and ~75 m deep Victoria crater in Meridiani Planum, Mars, is a degraded primary impact structure retaining a ~5 m raised rim consisting of 1-2 m of uplifted rocks overlain by ~3 m of ejecta at the rim crest. The rim is 120-220 m wide and is surrounded by a dark annulus reaching an average of 590 m beyond the raised rim. Comparison between observed morphology and that expected for pristine craters 500-750 m across indicates that the original, pristine crater was close to 600 m in diameter. Hence, the crater has been erosionally widened by ~150 m and infilled by ~50 m of sediments. Eolian processes are responsible for most crater modification, but lesser mass wasting or gully activity contributions cannot be ruled out. Erosion by prevailing winds is most significant along the exposed rim and upper walls and accounts for ~50 m widening across a WNW-ESE diameter. The volume of material eroded from the crater walls and rim is ~20% less than the volume of sediments partially filling the crater, indicating eolian infilling from sources outside the crater over time. The annulus formed when ~1 m deflation of the ejecta created a lag of more resistant hematite spherules that trapped <10-20 cm of darker, regional basaltic sands. Greater relief along the rim enabled meters of erosion. Comparison between Victoria and regional craters leads to definition of a crater degradation sequence dominated by eolian erosion and infilling over time.

  6. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S; Cabantous, Stephanie

    2014-04-01

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  7. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2015-07-14

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  8. Nucleic acid encoding a self-assembling split-fluorescent protein system

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-07

    The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

  9. Teaching Molecular Biology to Undergraduate Biology Students: An Illustration of Protein Expression and Purification

    ERIC Educational Resources Information Center

    Sommer, Cesar Adolfo; Silva, Flavio Henrique; Novo, Maria Teresa Marques

    2004-01-01

    Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of "Aequorea victoria" is an interesting system for didactic purposes because it can be viewed easily during…

  10. Molecular Determinants of Antiestrogen and Drug Sensitivity in Breast Carcinoma Cells

    DTIC Science & Technology

    1996-08-01

    the present project. A particularly attractive selectable marker is the Green Fluorescent Protein (GFP), originally isolated from fish Aequorea ... victoria (8). Cells expressing this protein can be isolated through a non-toxic process of fluorescence-activated cell sorting, very shortly after gene

  11. A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

    ERIC Educational Resources Information Center

    Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase…

  12. Monitoring transgenic plants using in vivo markers

    SciTech Connect

    Stewart, C.N. Jr.

    1996-06-01

    The gene coding for green fluorecent protein (GFP), isolated and cloned from the jellyfish Aequorea victoria, is an ideal transgene for the monitoring of any plant species. It has the ability to fluoresce without added substrate, enzyme, or cofactor; it does not introduce morphological or sexual aberrations when expressed. 7 refs., 1 fig.

  13. Sensitivity of Superfolder GFP to Ionic Agents

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K.

    2014-01-01

    Superfolder variant of the green fluorescent protein (sfGFP) became a favorite probe for examination of the unfolding–refolding processes of fluorescent proteins with beta-barrel structure owing to its reversible unfolding in comparison with other fluorescent proteins. Its benefit is the proper folding even in fusion constructions with poorly folded polypeptides. We noticed that guanidine thiocyanate affects not only the structure of protein but its chromophore directly. Therefore we studied the influence of ionic denaturants and salts including guanidine thiocyanate, guanidine hydrochloride, sodium chloride and sodium thiocyanate on spectral features of sfGFP. It was shown that moderate amounts of the studied agents do not disrupt sfGFP structure but provoke pronounced alteration of its spectral characteristics. Changes in absorption and CD spectra in visible spectral range indicate the specific binding of SCN− and Cl− anions in the sfGFP chromophore vicinity. The anion binding results in the redistribution of sfGFP molecules with neutral and anionic chromophores. This also hinders the proton transfer in the chromophore excited state, considerably decreasing the fluorescence intensity of sfGFP. Our results indicate that when ionic denaturants are used in the studies of fluorescent protein folding their effect on fluorophore charge state should be taken into account. PMID:25347822

  14. Degradation of Victoria crater, Mars

    USGS Publications Warehouse

    Grant, J. A.; Wilson, S.A.; Cohen, B. A.; Golombek, M.P.; Geissler, P.E.; Sullivan, R.J.; Kirk, R.L.; Parker, T.J.

    2008-01-01

    The ???750 m diameter and ???75 m deep Victoria crater in Meridiani Planum, Mars, is a degraded primary impact structure retaining a ???5 m raised rim consisting of 1-2 m of uplifted rocks overlain by ???3 m of ejecta at the rim crest. The rim is 120-220 m wide and is surrounded by a dark annulus reaching an average of 590 m beyond the raised rim. Comparison between observed morphology and that expected for pristine craters 500-750 m across indicates that the original, pristine crater was close to 600 m in diameter. Hence, the crater has been erosionally widened by ???150 m and infilled by ???50 m of sediments. Eolian processes are responsible for most crater modification, but lesser mass wasting or gully activity contributions cannot be ruled out. Erosion by prevailing winds is most significant along the exposed rim and upper walls and accounts for ???50 m widening across a WNW-ESE diameter. The volume of material eroded from the crater walls and rim is ???20% less than the volume of sediments partially filling the crater, indicating eolian infilling from sources outside the crater over time. The annulus formed when ???1 m deflation of the ejecta created a lag of more resistant hematite spherules that trapped <10-20 cm of darker, regional basaltic sands. Greater relief along the rim enabled meters of erosion. Comparison between Victoria and regional craters leads to definition of a crater degradation sequence dominated by eolian erosion and infilling over time. Copyright 2008 by the American Geophysical Union.

  15. Measles surveillance in Victoria, Australia.

    PubMed Central

    Wang, Yung-Hsuan J.; Andrews, Ross M.; Lambert, Stephen B.

    2006-01-01

    OBJECTIVE: Many countries are implementing measles elimination strategies. In Australia, the State of Victoria has conducted enhanced measles surveillance since 1997 using case interviews and home-based specimen collection for laboratory confirmation. We attempted to identify features of notified cases that would better target surveillance resources. METHODS: We retrospectively classified notifications received from 1998 to 2003 as having been received in an epidemic (one or more laboratory-confirmed cases) or an interepidemic period (no laboratory-confirmed cases). We labelled the first case notified in any epidemic period that was not laboratory-confirmed at the time of notification as a "sentinel case". To maximize detection of sentinel cases while minimizing the follow-up of eventually discarded notifications, we generated algorithms using sentinel cases and interepidemic notifications. FINDINGS: We identified 10 sentinel cases with 422 interepidemic notifications from 1281 Victorian notifications. Sentinel cases were more likely to report fever at rash onset (odds ratio (OR) 15.7, 95% confidence interval (CI) CI: 2.1-688.9), cough (OR 10.4, 95% CI: 1.4-456.7), conjunctivitis (OR 7.9, 95% CI: 1.8-39.1), or year of birth between 1968 and 1981 (OR 31.8, 95% CI: 6.7-162.3). Prospective application of an algorithm consisting of fever at rash onset or born between 1968 and 1981 in the review period would have detected all sentinel cases and avoided the need for enhanced follow-up of 162 of the 422 eventually discarded notifications. CONCLUSION: Elimination strategies should be refined to suit regional and local priorities. The prospective application of an algorithm in Victoria is likely to reduce enhanced measles surveillance resource use in interepidemic periods, while still detecting early cases during measles outbreaks. PMID:16501727

  16. 'Lyell' Panorama inside Victoria Crater

    NASA Technical Reports Server (NTRS)

    2008-01-01

    During four months prior to the fourth anniversary of its landing on Mars, NASA's Mars Exploration Rover Opportunity examined rocks inside an alcove called 'Duck Bay' in the western portion of Victoria Crater. The main body of the crater appears in the upper right of this stereo panorama, with the far side of the crater lying about 800 meters (half a mile) away. Bracketing that part of the view are two promontories on the crater's rim at either side of Duck Bay. They are 'Cape Verde,' about 6 meters (20 feet) tall, on the left, and 'Cabo Frio,' about 15 meters (50 feet) tall, on the right. The rest of the image, other than sky and portions of the rover, is ground within Duck Bay.

    Opportunity's targets of study during the last quarter of 2007 were rock layers within a band exposed around the interior of the crater, about 6 meters (20 feet) from the rim. Bright rocks within the band are visible in the foreground of the panorama. The rover science team assigned informal names to three subdivisions of the band: 'Steno,' 'Smith,' and 'Lyell.'

    This view combines many images taken by Opportunity's panoramic camera (Pancam) from the 1,332nd through 1,379th Martian days, or sols, of the mission (Oct. 23 to Dec. 11, 2007). Images taken through Pancam filters centered on wavelengths of 753 nanometers, 535 nanometers and 432 nanometers were mixed to produce an approximately true-color panorama. Some visible patterns in dark and light tones are the result of combining frames that were affected by dust on the front sapphire window of the rover's camera.

    Opportunity landed on Jan. 25, 2004, Universal Time, (Jan. 24, Pacific Time) inside a much smaller crater about 6 kilometers (4 miles) north of Victoria Crater, to begin a surface mission designed to last 3 months and drive about 600 meters (0.4 mile).

  17. Identification and Characterisation of a pH-stable GFP

    PubMed Central

    Roberts, Tania Michelle; Rudolf, Fabian; Meyer, Andreas; Pellaux, Rene; Whitehead, Ellis; Panke, Sven; Held, Martin

    2016-01-01

    Green fluorescent proteins (GFPs) are invaluable tools for modern cell biology. Even though many properties of GFP have been successfully engineered, a GFP retaining brightness at low pH has not emerged. This limits the use of GFP in quantitative studies performed in fluctuating or acidic conditions. We report the engineering and characterisation of tandem dimer GFP (pH-tdGFP), a bright and stable GFP that can be efficiently excited and maintains its fluorescence properties in acidic conditions. Therefore, pH-tdGFP could act as a quantitative marker for cellular processes that occur at low pH, such as endocytosis, autophagy or starvation. PMID:27324986

  18. Circular permutant GFP insertion folding reporters

    SciTech Connect

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2013-04-16

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  19. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2008-06-24

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  20. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S; Cabantous, Stephanie

    2013-02-12

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  1. Circular permutant GFP insertion folding reporters

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2011-06-14

    Provided are methods of assaying and improving protein folding using circular permutants of fluorescent proteins, including circular permutants of GFP variants and combinations thereof. The invention further provides various nucleic acid molecules and vectors incorporating such nucleic acid molecules, comprising polynucleotides encoding fluorescent protein circular permutants derived from superfolder GFP, which polynucleotides include an internal cloning site into which a heterologous polynucleotide may be inserted in-frame with the circular permutant coding sequence, and which when expressed are capable of reporting on the degree to which a polypeptide encoded by such an inserted heterologous polynucleotide is correctly folded by correlation with the degree of fluorescence exhibited.

  2. Regulation of Growth and Metastases in an Estrogen Independent Breast Cancer Cell by Vitamin D Compounds

    DTIC Science & Technology

    2000-08-01

    micrometastases in the nude mouse, we used the EGFP fluorescent marker gene, cloned from the bioluminescent jellyfish Aequorea Victoria (previously...Utilizing our SUM-159PT GFP model system in vivo we can more efficiently examine the effects of vitamin D compounds to modulate invasion and metastasis...cells in nude mice. We previously reported that use of SUM-159PT cells expressing GFP enabled visualization of cancer cells in the mammary fat pad as

  3. Regulation of Growth and Metastases in an Estrogen Independent Breast Cancer Cell by Vitamin D Compounds

    DTIC Science & Technology

    2001-08-01

    jellyfish Aequorea Victoria (previously described in original grant proposal). Orthotopic implantation of SUMp159PTGFP was carried out to allow for the...metastatic expression of the inoculated cells in nude mice. We previously reported that use of SUM-159PT cells expressing GFP enabled visualization of...the brain and kidney. Resolution of SUM- 159PTGF cells was down to the single cell level, and high-level GFP expression was maintained in primary

  4. Mandatory bicycle helmet use--Victoria, Australia.

    PubMed

    1993-05-14

    On July 1, 1990, the first statewide law in Australia requiring wearing of an approved safety helmet by all bicyclists became effective in Victoria (1989 population: approximately 4.3 million) (Figure 1). Implementation of the law was preceded by a decade-long campaign to promote helmet use among the estimated 2.2 million persons who ride bicycles; the campaign included educational programs; mass media publicity; financial incentives; and efforts by professional, community, and bicycle groups (1,2). This report assesses helmet law enforcement, helmet use, and injuries related to bicycling in Victoria.

  5. Opportunity at Work Inside Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA Mars Exploration Rover Opportunity used its front hazard-identification camera to capture this wide-angle view of its robotic arm extended to a rock in a bright-toned layer inside Victoria Crater.

    The image was taken during the rover's 1,322nd Martian day, or sol (Oct. 13, 2007).

    Victoria Crater has a scalloped shape of alternating alcoves and promontories around the crater's circumference. Opportunity descended into the crater two weeks earlier, within an alcove called 'Duck Bay.' Counterclockwise around the rim, just to the right of the arm in this image, is a promontory called 'Cabo Frio.'

  6. Use of GFP for in vivo imaging: concepts and misconceptions

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2008-02-01

    Although GFP and fluorescent proteins are used extensively for in vivo imaging, there are many misconceptions about GFP imaging especially compared to luciferase. GFP is not toxic, indeed, transgenic animals with GFP expressed in every cell (1) live as long as non-transgenic animals. Cancer cells with GFP are as aggressive and malignant as the cells without GFP (2-4). Cell lines can be made very bright with fluorescent proteins with no toxicity. The in vivo signal from fluorescent proteins is at least 1,000 times greater than luciferase (5). GFP is so bright that a single molecule of GFP can be seen in a bacterium (6). GFP can be observed through the skin on deep organs (7). Skin autofluorescence presents no problem for in vivo GFP imaging with proper filters (8). Fur can be rapidly clipped removing this autofluorescence (9). GFP is readily quantified by the image area which correlates to tumor volume (10). There are now numerous clones of GFP, RFP, YFP and proteins that change color (11) that can be used in vivo.

  7. Looking Forward: Community Gateways at Victoria University

    ERIC Educational Resources Information Center

    Mountford, Christine

    2011-01-01

    The mission and values of Victoria University provide the underlying criteria for the development and implementation of the Community Gateways initiative, which aims to transform the lives of those living in the west of Melbourne through the power of further education. Community Gateways takes the university into the community by providing career…

  8. View from Southwest of Victoria Crater

    NASA Technical Reports Server (NTRS)

    2009-01-01

    This mosaic of frames from the navigation camera on NASA's Mars Exploration Rover Opportunity gives a view to the northeast from the rover's position on its 1,687th Martian day, or sol (Oct. 22, 2008).

    By that date, Opportunity had driven southwestward from Victoria Crater, beginning a long trek toward a larger crater, Endeavour.

  9. Deselection: At the University of Victoria Library.

    ERIC Educational Resources Information Center

    Signori, Donna

    1985-01-01

    Presents a brief overview of weeding history at the University of Victoria library, an outline and evaluation of criteria and methodology proposed by a deselection task group and adopted for 1982 weeding project, and a summary of results and observations from pilot projects and policy proposals. Sixteen references are cited. (EJS)

  10. Salicylic acid interferes with GFP fluorescence in vivo.

    PubMed

    de Jonge, Jennifer; Hofius, Daniel; Hennig, Lars

    2017-03-29

    Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue-specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP-fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP-derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP-tagged proteins upon SA treatment should therefore be evaluated with caution.

  11. Homicide-suicide in Victoria, Australia.

    PubMed

    Milroy, C M; Dratsas, M; Ranson, D L

    1997-12-01

    Thirty-nine incidents of homicide-suicide occurring in Victoria, Australia between 1985 and 1989 were examined. In 33 cases the assailants were men. The victims were spouses or women living in a de facto marriage. The majority of the victims were shot, and this was also the most frequent method of suicide. Breakdown in a relationship was the most frequent reason for killing. Mental illness of the assailant accounted for the killing in approximately 20% of cases. Physical ill health and financial stress were identified as important associative factors, particularly in the elderly. The pattern of homicide-suicide in Victoria is similar to that observed in other jurisdictions and represents an important and distinct subgroup of homicide.

  12. Pancam Peek into 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Left-eye view of a stereo pair for PIA08776

    [figure removed for brevity, see original site] Right-eye view of a stereo pair for PIA08776

    A drive of about 60 meters (about 200 feet) on the 943rd Martian day, or sol, of Opportunity's exploration of Mars' Meridiani Planum region (Sept. 18, 2006) brought the NASA rover to within about 50 meters (about 160 feet) of the rim of 'Victoria Crater.' This crater has been the mission's long-term destination for the past 21 Earth months. Opportunity reached a location from which the cameras on top of the rover's mast could begin to see into the interior of Victoria. This stereo anaglyph was made from frames taken on sol 943 by the panoramic camera (Pancam) to offer a three-dimensional view when seen through red-blue glasses. It shows the upper portion of interior crater walls facing toward Opportunity from up to about 850 meters (half a mile) away. The amount of vertical relief visible at the top of the interior walls from this angle is about 15 meters (about 50 feet). The exposures were taken through a Pancam filter selecting wavelengths centered on 750 nanometers.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is the expectation that a thick stack of geological layers will be exposed in the crater walls, potentially several times the thickness that was previously studied at Endurance and therefore, potentially preserving several times the historical record.

  13. Hazardous thunderstorm intensification over Lake Victoria.

    PubMed

    Thiery, Wim; Davin, Edouard L; Seneviratne, Sonia I; Bedka, Kristopher; Lhermitte, Stef; van Lipzig, Nicole P M

    2016-09-23

    Weather extremes have harmful impacts on communities around Lake Victoria, where thousands of fishermen die every year because of intense night-time thunderstorms. Yet how these thunderstorms will evolve in a future warmer climate is still unknown. Here we show that Lake Victoria is projected to be a hotspot of future extreme precipitation intensification by using new satellite-based observations, a high-resolution climate projection for the African Great Lakes and coarser-scale ensemble projections. Land precipitation on the previous day exerts a control on night-time occurrence of extremes on the lake by enhancing atmospheric convergence (74%) and moisture availability (26%). The future increase in extremes over Lake Victoria is about twice as large relative to surrounding land under a high-emission scenario, as only over-lake moisture advection is high enough to sustain Clausius-Clapeyron scaling. Our results highlight a major hazard associated with climate change over East Africa and underline the need for high-resolution projections to assess local climate change.

  14. Hazardous thunderstorm intensification over Lake Victoria

    PubMed Central

    Thiery, Wim; Davin, Edouard L.; Seneviratne, Sonia I.; Bedka, Kristopher; Lhermitte, Stef; van Lipzig, Nicole P. M.

    2016-01-01

    Weather extremes have harmful impacts on communities around Lake Victoria, where thousands of fishermen die every year because of intense night-time thunderstorms. Yet how these thunderstorms will evolve in a future warmer climate is still unknown. Here we show that Lake Victoria is projected to be a hotspot of future extreme precipitation intensification by using new satellite-based observations, a high-resolution climate projection for the African Great Lakes and coarser-scale ensemble projections. Land precipitation on the previous day exerts a control on night-time occurrence of extremes on the lake by enhancing atmospheric convergence (74%) and moisture availability (26%). The future increase in extremes over Lake Victoria is about twice as large relative to surrounding land under a high-emission scenario, as only over-lake moisture advection is high enough to sustain Clausius–Clapeyron scaling. Our results highlight a major hazard associated with climate change over East Africa and underline the need for high-resolution projections to assess local climate change. PMID:27658848

  15. Opportunity Traverse Map, 'Eagle' to 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Annotated Image

    NASA's Mars Exploration Rover Opportunity reached the rim of 'Victoria Crater' on Sept. 27, 2006, during the 951st Martian day, or sol, of the rover's work in the Meridian Planum region of Mars. Opportunity drove 9.28 kilometers (5.77 miles) in the explorations that took it from 'Eagle Crater,' where it landed in January 2004, eastward to 'Endurance Crater,' which it investigated for about half of 2004, then southward to Victoria.

    This map of Opportunity's trek so far is overlaid onto images taken by the Mars Orbiter Camera on NASA's Mars Global Surveyor. Victoria is about 800 meters (one-half mile) in diameter, or about five times wider than Endurance and 40 times wider than Eagle. The scale bar at lower right shows the length of 800 meters (0.50 mile). North is up.

    The Martian sol dates in the annotated image are as follows: sol 58 was March 24, 2004 sol 315 was December 12, 2004 sol 446 was April 26, 2005 sol 654 was November 25, 2005 sol 833 was May 28, 2006 sol 898 was August 3, 2006 sol 952 was September 28, 2006

  16. Hazardous thunderstorm intensification over Lake Victoria

    NASA Astrophysics Data System (ADS)

    Thiery, Wim; Davin, Edouard L.; Seneviratne, Sonia I.; Bedka, Kristopher; Lhermitte, Stef; van Lipzig, Nicole P. M.

    2016-09-01

    Weather extremes have harmful impacts on communities around Lake Victoria, where thousands of fishermen die every year because of intense night-time thunderstorms. Yet how these thunderstorms will evolve in a future warmer climate is still unknown. Here we show that Lake Victoria is projected to be a hotspot of future extreme precipitation intensification by using new satellite-based observations, a high-resolution climate projection for the African Great Lakes and coarser-scale ensemble projections. Land precipitation on the previous day exerts a control on night-time occurrence of extremes on the lake by enhancing atmospheric convergence (74%) and moisture availability (26%). The future increase in extremes over Lake Victoria is about twice as large relative to surrounding land under a high-emission scenario, as only over-lake moisture advection is high enough to sustain Clausius-Clapeyron scaling. Our results highlight a major hazard associated with climate change over East Africa and underline the need for high-resolution projections to assess local climate change.

  17. Differential GFP expression patterns induced by different heavy metals in Tg(hsp70:gfp) transgenic medaka (Oryzias latipes).

    PubMed

    Ng, Grace Hwee Boon; Xu, Hongyan; Pi, Na; Kelly, Barry C; Gong, Zhiyuan

    2015-06-01

    Heat shock protein 70 (Hsp70) is one of the most widely used biomarker for monitoring environment perturbations in biological systems. To facilitate the analysis of hsp70 expression as a biomarker, we generated a Tg(hsp70:gfp) transgenic medaka line in which green fluorescence protein (GFP) reporter gene was driven by the medaka hsp70 promoter. Here, we characterized Tg(hsp70:gfp) medaka for inducible GFP expression by seven environment-relevant heavy metals, including mercury, arsenic, lead, cadmium, copper, chromium, and zinc. We found that four of them (mercury, arsenic, lead, and cadmium) induced GFP expression in multiple and different organs. In general, the liver, kidney, gut, and skin are among the most frequent organs to show induced GFP expression. In contrast, no detectable GFP induction was observed to copper, chromium, or zinc, indicating that the transgenic line was not responsive to all heavy metals. RT-qPCR determination of hsp70 mRNA showed similar induction and non-induction by these metals, which also correlated with the levels of metal uptake in medaka exposed to these metals. Our observations suggested that these heavy metals have different mechanisms of toxicity and/or differential bioaccumulation in various organs; different patterns of GFP expression induced by different metals may be used to determine or exclude metals in water samples tested. Furthermore, we also tested several non-metal toxicants such as bisphenol A, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 4-introphenol, and lindane; none of them induced significant GFP expression in Tg(hsp70:gfp) medaka, further suggesting that the inducibility of Tg(hsp70:gfp) for GFP expression is specific to a subset of heavy metals.

  18. A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici☆

    PubMed Central

    Kilaru, S.; Schuster, M.; Studholme, D.; Soanes, D.; Lin, C.; Talbot, N.J.; Steinberg, G.

    2015-01-01

    Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20–30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue. PMID:26092799

  19. A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.

    PubMed

    Kilaru, S; Schuster, M; Studholme, D; Soanes, D; Lin, C; Talbot, N J; Steinberg, G

    2015-06-01

    Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.

  20. GFP expression by intracellular gene delivery of GFP-coding fragments using nanocrystal quantum dots

    NASA Astrophysics Data System (ADS)

    Hoshino, Akiyoshi; Manabe, Noriyoshi; Fujioka, Kouki; Hanada, Sanshiro; Yasuhara, Masato; Kondo, Akihiko; Yamamoto, Kenji

    2008-12-01

    Gene therapy is an attractive approach to supplement a deficient gene function. Although there has been some success with specific gene delivery using various methods including viral vectors and liposomes, most of these methods have a limited efficiency or also carry a risk for oncogenesis. We herein report that quantum dots (QDs) conjugated with nuclear localizing signal peptides (NLSP) successfully introduced gene-fragments with promoter elements, which promoted the expression of the enhanced green fluorescent protein (eGFP) gene in mammalian cells. The expression of eGFP protein was observed when the QD/gene-construct was added to the culture media. The gene-expression efficiency varied depending on multiple factors around QDs, such as (1) the reading direction of the gene-fragments, (2) the quantity of gene-fragments attached on the surface of the QD-constructs, (3) the surface electronic charges varied according to the structure of the QD/gene-constructs, and (4) the particle size of QD/gene complex varied according to the structure and amounts of gene-fragments. Using this QD/gene-construct system, eGFP protein could be detected 28 days after the gene-introduction whereas the fluorescence of QDs had disappeared. This system therefore provides another method for the intracellular delivery of gene-fragments without using either viral vectors or specific liposomes.

  1. THE NEWARK VICTORIA PLAN, A REPORT TO THE VICTORIA FOUNDATION ON THE STATUS OF THE PLAN.

    ERIC Educational Resources Information Center

    FRIEDRICHS, ROBERT W.; AND OTHERS

    THIS EVALUATION IS CONCERNED WITH THE FIRST YEAR OF IMPLEMENTATION OF THE VICTORIA PLAN IN A NEWARK, N.J., ELEMENTARY SCHOOL. THE COMPONENTS OF THIS IMPROVEMENT PROGRAM WHICH ARE DISCUSSED ARE CLASS TRIPS AND ASSEMBLIES, THE ROLE OF THE TEACHER, THE NURSERY SCHOOL, AND MATERIALS AND SUPPLIES. MOST OF THE EVALUATION IS DEVOTED TO THE PROBLEMS WHICH…

  2. 'Victoria Crater' from 'Duck Bay' (Polar Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    This view is presented as a polar projection with geometric seam correction.

  3. 'Victoria Crater' from 'Duck Bay' (Vertical Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    This view is presented as a vertical projection with geometric seam correction.

  4. 'Victoria Crater' from 'Duck Bay' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Figure 1

    [figure removed for brevity, see original site] Figure 2

    NASA's Mars rover Opportunity edged 3.7 meters (12 feet) closer to the top of the 'Duck Bay' alcove along the rim of 'Victoria Crater' during the rover's 952nd Martian day, or sol (overnight Sept. 27 to Sept. 28), and gained this vista of the crater. The rover's navigation camera took the seven exposures combined into this mosaic view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    The far side of the crater is about 800 meters (one-half mile) away. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves, such as Duck Bay. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind. The rocky cliffs in the foreground have been informally named 'Cape Verde,' on the left, and 'Cabo Frio,' on the right.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is an expectation that the thick stack of geological layers exposed in the crater walls could reveal the record of past environmental conditions over a much greater span of time than Opportunity has read from rocks examined earlier in the mission.

    The stereo-anaglyph view presented here is a cylindrical projection with geometric seam correction.

  5. 'Victoria' After Sol 950 Drive (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Left-eye view of a stereo pair for PIA08778

    [figure removed for brevity, see original site] Right-eye view of a stereo pair for PIA08778 [figure removed for brevity, see original site] Cylindrical view for PIA08778

    A drive of about 30 meters (about 100 feet) on the 950th Martian day, or sol, of Opportunity's exploration of Mars' Meridiani Planum region (Sept. 25, 2006) brought the NASA rover to within about 20 meters (about 66 feet) of the rim of 'Victoria Crater.' From that position, the rover's navigation camera took the exposures combined into this stereo anaglyph, which appears three-dimensional when viewed through red-green glasses. The scalloped shape of the crater is visible on the left edge. Due to a small dune or ripple close to the nearest part of the rim, the scientists and engineers on the rover team planned on sol 951 to drive to the right of the ripple, but not quite all the way to the rim, then to proceed to the rim the following sol. The image is presented in cylindrical projection with geometric seam correction.

    Victoria Crater is about 800 meters (one-half mile) in diameter, about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed. The great lure of Victoria is the expectation that a thick stack of geological layers will be exposed in the crater walls, potentially several times the thickness that was previously studied at Endurance and therefore, potentially preserving several times the historical record.

  6. Glycosylatable GFP as a compartment-specific membrane topology reporter

    SciTech Connect

    Lee, Hunsang; Min, Jisoo; Heijne, Gunnar von; Kim, Hyun

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer An N-linked glycosylation site is introduced near the GFP fluorophore. Black-Right-Pointing-Pointer gGFP is not glycosylated and is fully fluorescent in the cytosol. Black-Right-Pointing-Pointer gGFP is glycosylated and non-fluorescent in the lumen of the ER. Black-Right-Pointing-Pointer gGFP is fused to membrane proteins of known topology. Black-Right-Pointing-Pointer Its applicability as a membrane topology reporter is demonstrated. -- Abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present a robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.

  7. Inside Victoria Crater for Extended Exploration

    NASA Technical Reports Server (NTRS)

    2007-01-01

    After a finishing an in-and-out maneuver to check wheel slippage near the rim of Victoria Crater, NASA's Mars Exploration Rover Opportunity re-entered the crater during the rover's 1,293rd Martian day, or sol, (Sept. 13, 2007) to begin a weeks-long exploration of the inner slope.

    Opportunity's front hazard-identification camera recorded this wide-angle view looking down into and across the crater at the end of the day's drive. The rover's position was about six meters (20 feet) inside the rim, in the 'Duck Bay' alcove of the crater.

  8. Uranium mineralization in southern Victoria Land, Antarctica

    SciTech Connect

    Dreschhoff, G.A.M.; Zeller, E.J.

    1986-01-01

    For the past 10 antarctic field seasons, an airborne gamma-ray spectrometric survey has been conducted over widely separated parts of the continent. Localized accumulations of both primary and secondary uranium minerals have been discovered at several localities scattered along the Transantarctic Mountains from the Scott Glacier to northern Victoria Land. A number of highly significant radiation anomalies have been discovered in the area between the Koettlitz Glacier and the Pyramid Trough. The occurrences consist of pegmatite vein complexes which contain an association of primary uranium and thorium minerals. Of still greater significance is the fact that abundant secondary uranium minerals were found in association with the primary deposits, and they indicate clearly that uranium is geochemically mobile under the conditions imposed by the arid polar climate that now exists in southern Victoria Land. Preliminary results of a uranium analysis performed by neutron activation indicate a concentration of 0.12% uranium in a composite sample from the two veins. Even higher levels of thorium are present. The nature of the primary uranium mineralization is currently under investigation. Preliminary results are discussed.

  9. At Bright Band Inside Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    A layer of light-toned rock exposed inside Victoria Crater in the Meridiani Planum region of Mars appears to mark where the surface was at the time, many millions of years ago, when an impact excavated the crater. NASA's Mars Exploration Rover Opportunity drove to this bright band as the science team's first destination for the rover during investigations inside the crater.

    Opportunity's left front hazard-identification camera took this image just after the rover finished a drive of 2.25 meters (7 feet, 5 inches) during the rover's 1,305th Martian day, or sol, (Sept. 25, 2007). The rocks beneath the rover and its extended robotic arm are part of the bright band.

    Victoria Crater has a scalloped shape of alternating alcoves and promontories around the crater's circumference. Opportunity descended into the crater two weeks earlier, within an alcove called 'Duck Bay.' Counterclockwise around the rim, just to the right of the arm in this image, is a promontory called 'Cabo Frio.'

  10. On the Rim of 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a cylindrical projection with geometric seam correction.

  11. Degradation of Victoria Crater, Meridiani Planum, Mars

    NASA Astrophysics Data System (ADS)

    Grant, J. A.; Wilson, S. A.; Cohen, B. A.; Golombek, M. P.; Geissler, P. E.; Sullivan, R. J.

    2007-12-01

    Victoria crater (2.05N, 354.51E) is ~750 m in diameter and the largest crater on Mars observed in situ. The Mars Exploration Rover Opportunity traversed NW to SE across a broad annulus dominated by dark sand that at least partially surrounds the crater before navigating the northern crater rim. Rover observations of the crater and ejecta deposits are complemented by images with 26-52 cm/pixel scales from the High Resolution Imaging Science Experiment (HiRISE) on Mars Reconnaissance Orbiter and enable assessment of degradation state. The present depth/diameter ratio for Victoria is 0.1, less than the 0.2 expected for a pristine primary impact structure. Together with the eroded, serrated rim, this implies an originally smaller crater diameter and/or considerable infilling consistent with occurrence of a large dune field and few exposed rocks on the crater floor. The height and width of the raised rim is generally 4-5 m and 150-225 m, respectively, less than the 30 m and 500-600 m, respectively, expected for a pristine 750 m diameter crater. Ejecta thicknesses around the rim were derived using rover-based and HiRISE images and yield consistent estimates averaging ~3 m. The serrated rim plan creates a series of promontories extending up to 50 m into the crater and generally fronted by 30-60 degree slopes that are locally vertical and are separated by bays whose floors typically slope 15-25 degrees. A crater originally on order of 600-650 m in diameter and subsequently enlarged by mass wasting and aeolian erosion may yield a structure resembling Victoria today. The steep expression of the promontories and local outcroppings of rocks in the ejecta blanket points to some ongoing mass wasting, but the relative paucity of associated flanking talus indicates derived blocks of sulfate sandstone are not resistant to saltating sand and are rapidly broken down by the wind or are completely covered/filled in by aeolian drift. At Cape St. Vincent, the promontory appears undercut

  12. Imaging individual green fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Pierce, Daniel W.; Hom-Booher, Nora; Vale, Ronald D.

    1997-07-01

    Recent advances in fluorescence microscopy techniques have allowed the video-time imaging of single molecules of fluorescent dyes covalently bound to proteins in aqueous environments. However, the techniques have not been exploited fully because proteins can be difficult to label, and dye modification may cause partial or complete loss of activity. These difficulties could be circumvented by fusing proteins to green fluorescent protein (GFP) of the jellyfish Aequorea victoria. Here we report that single S65T mutant GFP molecules can be imaged using total internal reflection microscopy, and that ATP-driven movement of an individual kinesin molecule (a microtubule motor protein) fused to GFP can be readily observed.

  13. A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification.

    PubMed

    Fernandes, Cláudia S M; Pina, Ana Sofia; Dias, Ana M G C; Branco, Ricardo J F; Roque, Ana Cecília Afonso

    2014-09-30

    The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions.

  14. Prokaryotic Abundance and Activity in Permafrost of the Northern Victoria Land and Upper Victoria Valley (Antarctica).

    PubMed

    La Ferla, Rosabruna; Azzaro, Maurizio; Michaud, Luigi; Caruso, Gabriella; Lo Giudice, Angelina; Paranhos, Rodolfo; Cabral, Anderson S; Conte, Antonella; Cosenza, Alessandro; Maimone, Giovanna; Papale, Maria; Rappazzo, Alessandro Ciro; Guglielmin, Mauro

    2017-03-13

    Victoria Land permafrost harbours a potentially large pool of cold-affected microorganisms whose metabolic potential still remains underestimated. Three cores (BC-1, BC-2 and BC-3) drilled at different depths in Boulder Clay (Northern Victoria Land) and one sample (DY) collected from a core in the Dry Valleys (Upper Victoria Valley) were analysed to assess the prokaryotic abundance, viability, physiological profiles and potential metabolic rates. The cores drilled at Boulder Clay were a template of different ecological conditions (different temperature regime, ice content, exchanges with atmosphere and with liquid water) in the same small basin while the Dry Valleys site was very similar to BC-2 conditions but with a complete different geological history and ground ice type. Image analysis was adopted to determine cell abundance, size and shape as well as to quantify the potential viable and respiring cells by live/dead and 5-cyano-2,3-ditolyl-tetrazolium chloride staining, respectively. Subpopulation recognition by apparent nucleic acid contents was obtained by flow cytometry. Moreover, the physiological profiles at community level by Biolog-Ecoplate™ as well as the ectoenzymatic potential rates on proteinaceous (leucine-aminopeptidase) and glucidic (ß-glucosidase) organic matter and on organic phosphates (alkaline-phosphatase) by fluorogenic substrates were tested. The adopted methodological approach gave useful information regarding viability and metabolic performances of microbial community in permafrost. The occurrence of a multifaceted prokaryotic community in the Victoria Land permafrost and a large number of potentially viable and respiring cells (in the order of 10(4)-10(5)) were recognised. Subpopulations with a different apparent DNA content within the different samples were observed. The physiological profiles stressed various potential metabolic pathways among the samples and intense utilisation rates of polymeric carbon compounds and carbohydrates

  15. The Bands Culture in Victoria, Australia: Live Music Benefits Career Paths, Employment and Community

    ERIC Educational Resources Information Center

    Watson, Amanda; Forrest, David

    2012-01-01

    This study explores the career paths, employment, business opportunities and community contributions made available through the provision and development of the contemporary performance bands' culture in the State of Victoria. It is framed with the support given to live music performers by Arts Victoria, Small Business Victoria and Music Victoria.…

  16. Forty Meters from Entry to Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view is presented as a cylindrical projection with geometric seam correction.

  17. Layers of 'Cape Verde' in 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is an approximately true color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  18. Layers of 'Cabo Frio' in 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of 'Victoria crater' is looking southeast from 'Duck Bay' towards the dramatic promontory called 'Cabo Frio.' The small crater in the right foreground, informally known as 'Sputnik,' is about 20 meters (about 65 feet) away from the rover, the tip of the spectacular, layered, Cabo Frio promontory itself is about 200 meters (about 650 feet) away from the rover, and the exposed rock layers are about 15 meters (about 50 feet) tall. This is an approximately true color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  19. GFP-based FRET analysis in live cells.

    PubMed

    Takanishi, Christina L; Bykova, Ekaterina A; Cheng, Wei; Zheng, Jie

    2006-05-26

    Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1-10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor-acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest. The combination of easy access to the GFP-based FRET technique and its obvious usefulness in many applications can lead to complacency. Potential problems such as light contaminants, e.g., bleed-through and cross-talk, and inconsistent donor and acceptor concentrations are easily overlooked and can lead to errors in FRET calculation and data interpretation. In this article, we outline possible pitfalls of GFP-based FRET and approaches that address these issues, including a "Spectra FRET" technique that can be easily applied to live cell studies.

  20. Energy profile of nanobody-GFP complex under force

    NASA Astrophysics Data System (ADS)

    Klamecka, Kamila; Severin, Philip M.; Milles, Lukas F.; Gaub, Hermann E.; Leonhardt, Heinrich

    2015-10-01

    Nanobodies (Nbs)—the smallest known fully functional and naturally occuring antigen-binding fragments—have attracted a lot of attention throughout the last two decades. Exploring their potential beyond the current use requires more detailed characterization of their binding forces as those cannot be directly derived from the binding affinities. Here we used atomic force microscope to measure rupture force of the Nb-green fluorescent protein (GFP) complex in various pulling geometries and derived the energy profile characterizing the interaction along the direction of the pulling force. We found that—despite identical epitopes—the Nb binds stronger (41-56 pN) to enhanced GFP than to wild-type GFP (28-45 pN). Measured forces make the Nb-GFP pair a potent reference for investigating molecular forces in living systems both in and ex vivo.

  1. Satellite View of Opportunity's Journey around 'Victoria Crater'

    NASA Technical Reports Server (NTRS)

    2007-01-01

    Three years after embarking on a historic exploration of the red planet and six miles away from its landing site, NASA's Mars Exploration Rover Opportunity is traversing 'Victoria Crater' ridge by ridge, peering at layered cliffs in the interior. To identify various alcoves and cliffs along the way, science team members are using names of places visited by the 16th-century Earth explorer Ferdinand Magellan and his crew aboard the ship Victoria, who proved the Earth is round. (All names are unofficial unless approved by the International Astronomical Union.) This orbital view of 'Victoria Crater' was taken by NASA's Mars Reconnaissance Orbiter.

  2. Space Radar Image of Victoria, Canada

    NASA Technical Reports Server (NTRS)

    1994-01-01

    This three-frequency spaceborne radar image shows the southern end of Vancouver Island on the west coast of Canada. The white area in the lower right is the city of Victoria, the capital of the province of British Columbia. The three radar frequencies help to distinguish different land use patterns. The bright pink areas are suburban regions, the brownish areas are forested regions, and blue areas are agricultural fields or forest clear-cuts. Founded in 1843 as a fur trading post, Victoria has grown to become one of western Canada's largest commercial centers. In the upper right is San Juan Island, in the state of Washington. The Canada/U.S. border runs through Haro Strait, on the right side of the image, between San Juan Island and Vancouver Island. The image was acquired by the Spaceborne Imaging Radar-C/X-band Synthetic Aperture Radar (SIR-C/X-SAR) on October 6, 1994, onboard the space shuttle Endeavour. The area shown is 37 kilometers by 42 kilometers (23 miles by 26 miles) and is centered at 48.5 degrees north latitude, 123.3 degrees west longitude. North is toward the upper left. The colors are assigned to different radar frequencies and polarizations as follows: red is L-band horizontally transmitted and received; green is C-band, vertically transmitted and received; and blue is X-band, vertically transmitted and received. SIR-C/X-SAR, a joint mission of the German, Italian and United States space agencies, is part of NASA's Mission to Planet Earth program.

  3. 'Lyell' Panorama inside Victoria Crater (Stereo)

    NASA Technical Reports Server (NTRS)

    2008-01-01

    During four months prior to the fourth anniversary of its landing on Mars, NASA's Mars Exploration Rover Opportunity examined rocks inside an alcove called 'Duck Bay' in the western portion of Victoria Crater. The main body of the crater appears in the upper right of this stereo panorama, with the far side of the crater lying about 800 meters (half a mile) away. Bracketing that part of the view are two promontories on the crater's rim at either side of Duck Bay. They are 'Cape Verde,' about 6 meters (20 feet) tall, on the left, and 'Cabo Frio,' about 15 meters (50 feet) tall, on the right. The rest of the image, other than sky and portions of the rover, is ground within Duck Bay.

    Opportunity's targets of study during the last quarter of 2007 were rock layers within a band exposed around the interior of the crater, about 6 meters (20 feet) from the rim. Bright rocks within the band are visible in the foreground of the panorama. The rover science team assigned informal names to three subdivisions of the band: 'Steno,' 'Smith,' and 'Lyell.'

    This view incorporates many images taken by Opportunity's panoramic camera (Pancam) from the 1,332nd through 1,379th Martian days, or sols, of the mission (Oct. 23 to Dec. 11, 2007). It combines a stereo pair so that it appears three-dimensional when seen through blue-red glasses. Some visible patterns in dark and light tones are the result of combining frames that were affected by dust on the front sapphire window of the rover's camera.

    Opportunity landed on Jan. 25, 2004, Universal Time, (Jan. 24, Pacific Time) inside a much smaller crater about 6 kilometers (4 miles) north of Victoria Crater, to begin a surface mission designed to last 3 months and drive about 600 meters (0.4 mile).

  4. On the Rim of 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Left-eye view of a stereo pair for PIA08780

    [figure removed for brevity, see original site] Right-eye view of a stereo pair for PIA08780

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a cylindrical-perspective projection with geometric seam correction.

  5. Ecological Biogeography of the Terrestrial Nematodes of Victoria Land, Antarctica

    PubMed Central

    Adams, Byron J.; Wall, Diana H.; Virginia, Ross A.; Broos, Emma; Knox, Matthew A.

    2014-01-01

    Abstract The terrestrial ecosystems of Victoria Land, Antarctica are characteristically simple in terms of biological diversity and ecological functioning. Nematodes are the most commonly encountered and abundant metazoans of Victoria Land soils, yet little is known of their diversity and distribution. Herein we present a summary of the geographic distribution, habitats and ecology of the terrestrial nematodes of Victoria Land from published and unpublished sources. All Victoria Land nematodes are endemic to Antarctica, and many are common and widely distributed at landscape scales. However, at smaller spatial scales, populations can have patchy distributions, with the presence or absence of each species strongly influenced by specific habitat requirements. As the frequency of nematode introductions to Antarctica increases, and soil habitats are altered in response to climate change, our current understanding of the environmental parameters associated with the biogeography of Antarctic nematofauna will be crucial to monitoring and possibly mitigating changes to these unique soil ecosystems. PMID:25061360

  6. Victoria Land, Ross Sea, and Ross Ice Shelf, Antarctica

    NASA Technical Reports Server (NTRS)

    2002-01-01

    On December 19, 2001, MODIS acquired data that produced this image of Antarctica's Victoria Land, Ross Ice Shelf, and the Ross Sea. The coastline that runs up and down along the left side of the image denotes where Victoria Land (left) meets the Ross Ice Shelf (right). The Ross Ice Shelf is the world's largest floating body of ice, approximately the same size as France. Credit: Jacques Descloitres, MODIS Land Rapid Response Team, NASA/GSFC

  7. A review of the Victoria, Australia granny flat program.

    PubMed

    Lazarowich, N M

    1990-04-01

    The 10-year-old Victoria, Australia granny flat program has about 3000 units in place. Granny flats, designed as standardized units using panels, offer privacy yet proximity to family members. There are suggestions they be considered as affordable housing for younger people. This review of the Victoria program suggests that state initiatives are more successful than local ones, and that decentralized administration is associated with increased ability to delivery the granny flat units.

  8. 'Lyell' Panorama inside Victoria Crater (False Color)

    NASA Technical Reports Server (NTRS)

    2008-01-01

    During four months prior to the fourth anniversary of its landing on Mars, NASA's Mars Exploration Rover Opportunity examined rocks inside an alcove called 'Duck Bay' in the western portion of Victoria Crater. The main body of the crater appears in the upper right of this stereo panorama, with the far side of the crater lying about 800 meters (half a mile) away. Bracketing that part of the view are two promontories on the crater's rim at either side of Duck Bay. They are 'Cape Verde,' about 6 meters (20 feet) tall, on the left, and 'Cabo Frio,' about 15 meters (50 feet) tall, on the right. The rest of the image, other than sky and portions of the rover, is ground within Duck Bay.

    Opportunity's targets of study during the last quarter of 2007 were rock layers within a band exposed around the interior of the crater, about 6 meters (20 feet) from the rim. Bright rocks within the band are visible in the foreground of the panorama. The rover science team assigned informal names to three subdivisions of the band: 'Steno,' 'Smith,' and 'Lyell.'

    This view combines many images taken by Opportunity's panoramic camera (Pancam) from the 1,332nd through 1,379th Martian days, or sols, of the mission (Oct. 23 to Dec. 11, 2007). Images taken through Pancam filters centered on wavelengths of 753 nanometers, 535 nanometers and 432 nanometers were mixed to produce this view, which is presented in a false-color stretch to bring out subtle color differences in the scene. Some visible patterns in dark and light tones are the result of combining frames that were affected by dust on the front sapphire window of the rover's camera.

  9. Opportunity's First Dip into Victoria Crater

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity entered Victoria Crater during the rover's 1,291st Martian day, or sol, (Sept. 11, 2007). The rover team commanded Opportunity to drive just far enough into the crater to get all six wheels onto the inner slope, and then to back out again and assess how much the wheels slipped on the slope. The driving commands for the day included a precaution for the rover to stop driving if the wheels were slipping more than 40 percent. Slippage exceeded that amount on the last step of the drive, so Opportunity stopped with its front pair of wheels still inside the crater. The rover team planned to assess results of the drive, then start Opportunity on an extended exploration inside the crater.

    This wide-angle view taken by Opportunity's front hazard-identification camera at the end of the day's driving shows the wheel tracks created by the short dip into the crater. The left half of the image looks across an alcove informally named 'Duck Bay' toward a promontory called 'Cape Verde' clockwise around the crater wall. The right half of the image looks across the main body of the crater, which is 800 meters (half a mile) in diameter.

  10. A Suite of Engineered GFP Molecules for Oligomeric Scaffolding

    PubMed Central

    Leibly, David J.; Arbing, Mark A.; Pashkov, Inna; DeVore, Natasha; Waldo, Geoffrey S.; Terwilliger, Thomas C.

    2015-01-01

    SUMMARY Applications ranging from synthetic biology to protein crystallization could be advanced by facile systems for connecting multiple proteins together in predefined spatial relationships. One approach to this goal is to engineer many distinct assembly forms of a single carrier protein or scaffold, to which other proteins of interest can then be readily attached. In this work we chose green fluorescent protein (GFP) as a scaffold, and engineered many alternate oligomeric forms, driven by either specific disulfide bond formation or metal ion addition. We generated a wide range of spatial arrangements of GFP subunits from 11 different oligomeric variants, and determined their X-ray structures in a total of 33 distinct crystal forms. Some of the oligomeric GFP variants show geometric polymorphism depending on conditions while others show considerable geometric rigidity. Potential future applications of this system are discussed. PMID:26278175

  11. Coherent Neutron Scattering and Collective Dynamics in the Protein, GFP

    PubMed Central

    Nickels, Jonathan D.; Perticaroli, Stefania; O’Neill, Hugh; Zhang, Qiu; Ehlers, Georg; Sokolov, Alexei P.

    2013-01-01

    Collective dynamics are considered to be one of the major properties of soft materials, including biological macromolecules. We present coherent neutron scattering studies of the low-frequency vibrations, the so-called boson peak, in fully deuterated green fluorescent protein (GFP). Our analysis revealed unexpectedly low coherence of the atomic motions in GFP. This result implies a low amount of in-phase collective motion of the secondary structural units contributing to the boson peak vibrations and fast conformational fluctuations on the picosecond timescale. These observations are in contrast to earlier studies of polymers and glass-forming systems, and suggest that random or out-of-phase motions of the β-strands contribute greater than two-thirds of the intensity to the low-frequency vibrational spectra of GFP. PMID:24209864

  12. Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto; Krol, Silke; Campanini, Barbara; Cannone, Fabio; Chirico, Giuseppe

    2006-10-01

    Discovery of Green Fluorescent Protein (GFP) constituted an important improvement for living cell studies on submicron resolution allowing in vivo fluorescence labeling. We studied the photo-physical properties of single GFP molecules incorporated in a charged polyelectrolyte environment by means of single molecule spectroscopy. The fluorescence characteristics change dramatically in terms of photo-stability,lifetime and blinking behavior so that the proteins scale up to quantum dots. The reported results highlight interesting applications in the design of fluorescent markers and in the development of optical data storage architectures.

  13. Are "g" and the General Factor of Personality (GFP) Correlated?

    ERIC Educational Resources Information Center

    Irwing, Paul; Booth, Tom; Nyborg, Helmuth; Rushton, J. Philippe

    2012-01-01

    We examined whether the General Factor of Personality (GFP) is related to the "g" factor of cognitive ability using data from the Vietnam Experience Study which randomly sampled 4462 Vietnam War veterans from a total sample of about five million Vietnam era army veterans. Exclusionary criteria included passing a fitness test, achieving a…

  14. Age- and light-dependent development of localised retinal atrophy in CCL2(-/-)CX3CR1(GFP/GFP) mice.

    PubMed

    Chen, Mei; Hombrebueno, Jose R; Luo, Chang; Penalva, Rosana; Zhao, Jiawu; Colhoun, Liza; Pandi, Sudha Pirya Soundara; Forrester, John V; Xu, Heping

    2013-01-01

    Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2(-/-)CX3CR1(GFP/GFP) mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2(-/-)CX3CR1(GFP/GFP) mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17∼60% of 12-month, and 30∼100% of 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice respectively, but not in wild-type mice. All CCL2(-/-)CX3CR1(GFP/GFP) mice exposed to extra-light (∼800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20-25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2(-/-)CX3CR1(GFP/GFP) mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2(-/-)CX3CR1(GFP/GFP) mice. GABA expression was reduced in the inner retina of aged CCL2(-/-)CX3CR1(GFP/GFP) mice. Significantly increased Müller glial and microglial activation was observed in CCL2(-/-)CX3CR1(GFP/GFP) mice compared to age-matched WT mice. Macrophages from CCL2(-/-)CX3CR1(GFP/GFP) mice were less phagocytic, but expressed higher levels of iNOS, IL-1β, IL-12 and TNF-α under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.

  15. The Victoria (Australia) Certificate of Education (VCE) in Geology for Senior Secondary-School Students.

    ERIC Educational Resources Information Center

    Markovics, Gabor

    1990-01-01

    The state of precollege earth science education in Victoria is described. The unit structure and content for the Victoria Certificate of Education in geology is detailed. The relationship between society and technological development is stressed in this curriculum. (CW)

  16. Geologic map and structural analysis of the Victoria quadrangle, Mercury

    NASA Astrophysics Data System (ADS)

    Galluzzi, Valentina; Di Achille, Gaetano; Ferranti, Luigi; Rothery, David A.; Palumbo, Pasquale

    2015-04-01

    In this work we present a new geologic map and structural analysis of the Victoria quadrangle (H2) of Mercury, along with a reconnaissance study of the geometry and kinematics of lobate scarps in this area. To this end, we produced a 1:3,000,000 geologic map of the area using the images provided by the NASA spacecraft MESSENGER, which has been orbiting the planet since March, 2011. The geologic map shows the distribution of smooth plains, intermediate plains, intercrater plains units and a classification of crater materials based on an empirical distinction among three stages of degradation. Structural mapping shows that the H2 quadrangle is dominated by N-S faults (here grouped into the Victoria system) to the east and NE-SW faults (Larrocha system) to the west, with the secondary existence of NW-SE-trending faults (Carnegie system) in the north-western area of the quadrangle. A systematic analysis of these systems has led to the following results. 1) the Victoria system is characterized by a main array of faults located along Victoria Rupes - Endeavour Rupes - Antoniadi Dorsum. The segmentation of this array into three different sectors changes from north to south and is spatially linked to the presence of three volcanic vents located at the boundaries between each sector and at the northern end of the Victoria Rupes sector , suggesting that volcanism and faulting are interrelated 2) The main array of Carnegie system is kinematically linked and antithetical to the Victoria system. Both systems have arguably controlled the growth of a longitudinal, fault-free, crustal and gravimetric bulge in the central area of the Victoria quadrangle, which is interpreted as a regional contractional pop-up. 3) The Larrocha system is interrupted against the central bulge and thus is probably older than the Victoria and Carnegie systems. Buffered crater counting performed on the Victoria system confirms the young relative age of its fault segments with respect to the map units

  17. Hazardous thunderstorms over Lake Victoria: climate change and early warnings

    NASA Astrophysics Data System (ADS)

    Thiery, Wim; Davin, Edouard L.; Seneviratne, Sonia I.; Bedka, Kristopher; Lhermitte, Stef; van Lipzig, Nicole

    2016-04-01

    Severe thunderstorms and associated high waves represent a constant threat to the 200,000 fishermen operating on Lake Victoria. According to the International Red Cross, presumably 3000 to 5000 fishermen die every year on the lake, thereby substantially contributing to the global death toll from natural disasters. Despite the long-known bad reputation of Lake Victoria, operational early warning systems are lacking and possible future changes of these extreme thunderstorms are unknown. Here we present the first dedicated high-resolution, coupled lake-land-atmosphere climate projection for the African Great Lakes region and analyse it in combination with new satellite data and coarser-scale ensemble projections. Our model projections for the end-of-the-century indicate that Lake Victoria amplifies the future intensification of extreme precipitation seen over the surrounding land. Under a high-emission scenario (RCP8.5), the 1% most extreme over-lake precipitation may intensify up to four times faster compared to surrounding land. Our findings are consistent with an ensemble of coarser-scale climate projections for Africa, but the lower skill of the ensemble over Lake Victoria constrains its applicability. Interestingly, the change in extremes contrasts to the change in average over-lake precipitation, which is projected to decrease by -6% for the same period. By further analyzing the high-resolution output we are able to explain this different response: while mesoscale circulation changes cause the average precipitation decline, the response of extremes is essentially thermodynamic. Finally, the study of the satellite-based detection of severe thunderstorms revealed a strong dependency of the nighttime storm intensity over Lake Victoria on the antecedent daytime land storm activity. This highlights the potential of this new satellite product for predicting intense storms over Lake Victoria. Overall, our results indicate a new major hazard associated with climate

  18. A practical teaching course in directed protein evolution using the green fluorescent protein as a model.

    PubMed

    Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Cruz Schneider, Maria Paula; Ward, Richard John

    2011-01-01

    Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility.

  19. 78 FR 55215 - Drawbridge Operation Regulation; Old River, Between Victoria Island and Byron Tract, CA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-10

    ... SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulation; Old River, Between Victoria Island... State Highway 4 Drawbridge across Old River, mile 14.8 between Victoria Island and Byron Tract, CA. The..., between Victoria Island and Byron Tract, CA. The drawbridge navigation span provides 12 feet...

  20. 77 FR 61645 - Exelon Generation Company, LLC, Victoria County Station Site; Notice of Withdrawal of Application...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-10

    ... COMMISSION Exelon Generation Company, LLC, Victoria County Station Site; Notice of Withdrawal of Application...) submitted an application for an Early Site Permit (ESP) for the Victoria County Station (VCS) site located in Victoria County, Texas to the U.S. Nuclear Regulatory Commission (NRC or the Commission)...

  1. 40 CFR 81.136 - Corpus Christi-Victoria Intrastate Air Quality Control Region.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 17 2010-07-01 2010-07-01 false Corpus Christi-Victoria Intrastate Air... Air Quality Control Regions § 81.136 Corpus Christi-Victoria Intrastate Air Quality Control Region. The Corpus Christi-Victoria Intrastate Air Quality Control Region (Texas) consists of the...

  2. 78 FR 65873 - Drawbridge Operation Regulation; Old River, Between Victoria Island and Byron Tract, CA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-04

    ... SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulation; Old River, Between Victoria Island... State Highway 4 Drawbridge across Old River, mile 14.8 between Victoria Island and Byron Tract, CA. The... Highway 4 Drawbridge, mile 14.8, over Old River, between Victoria Island and Byron Tract, CA....

  3. Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments.

    PubMed

    Ansari, Amir Mehdi; Ahmed, A Karim; Matsangos, Aerielle E; Lay, Frank; Born, Louis J; Marti, Guy; Harmon, John W; Sun, Zhaoli

    2016-10-01

    Green Fluorescent protein (GFP), used as a cellular tag, provides researchers with a valuable method of measuring gene expression and cell tracking. However, there is evidence to suggest that the immunogenicity and cytotoxicity of GFP potentially confounds the interpretation of in vivo experimental data. Studies have shown that GFP expression can deteriorate over time as GFP tagged cells are prone to death. Therefore, the cells that were originally marked with GFP do not survive and cannot be accurately traced over time. This review will present current evidence for the immunogenicity and cytotoxicity of GFP in in vivo studies by characterizing these responses.

  4. Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1(gfp/gfp) mice.

    PubMed

    Chrobok, Navina L; Jaouen, Alexandre; Fenrich, Keith K; Bol, John G J M; Wilhelmus, Micha M M; Drukarch, Benjamin; Debarbieux, Franck; van Dam, Anne-Marie

    2017-03-01

    Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1(gfp/gfp) mice during EAE, visualizing CX3CR1-GFP(+) monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP(+) monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP(+) monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology.

  5. Epilithic lichens in the Beacon sandstone formation, Victoria Land, Antarctica

    NASA Technical Reports Server (NTRS)

    Hale, M. E.; Friedmann, E. I. (Principal Investigator)

    1987-01-01

    The epilithic lichen flora on the Beacon sandstone formation in Victoria Land consists of seven species: Acarospora gwynnii Dodge & Rudolph, Buellia grisea Dodge & Baker, B. pallida Dodge & Baker, Carbonea capsulata (Dodge & Baker) Hale comb. nov., Lecanora fuscobrunnea Dodge & Baker, Lecidea cancriformis Dodge & Baker, and L. siplei Dodge & Baker. The typification of the species is given along with descriptions and distribution in Antarctica.

  6. Persistence of neutral polymorphisms in Lake Victoria cichlid fish

    PubMed Central

    Nagl, Sandra; Tichy, Herbert; Mayer, Werner E.; Takahata, Naoyuki; Klein, Jan

    1998-01-01

    Phylogenetic trees for groups of closely related species often have different topologies, depending on the genes used. One explanation for the discordant topologies is the persistence of polymorphisms through the speciation phase, followed by differential fixation of alleles in the resulting species. The existence of transspecies polymorphisms has been documented for alleles maintained by balancing selection but not for neutral alleles. In the present study, transspecific persistence of neutral polymorphisms was tested in the endemic haplochromine species flock of Lake Victoria cichlid fish. Putative noncoding region polymorphisms were identified at four randomly selected nuclear loci and tested on a collection of 12 Lake Victoria species and their putative riverine ancestors. At all loci, the same polymorphism was found to be present in nearly all the tested species, both lacustrine and riverine. Different polymorphisms at these loci were found in cichlids of other East African lakes (Malawi and Tanganyika). The Lake Victoria polymorphisms must have therefore arisen after the flocks now inhabiting the three great lakes diverged from one another, but before the riverine ancestors of the Lake Victoria flock colonized the Lake. Calculations based on the mtDNA clock suggest that the polymorphisms have persisted for about 1.4 million years. To maintain neutral polymorphisms for such a long time, the population size must have remained large throughout the entire period. PMID:9826684

  7. Persistence of neutral polymorphisms in Lake Victoria cichlid fish.

    PubMed

    Nagl, S; Tichy, H; Mayer, W E; Takahata, N; Klein, J

    1998-11-24

    Phylogenetic trees for groups of closely related species often have different topologies, depending on the genes used. One explanation for the discordant topologies is the persistence of polymorphisms through the speciation phase, followed by differential fixation of alleles in the resulting species. The existence of transspecies polymorphisms has been documented for alleles maintained by balancing selection but not for neutral alleles. In the present study, transspecific persistence of neutral polymorphisms was tested in the endemic haplochromine species flock of Lake Victoria cichlid fish. Putative noncoding region polymorphisms were identified at four randomly selected nuclear loci and tested on a collection of 12 Lake Victoria species and their putative riverine ancestors. At all loci, the same polymorphism was found to be present in nearly all the tested species, both lacustrine and riverine. Different polymorphisms at these loci were found in cichlids of other East African lakes (Malawi and Tanganyika). The Lake Victoria polymorphisms must have therefore arisen after the flocks now inhabiting the three great lakes diverged from one another, but before the riverine ancestors of the Lake Victoria flock colonized the Lake. Calculations based on the mtDNA clock suggest that the polymorphisms have persisted for about 1.4 million years. To maintain neutral polymorphisms for such a long time, the population size must have remained large throughout the entire period.

  8. Vocabulary Size Research at Victoria University of Wellington, New Zealand

    ERIC Educational Resources Information Center

    Nation, Paul; Coxhead, Averil

    2014-01-01

    The English Language Institute (now the School of Linguistics and Applied Language Studies) at Victoria University of Wellington has a long history of corpus-based vocabulary research, especially after the arrival of the second director of the institute, H. V. George, and the appointment of Helen Barnard, whom George knew in India. George's…

  9. The Free Kindergarten Union of Victoria, 1908-80.

    ERIC Educational Resources Information Center

    Gardiner, Lyndsay

    The social history of the Free Kindergarten Union of the State of Victoria, Australia, from inception in 1908 to the year 1980 is recorded in this book. Growth of the union is described within the context of the World Wars, the Depression, and urbanization and industrialization. The story begins with volunteerism and philanthropy, and with four…

  10. Molecular epidemiology and phylogeography of Schistosoma mansoni around Lake Victoria.

    PubMed

    Standley, C J; Kabatereine, N B; Lange, C N; Lwambo, N J S; Stothard, J R

    2010-11-01

    Intestinal schistosomiasis continues to be a major public health problem in sub-Saharan Africa, and is endemic in communities around Lake Victoria. Interest is growing in the molecular evolution and population genetic structure of Schistosoma mansoni and we describe a detailed analysis of the molecular epidemiology and phylogeography of S. mansoni from Lake Victoria. In total, 388 cytochrome oxidase 1 (COI) sequences were obtained from 25 sites along the Ugandan, Tanzanian and Kenyan shorelines of Lake Victoria, and 122 unique barcodes were identified; 9 corresponded to previously discovered barcodes from Lakes Victoria and Albert. A subset of the data, composed of COI sequences from miracidia from 10 individual children, was used for population genetics analyses; these results were corroborated by microsatellite analysis of 4 isolates of lab-passaged adult worms. Overall, 12 barcodes were found to be shared across all 3 countries, whereas the majority occurred singly and were locally restricted. The population genetics analyses were in agreement in revealing high diversity at the level of the human host and negligible population structuring by location. The lack of correlation between genetic distance and geographical distance in these data may be attributed to the confounding influence of high intra-individual diversity as well as human migration between communities.

  11. Catalogue Use at the State Library of Victoria

    ERIC Educational Resources Information Center

    Hider, Philip

    2008-01-01

    A questionnaire survey conducted at the State Library of Victoria indicates that users in general, and not just scholars, value the standard elements of bibliographic data found in the Library's catalogues, and consider all these elements useful for finding, identifying and selecting items. Rather than making do with less, users wanted more…

  12. Reaching Year 12 in Victoria, Australia: Student and School Influences

    ERIC Educational Resources Information Center

    Marks, Gary

    2014-01-01

    This paper examines student and school influences on reaching Year 12, the final year of schooling in Victoria, Australia. It analyses data from the population of students who were in Year 9 in 2008. Male, English-speaking background, government school, and especially Indigenous students were less likely to reach Year 12 than comparison groups.…

  13. Information Brokers in Victoria: Doing What, for Whom and How.

    ERIC Educational Resources Information Center

    Broadbent, Marianne; Kelson, Deborah

    1984-01-01

    Reports findings of study of information brokers in Victoria, Australia, which identified services offered by individual brokers and information brokerage businesses, resources used to provide those services, their clientele and pricing strategies, the attributes needed for a successful broker, and relationships between brokers and traditional…

  14. Metal-enhanced fluorescence of single green fluorescent protein (GFP).

    PubMed

    Fu, Yi; Zhang, Jian; Lakowicz, Joseph R

    2008-11-28

    The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer duration time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.

  15. Metal-enhanced fluorescence of single green fluorescent protein (GFP)

    SciTech Connect

    Fu Yi; Zhang Jian; Lakowicz, Joseph R.

    2008-11-28

    The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer duration time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.

  16. Investigation of a steam generator tube rupture sequence using VICTORIA

    SciTech Connect

    Bixler, N.E.; Erickson, C.M.; Schaperow, J.H.

    1995-12-31

    VICTORIA-92 is a mechanistic computer code for analyzing fission product behavior within the reactor coolant system (RCS) during a severe reactor accident. It provides detailed predictions of the release of radionuclides and nonradioactive materials from the core and transport of these materials within the RCS. The modeling accounts for the chemical and aerosol processes that affect radionuclide behavior. Coupling of detailed chemistry and aerosol packages is a unique feature of VICTORIA; it allows exploration of phenomena involving deposition, revaporization, and re-entrainment that cannot be resolved with other codes. The purpose of this work is to determine the attenuation of fission products in the RCS and on the secondary side of the steam generator in an accident initiated by a steam generator tube rupture (SGTR). As a class, bypass sequences have been identified in NUREG-1150 as being risk dominant for the Surry and Sequoyah pressurized water reactor (PWR) plants.

  17. Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes

    PubMed Central

    Stepanenko, Olesya V.; Verkhusha, Vladislav V.; Kuznetsova, Irina M.; Uversky, Vladimir N.; Turoverov, K.K.

    2010-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology. PMID:18691124

  18. Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes.

    PubMed

    Stepanenko, Olesya V; Verkhusha, Vladislav V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, K K

    2008-08-01

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

  19. Regional climate model performance in the Lake Victoria basin

    NASA Astrophysics Data System (ADS)

    Williams, Karina; Chamberlain, Jill; Buontempo, Carlo; Bain, Caroline

    2015-03-01

    Lake Victoria, the second largest freshwater lake in the world, plays a crucial role in the hydrology of equatorial eastern Africa. Understanding how climate change may alter rainfall and evaporation patterns is thus of vital importance for the economic development and the livelihood of the region. Regional rainfall distribution appears, up to a large extent, to be controlled by local drivers which may be not well resolved in general circulation model simulations. We investigate the performance over the Lake Victoria basin of an ensemble of UK Met Office Hadley Centre regional climate model (HadRM3P) simulations at 50 km, driven by five members of the Hadley Centre global perturbed-physics ensemble (QUMP). This is part of the validation of an ensemble of simulations that has been used to assess the impacts of climate change over the continent over the period 1950-2099. We find that the regional climate model is able to simulate a lake/land breeze over Lake Victoria, which is a significant improvement over the driving global climate model and a vital step towards reproducing precipitation characteristics in the region. The local precipitation correlates well with large-scale processes in the Pacific Ocean and Indian Ocean, which is in agreement with observations. We find that the spatial pattern of precipitation in the region and the diurnal cycle of convection is well represented although the amount of rainfall over the lake appears to be overestimated in most seasons. Reducing the observational uncertainty in precipitation over the lake through future field campaigns would enable this model bias to be better quantified. We conclude that increasing the spatial resolution of the model significantly improves its ability to simulate the current climate of the Lake Victoria basin. We suggest that, despite the higher computational costs, the inclusion of a model which allows two-way interactions between the lake and its surroundings should be seriously considered for

  20. View of 'Bottomless Bay' on Rim of 'Victoria'

    NASA Technical Reports Server (NTRS)

    2006-01-01

    As part of its investigation of 'Victoria Crater,' NASA's Mars Exploration Rover Opportunity examined a section of the scalloped rim called 'Bottomless Bay' (or 'Bahia sin Fondo'). This view shows the northeastern side of Bottomless Bay as seen from the southwest. The exposures combined into this mosaic were taken by the rover's panoramic camera through a 750-nanometer filter during the 1,019th Martian day, or sol, of Opportunity's Mars-surface mission (Dec. 5, 2006).

  1. Transcriptome pyrosequencing of the Antarctic brittle star Ophionotus victoriae.

    PubMed

    Burns, Gavin; Thorndyke, Michael C; Peck, Lloyd S; Clark, Melody S

    2013-03-01

    Brittle stars are included within a whole range of species, which contribute to knowledge in the medically important area of tissue regeneration. All brittle stars regenerate lose limbs, but the rate at which this occurs is highly variable and species-specific. One of the slowest rates of arm regeneration reported so far is that of the Antarctic Ophionotus victoriae. Additionally, O. victoriae also has an unusual delay in the onset of regeneration of about 5months. Both processes are of interest for the areas of regeneration biology and adaptation to cold environments. One method of understanding the details of regeneration events in brittle stars is to characterise the genes involved. In the largest transcriptome study of any ophiuroid to date, we describe the results of mRNA pyrosequencing from pooled samples of regenerating arms of O. victoriae. The sequencing reads resulted in 18,000 assembled contiguous sequences of which 19% were putatively annotated by blast sequence similarity searching. We focus on the identification of major gene families and pathways with potential relevance to the regenerative processes including the Wnt/β-catenin pathway, Hox genes, the SOX gene family and the TGF beta signalling pathways. These data significantly increase the amount of ophiuroid sequences publicly available and provide candidate transcripts for the further investigation of the unusual regenerative process in this Antarctic ophiuroid.

  2. Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag

    PubMed Central

    Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

    2015-01-01

    With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as

  3. Recent MELCOR and VICTORIA Fission Product Research at the NRC

    SciTech Connect

    Bixler, N.E.; Cole, R.K.; Gauntt, R.O.; Schaperow, J.H.; Young, M.F.

    1999-01-21

    The MELCOR and VICTORIA severe accident analysis codes, which were developed at Sandia National Laboratories for the U. S. Nuclear Regulatory Commission, are designed to estimate fission product releases during nuclear reactor accidents in light water reactors. MELCOR is an integrated plant-assessment code that models the key phenomena in adequate detail for risk-assessment purposes. VICTORIA is a more specialized fission- product code that provides detailed modeling of chemical reactions and aerosol processes under the high-temperature conditions encountered in the reactor coolant system during a severe reactor accident. This paper focuses on recent enhancements and assessments of the two codes in the area of fission product chemistry modeling. Recently, a model for iodine chemistry in aqueous pools in the containment building was incorporated into the MELCOR code. The model calculates dissolution of iodine into the pool and releases of organic and inorganic iodine vapors from the pool into the containment atmosphere. The main purpose of this model is to evaluate the effect of long-term revolatilization of dissolved iodine. Inputs to the model include dose rate in the pool, the amount of chloride-containing polymer, such as Hypalon, and the amount of buffering agents in the containment. Model predictions are compared against the Radioiodine Test Facility (RTF) experiments conduced by Atomic Energy of Canada Limited (AECL), specifically International Standard Problem 41. Improvements to VICTORIA's chemical reactions models were implemented as a result of recommendations from a peer review of VICTORIA that was completed last year. Specifically, an option is now included to model aerosols and deposited fission products as three condensed phases in addition to the original option of a single condensed phase. The three-condensed-phase model results in somewhat higher predicted fission product volatilities than does the single-condensed-phase model. Modeling of U02

  4. Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

    SciTech Connect

    Wang Hongmin; Monteiro, Mervyn J. . E-mail: monteiro@umbi.umd.edu

    2007-08-01

    Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases.

  5. Illumina MiSeq sequencing disfavours a sequence motif in the GFP reporter gene

    PubMed Central

    Van den Hoecke, Silvie; Verhelst, Judith; Saelens, Xavier

    2016-01-01

    Green fluorescent protein (GFP) is one of the most used reporter genes. We have used next-generation sequencing (NGS) to analyse the genetic diversity of a recombinant influenza A virus that expresses GFP and found a remarkable coverage dip in the GFP coding sequence. This coverage dip was present when virus-derived RT-PCR product or the parental plasmid DNA was used as starting material for NGS and regardless of whether Nextera XT transposase or Covaris shearing was used for DNA fragmentation. Therefore, the sequence coverage dip in the GFP coding sequence was not the result of emerging GFP mutant viruses or a bias introduced by Nextera XT fragmentation. Instead, we found that the Illumina MiSeq sequencing method disfavours the ‘CCCGCC’ motif in the GFP coding sequence. PMID:27193250

  6. Development of Neutralization Assay Using an eGFP Chikungunya Virus

    PubMed Central

    Deng, Cheng-Lin; Liu, Si-Qing; Zhou, Dong-Gen; Xu, Lin-Lin; Li, Xiao-Dan; Zhang, Pan-Tao; Li, Peng-Hui; Ye, Han-Qing; Wei, Hong-Ping; Yuan, Zhi-Ming; Qin, Cheng-Feng; Zhang, Bo

    2016-01-01

    Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV. PMID:27367716

  7. Live imaging of GFP-labeled proteins in Drosophila oocytes.

    PubMed

    Pokrywka, Nancy Jo

    2013-03-29

    The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes. The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent and provides a useful system for investigating cytoskeleton function at these stages. Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

  8. Making genes green: creating green fluorescent protein (GFP) fusions with blunt-end PCR products.

    PubMed

    Lo, W; Rodgers, W; Hughes, T

    1998-07-01

    The jellyfish green fluorescent protein (GFP) has proven to be a useful tool in protein localization and trafficking studies. Fused to GFP, a protein of interest can be visualized and tracked in vivo through fluorescence microscopy. However, the process of making these fusion proteins is often tedious and painstaking. Here, we describe a simple and quick method for creating GFP fusion proteins using blunt-end PCR product ligation.

  9. Construction of ICAM-1-GFP and its binding with Molt-4 cells.

    PubMed

    Chen, Wei-Hua; DA, Wan-Ming; Gao, Chun-Ji

    2009-06-01

    This study was aimed to clone human intercellular adhesion molecule-1 (ICAM-1) gene, to transfect the constructed eukaryotic expression vector ICAM-1-GFP into CHO cells, as well as to detect ICAM-1-GFP expression in CHO cells binding with Molt-4 cells. ICAM-1 cDNA gene was amplified by RT-PCR and inserted in PMD(18)-T vector. Then ICAM-1 cDNA from pMD18-ICAM-1 vector was subcloned into eukaryotic expression vector pEGFP-C1 to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing were used to confirm the recombinant vector. After stable transfection of CHO-K1 cells with the recombinant vector, the expression and subcellular localization of ICAM-1-GFP were detected by RT-PCR, flow cytometry and fluorescence microscopy. The function of ICAM-1-GFP fusion protein was assessed by the binding of ICAM-1-GFP/CHO cells to Molt-4 cells. The results showed that 1622 bp full-length ICAM-1 cDNA obtained and was successfully ligated with pMD(18)-T-vector, subcloned to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing indicated that recombinant ICAM-1-GFP was successfully constructed and ICAM-1-GFP was expressed stably in CHO cells. ICAM-1-GFP expression was only observed in the cytoplasm of ICAM-1-GFP/CHO cells by fluorescence microscopy. The ICAM-1-GFP/CHO cells were bound to PMA-treated Molt-4 cells. The expression of MEM-148 was very weak in PMA-treated Molt-4 cells. It is concluded that the ICAM-1-GFP eukaryotic expression vector has been constructed successfully and expresses stably in CHO cells. PMA can increase the binding of Molt-4 cells to ICAM-1-GFP/CHO cells by inducing specialized form of ICAM-1 clustering.

  10. A New Protein-Protein Interaction Sensor Based on Tripartite Split-GFP Association

    PubMed Central

    Cabantous, Stéphanie; Nguyen, Hau B.; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A.; Favre, Gilles; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence. PMID:24092409

  11. A new protein-protein interaction sensor based on tripartite split-GFP association.

    PubMed

    Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-10-04

    Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

  12. Oxygen-dependent secretion of a bioactive hepcidin-GFP chimera.

    PubMed

    Chachami, Georgia; Lyberopoulou, Aggeliki; Kalousi, Alkmini; Paraskeva, Efrosyni; Pantopoulos, Kostas; Simos, George

    2013-06-14

    Hepcidin, a hepatic hormone, regulates serum iron levels by controlling both intestinal iron absorption and iron release from macrophages. Although transcription of hepcidin is controlled by diverse stimuli, it remains elusive if post-transcriptional steps of its production are also regulated. To address this issue, GFP was fused to the C-terminus of hepcidin and the chimeric hepcidin-GFP protein was expressed in hepatoma Huh7 cells. Expression and secretion of hepcidin-GFP were analyzed by fluorescence microscopy or western blotting and its activity was assessed by in vitro biological assays. Transient over-expression of hepcidin-GFP resulted in production and secretion of premature forms. On the other hand, stable low-level expression led to synthesis and secretion of a properly matured hepcidin-GFP. This form was biologically active since it affected appropriately the levels of IRP2 and ferritin in human THP1 monocytes and targeted ferroportin in mouse J774 macrophages. Treatment of hepcidin-GFP expressing cells with hypoxia (0.1% O2) altered the subcellular distribution of pro-hepcidin-GFP and significantly reduced the secretion of mature hepcidin-GFP. Our hepcidin-GFP expression system allows the investigation of post-transcriptional processing of hepcidin and implicates hypoxia in its secretion control.

  13. Drowning deaths between 1861 and 2000 in Victoria, Australia

    PubMed Central

    Ozanne-Smith, Joan

    2017-01-01

    Abstract Objective To identify the long-term patterns of drowning mortality in the state of Victoria, Australia, and to describe the historical context in which the decrease occurred. Methods We obtained data on drowning deaths and population statistics from the Australian Bureau of Statistics and its predecessors for the period 1861 to 2000. From these data, we calculated drowning death rates per 100 000 population per year, by gender and age. We reviewed primary and secondary historical resources, such as government and newspaper archives, books and the Internet, to identify changes or events in the state that may have affected drowning mortality. Findings From 1861 to 2000, at least 18 070 people drowned in Victoria. Male drowning rates were higher than those for females in all years and for all ages. Both sexes experienced the highest drowning rate in 1863 (79.5 male deaths per 100 000 population and 18.8 female death per 100 000 population). The lowest drowning rate was documented in 2000 (1.4 male deaths per 100 000 population and 0.3 female deaths per 100 000 population). The reduction patterns of drowning mortality occurred within a historical context of factors that directly affected drowning mortality, such as the improvement in people’s water safety skills, or those that incidentally affected drowning mortality, like infrastructure development. Conclusion We identified patterns of reduction in drowning mortality, both in males and females and across age groups. These patterns could be linked to events and factors that happened in Victoria during this period. These findings may have relevance to current developing communities. PMID:28250530

  14. Details of Layers in Victoria Crater's Cape St. Vincent

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity rover spent about 300 sols (Martian days) during 2006 and 2007 traversing the rim of Victoria Crater. Besides looking for a good place to enter the crater, the rover obtained images of rock outcrops exposed at several cliffs along the way.

    The cliff in this image from Opportunity's panoramic camera (Pancam) is informally named Cape St. Vincent. It is a promontory approximately 12 meters (39 feet) tall on the northern rim of Victoria crater, near the farthest point along the rover's traverse around the rim. Layers seen in Cape St. Vincent have proven to be among the best examples of meter scale cross-bedding observed on Mars to date. Cross-bedding is a geologic term for rock layers which are inclined relative to the horizontal and which are indicative of ancient sand dune deposits. In order to get a better look at these outcrops, Pancam 'super-resolution' imaging techniques were utilized. Super-resolution is a type of imaging mode which acquires many pictures of the same target to reconstruct a digital image at a higher resolution than is native to the camera. These super-resolution images have allowed scientists to discern that the rocks at Victoria Crater once represented a large dune field, not unlike the Sahara desert on Earth, and that this dune field migrated with an ancient wind flowing from the north to the south across the region. Other rover chemical and mineral measurements have shown that many of the ancient sand dunes studied in Meridiani Planum were modified by surface and subsurface liquid water long ago.

    This is a Mars Exploration Rover Opportunity Panoramic Camera image acquired on sol 1167 (May 7, 2007), and was constructed from a mathematical combination of 16 different blue filter (480 nm) images.

  15. Exploration of Victoria crater by the Mars rover Opportunity.

    PubMed

    Squyres, S W; Knoll, A H; Arvidson, R E; Ashley, J W; Bell, J F; Calvin, W M; Christensen, P R; Clark, B C; Cohen, B A; de Souza, P A; Edgar, L; Farrand, W H; Fleischer, I; Gellert, R; Golombek, M P; Grant, J; Grotzinger, J; Hayes, A; Herkenhoff, K E; Johnson, J R; Jolliff, B; Klingelhöfer, G; Knudson, A; Li, R; McCoy, T J; McLennan, S M; Ming, D W; Mittlefehldt, D W; Morris, R V; Rice, J W; Schröder, C; Sullivan, R J; Yen, A; Yingst, R A

    2009-05-22

    The Mars rover Opportunity has explored Victoria crater, an approximately 750-meter eroded impact crater formed in sulfate-rich sedimentary rocks. Impact-related stratigraphy is preserved in the crater walls, and meteoritic debris is present near the crater rim. The size of hematite-rich concretions decreases up-section, documenting variation in the intensity of groundwater processes. Layering in the crater walls preserves evidence of ancient wind-blown dunes. Compositional variations with depth mimic those approximately 6 kilometers to the north and demonstrate that water-induced alteration at Meridiani Planum was regional in scope.

  16. Exploration of Victoria crater by the mars rover opportunity

    USGS Publications Warehouse

    Squyres, S. W.; Knoll, A.H.; Arvidson, R. E.; Ashley, James W.; Bell, J.F.; Calvin, W.M.; Christensen, P.R.; Clark, B. C.; Cohen, B. A.; De Souza, P.A.; Edgar, L.; Farrand, W. H.; Fleischer, I.; Gellert, Ralf; Golombek, M.P.; Grant, J.; Grotzinger, J.; Hayes, A.; Herkenhoff, K. E.; Johnson, J. R.; Jolliff, B.; Klingelhofer, G.; Knudson, A.; Li, R.; McCoy, T.J.; McLennan, S.M.; Ming, D. W.; Mittlefehldt, D. W.; Morris, R.V.; Rice, J. W.; Schroder, C.; Sullivan, R.J.; Yen, A.; Yingst, R.A.

    2009-01-01

    The Mars rover Opportunity has explored Victoria crater, a ???750-meter eroded impact crater formed in sulfate-rich sedimentary rocks. Impact-related stratigraphy is preserved in the crater walls, and meteoritic debris is present near the crater rim. The size of hematite-rich concretions decreases up-section, documenting variation in the intensity of groundwater processes. Layering in the crater walls preserves evidence of ancient wind-blown dunes. Compositional variations with depth mimic those ???6 kilometers to the north and demonstrate that water-induced alteration at Meridiani Planum was regional in scope.

  17. View of 'Bottomless Bay' on Rim of 'Victoria' (Altered Contrast)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    As part of its investigation of 'Victoria Crater,' NASA's Mars Exploration Rover Opportunity examined a section of the scalloped rim called 'Bottomless Bay' (or 'Bahia sin Fondo'). This view shows the northeastern side of Bottomless Bay as seen from the southwest. The exposures combined into this mosaic were taken by the rover's panoramic camera through a 750-nanometer filter during the 1,019th Martian day, or sol, of Opportunity's Mars-surface mission (Dec. 5, 2006). Contrast has been altered to improve the visibility of details in shadowed areas.

  18. Bigger Crater Farther South of 'Victoria' on Mars

    NASA Technical Reports Server (NTRS)

    2008-01-01

    [figure removed for brevity, see original site] Annotated version

    The team operating NASA's Mars Exploration Rover Opportunity has chosen southeast as the direction for the rover's next extended journey, toward a crater more than 20 times wider than 'Victoria Crater.' Opportunity exited Victoria Crater on Aug. 28, 2008, after nearly a year investigating the interior.

    The crater to the southeast is about 22 kilometers (13.7 miles) in diameter and about 300 meters (1,000 feet) deep, exposing a much thicker stack of rock layers than those examined in Victoria Crater.

    The rover team informally calls the bigger crater 'Endeavour' and emphasizes that Opportunity may well never reach it. The rover has already operated more than 18 times longer than originally planned, and the distance to the big crater, about 12 kilometers (7 miles) matches the total distance Opportunity has driven since landing in early 2004. Driving southeastward is expected to take Opportunity to exposures of younger rock layers than is has previously seen and to provide access to rocks on the plain that were thrown long distances by impacts that excavated even deeper, more distant craters.

    The crater that Opportunity will drive toward dominates this orbital view from the Thermal Emission Imaging System (THEMIS) camera on NASA's Mars Odyssey orbiter. The much smaller Victoria Crater is the most prominent circle near the upper left corner of the image. This view is a mosaic of about 50 separate visible-light images taken by THEMIS.

    NASA's Jet Propulsion Laboratory manages the Mars Odyssey and Mars Exploration Rover missions for the NASA Science Mission Directorate, Washington, D.C. THEMIS was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the

  19. Reforming Victoria's primary health and community service sector: rural implications.

    PubMed

    Alford, K

    2000-01-01

    In 1999 the Victorian primary care and community support system began a process of substantial reform, involving purchasing reforms and a contested selection process between providers in large catchment areas across the State. The Liberal Government's electoral defeat in September 1999 led to a review of these reforms. This paper questions the reforms from a rural perspective. They were based on a generic template that did not consider rural-urban differences in health needs or other differences including socio-economic status, and may have reinforced if not aggravated rural-urban differences in the quality of and access to primary health care in Victoria.

  20. Formula-Based Public School Funding System in Victoria: An Empirical Analysis of Equity

    ERIC Educational Resources Information Center

    Bandaranayake, Bandara

    2013-01-01

    This article explores the formula-based school funding system in the state of Victoria, Australia, where state funds are directly allocated to schools based on a range of equity measures. The impact of Victoria' funding system for education in terms of alleviating inequality and disadvantage is contentious, to say the least. It is difficult to…

  1. A Snapshot: Multicultural Music Teaching in Schools in Victoria, Australia, Portrayed by School Teachers

    ERIC Educational Resources Information Center

    Nethsinghe, Rohan Nishantha

    2012-01-01

    Due to the changing demographic factors and as demanded by the governmental policies and regulations, schools in Victoria, Australia, are expected to foster multicultural educational programs that address the diverse needs of students. Research has found that school teachers in Victoria struggle to provide the aspired to multicultural education…

  2. Events Management Education through CD-ROM Simulation at Victoria University of Technology.

    ERIC Educational Resources Information Center

    Perry, Marcia; And Others

    There has been a rapid growth in the events industry in Victoria and Australia over the past five years with an increase in large scale events--resulting in substantive economic impact. The growth in events in Australia is projected to continue to beyond 2001. The Department of Management at Victoria University of Technology (VU) received a…

  3. 78 FR 48373 - Approval and Promulgation of Air Quality Implementation Plans; Texas; Victoria County; 1997 8...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-08

    ... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY 40 CFR Part 52 Approval and Promulgation of Air Quality Implementation Plans; Texas; Victoria... Implementation Plan (SIP). The revision consists of a maintenance plan for Victoria County developed to...

  4. 78 FR 76403 - Requested Administrative Waiver of the Coastwise Trade Laws: Vessel VICTORIA; Invitation for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-17

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF TRANSPORTATION Maritime Administration Requested Administrative Waiver of the Coastwise Trade Laws: Vessel VICTORIA... of the vessel VICTORIA is: Intended Commercial Use Of Vessel: ``Carrying up to 12 passengers for...

  5. Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells

    PubMed Central

    Noda, Asao; Suemori, Hirofumi; Hirai, Yuko; Hamasaki, Kanya; Kodama, Yoshiaki; Mitani, Hiroshi; Landes, Reid D.; Nakamura, Nori

    2015-01-01

    It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3’ portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation

  6. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-12-29

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

  7. Expression of GFP in nuclear transplants generated by transplantation of embryonic cell nuclei from GFP-transgenic fish into nonenucleated eggs of medaka, Oryzias latipes.

    PubMed

    Niwa, K; Kani, S; Kinoshita, M; Ozato, K; Wakamatsu, Y

    2000-01-01

    In order to investigate whether foreign genes can be used as genetic markers of donor nuclei in fish nuclear transplantation, expression of the GFP gene derived from donor nuclei was examined in nuclear transplants in medaka (Oryzias latipes). Embryonic nuclei were obtained from blastula embryos produced by crossing of transgenic fish of the wild-type strain heterozygous for the GFP gene with nontransgenic ones or by mutual crossing between transgenic fish. The GFP gene was driven by the promoter of the medaka elongation factor gene, EF-1alpha-A, which is known to induce GFP expression in many tissues except for the muscle in the transgenic fish. The nuclei were transplanted into nonenucleated unfertilized eggs of the orange-red strain. Adult nuclear transplants were successfully obtained at the rate of about 2% of the operated eggs. They were triploid and had no reproductive potential. The GFP gene was expressed in embryos, fry, and adults of nuclear transplants in a pattern similar to that in the transgenic fish. These results indicate that GFP is useful as a foreign genetic marker of donor nuclei in fish nuclear transplantation.

  8. Analysis of continuous GPS measurements from southern Victoria Land, Antarctica

    USGS Publications Warehouse

    Willis, Michael J.

    2007-01-01

    Several years of continuous data have been collected at remote bedrock Global Positioning System (GPS) sites in southern Victoria Land, Antarctica. Annual to sub-annual variations are observed in the position time-series. An atmospheric pressure loading (APL) effect is calculated from pressure field anomalies supplied by the European Centre for Medium-Range Weather Forecasts (ECMWF) model loading an elastic Earth model. The predicted APL signal has a moderate correlation with the vertical position time-series at McMurdo, Ross Island (International Global Navigation Satellite System Service (IGS) station MCM4), produced using a global solution. In contrast, a local solution in which MCM4 is the fiducial site generates a vertical time series for a remote site in Victoria Land (Cape Roberts, ROB4) which exhibits a low, inverse correlation with the predicted atmospheric pressure loading signal. If, in the future, known and well modeled geophysical loads can be separated from the time-series, then local hydrological loading, of interest for glaciological and climate applications, can potentially be extracted from the GPS time-series.

  9. On the Rim of 'Victoria Crater' (Vertical Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a vertical projection with geometric seam correction.

  10. On the Rim of 'Victoria Crater' (Polar Projection)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    NASA's Mars rover Opportunity reached the rim of 'Victoria Crater' in Mars' Meridiani Planum region with a 26-meter (85-foot) drive during the rover's 951st Martian day, or sol (Sept. 26, 2006). After the drive, the rover's navigation camera took the three exposures combined into this view of the crater's interior. This crater has been the mission's long-term destination for the past 21 Earth months.

    A half mile in the distance one can see about 20 percent of the far side of the crater framed by the rocky cliffs in the foreground to the left and right of the image. The rim of the crater is composed of alternating promontories, rocky points towering approximately 70 meters (230 feet) above the crater floor, and recessed alcoves. The bottom of the crater is covered by sand that has been shaped into ripples by the Martian wind.

    The position at the end of the sol 951 drive is about six meters from the lip of an alcove called 'Duck Bay.' The rover team planned a drive for sol 952 that would move a few more meters forward, plus more imaging of the near and far walls of the crater.

    Victoria Crater is about five times wider than 'Endurance Crater,' which Opportunity spent six months examining in 2004, and about 40 times wider than 'Eagle Crater,' where Opportunity first landed.

    This view is presented as a polar projection with geometric seam correction.

  11. Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

    PubMed

    Marr, Lisa; Lülf, Anna-Theresa; Freudenstein, Astrid; Sutter, Gerd; Volz, Asisa

    2016-04-01

    Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens.

  12. Synthesis and photochemistry of pH-sensitive GFP chromophore analogues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nobel GFP chromophore analogues containing 2-thienyl-, 5-methyl-2-furyl-, 2-pyrryl, and 6-methyl-2-pyridyl-groups were synthesized, and their fluorescence spectra were recorded across pH range of 1 to 7. The GFP chromophores prevent photoisomerizaiton in acidic media and increase their fluorescent a...

  13. Disappearance of GFP-Positive Hepatocytes Transplanted into the Liver of Syngeneic Wild-Type Rats Pretreated with Retrorsine

    PubMed Central

    Maeda, Hiromichi; Shigoka, Masatoshi; Wang, Yongchun; Fu, Yingxin; Wesson, Russell N.; Lin, Qing; Montgomery, Robert A.; Enzan, Hideaki; Sun, Zhaoli

    2014-01-01

    Background and Aim Green fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP. Methods Hepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison. Results All biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14. Conclusion GFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be

  14. A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants

    PubMed Central

    Xie, Wenjun; Nielsen, Mads Eggert; Pedersen, Carsten

    2017-01-01

    To understand the function of membrane proteins, it is imperative to know their topology. For such studies, a split green fluorescent protein (GFP) method is useful. GFP is barrel-shaped, consisting of 11 β-sheets. When the first ten β-sheets (GFP1-10) and the 11th β-sheet (GFP11) are expressed from separate genes they will self-assembly and reconstitute a fluorescent GFP protein. However, this will only occur when the two domains co-localize in the same cellular compartment. We have developed an easy-to-use Gateway vector set for determining on which side of the membrane the N- and C-termini are located. Two vectors were designed for making N- and C-terminal fusions between the membrane proteins-of-interest and GFP11, while another three plasmids were designed to express GFP1-10 in either the cytosol, the endoplasmic reticulum (ER) lumen or the apoplast. We tested functionality of the system by applying the vector set for the transmembrane domain, CNXTM, of the ER membrane protein, calnexin, after transient expression in Nicotiana benthamiana leaves. We observed GFP signal from the ER when we reciprocally co-expressed GFP11-CNXTM with GFP1-10-HDEL and CNXTM-GFP with cytosolic GFP1-10. The opposite combinations did not result in GFP signal emission. This test using the calnexin ER-membrane domain demonstrated its C-terminus to be in the cytosol and its N-terminus in the ER lumen. This result confirmed the known topology of calnexin, and we therefore consider this split-GFP system highly useful for ER membrane topology studies. Furthermore, the vector set provided is useful for detecting the topology of proteins on other membranes in the cell, which we confirmed for a plasma membrane syntaxin. The set of five Ti-plasmids are easily and efficiently used for Gateway cloning and transient transformation of N. benthamiana leaves. PMID:28085941

  15. Development of a stable ERroGFP variant suitable for monitoring redox dynamics in the ER

    PubMed Central

    Hoseki, Jun; Oishi, Asami; Fujimura, Takaaki; Sakai, Yasuyoshi

    2016-01-01

    The endoplasmic reticulum (ER) is an essential organelle for cellular metabolic homeostasis including folding and maturation of secretory and membrane proteins. Disruption of ER proteostasis has been implicated in the pathogenesis of various diseases such as diabetes and neurodegenerative diseases. The ER redox state, which is an oxidative environment suitable for disulfide-bond formation, is essential for ER protein quality control. Hence, detection of the ER redox state, especially in living cells, is essential to understand the mechanism by which the redox state of the ER is maintained. However, methods to detect the redox state of the ER have not been well-established because of inefficient folding and stability of roGFP variants with oxidative redox potential like roGFP-iL. Here we have improved the folding efficiency of ER-targeted roGFP-iL (ERroGFP-iL) in cells by introducing superfolder GFP (sfGFP) mutations. Four specific amino acid substitutions (S30R, Y39N, T105N and I171V) greatly improved folding efficiency in Escherichia coli and in the ER of HeLa cells, as well as the thermostability of the purified proteins. Introduction of these mutations also enhanced the dynamic range for redox change both in vitro and in the ER of living cells. ER-targeted roGFP-S4 (ERroGFP-S4) possessing these four mutations could detect physiological redox changes within the ER. ERroGFP-S4 is therefore a novel probe suitable for monitoring redox change in the ER. ERroGFP-S4 can be applied to detect aberrant ER redox states associated with various pathological conditions and to identify the mechanisms used to maintain the redox state of the ER. PMID:26934978

  16. Plasmids for C-terminal tagging in Saccharomyces cerevisiae that contain improved GFP proteins, Envy and Ivy.

    PubMed

    Slubowski, Christian J; Funk, Alyssa D; Roesner, Joseph M; Paulissen, Scott M; Huang, Linda S

    2015-04-01

    Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance.

  17. Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

    PubMed Central

    Limon, Agenor; Reyes-Ruiz, Jorge Mauricio; Eusebi, Fabrizio; Miledi, Ricardo

    2007-01-01

    Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oocytes, with similar EC50 values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate- to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins. PMID:17881566

  18. Forty Meters from Entry to Victoria Crater (Vertical)

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view is presented as a vertical projection with geometric seam correction.

  19. Forty Meters from Entry to Victoria Crater (Polar)

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view is presented as a polar projection with geometric seam correction.

  20. Layers of 'Cabo Frio' in 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of 'Victoria crater' is looking southeast from 'Duck Bay' towards the dramatic promontory called 'Cabo Frio.' The small crater in the right foreground, informally known as 'Sputnik,' is about 20 meters (about 65 feet) away from the rover, the tip of the spectacular, layered, Cabo Frio promontory itself is about 200 meters (about 650 feet) away from the rover, and the exposed rock layers are about 15 meters (about 50 feet) tall. This is a red-blue stereo anaglyph generated from images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 430-nanometer filters.

  1. Layers of 'Cape Verde' in 'Victoria Crater' (Enhanced)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is a false color rendering (enhanced to bring out details from within the shadowed regions of the scene) of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  2. Layers of 'Cape Verde' in 'Victoria Crater' (False Color)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is an enhanced false color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  3. Layers of 'Cape Verde' in 'Victoria Crater' (Stereo)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of Victoria crater is looking north from 'Duck Bay' towards the dramatic promontory called 'Cape Verde.' The dramatic cliff of layered rocks is about 50 meters (about 165 feet) away from the rover and is about 6 meters (about 20 feet) tall. The taller promontory beyond that is about 100 meters (about 325 feet) away, and the vista beyond that extends away for more than 400 meters (about 1300 feet) into the distance. This is a red-blue stereo anaglyph generated from images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 430-nanometer filters.

  4. Layers of 'Cabo Frio' in 'Victoria Crater' (False Color)

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This view of 'Victoria crater' is looking southeast from 'Duck Bay' towards the dramatic promontory called 'Cabo Frio.' The small crater in the right foreground, informally known as 'Sputnik,' is about 20 meters (about 65 feet) away from the rover, the tip of the spectacular, layered, Cabo Frio promontory itself is about 200 meters (about 650 feet) away from the rover, and the exposed rock layers are about 15 meters (about 50 feet) tall. This is an enhanced false color rendering of images taken by the panoramic camera (Pancam) on NASA's Mars Exploration Rover Opportunity during the rover's 952nd sol, or Martian day, (Sept. 28, 2006) using the camera's 750-nanometer, 530-nanometer and 430-nanometer filters.

  5. Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs.

    PubMed

    Millwood, R J; Halfhill, M D; Harkins, D; Russotti, R; Stewart, C N

    2003-03-01

    The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes.

  6. Cnidarian and bilaterian promoters can direct GFP expression in transfected hydra.

    PubMed

    Miljkovic, Marijana; Mazet, Françoise; Galliot, Brigitte

    2002-06-15

    Complete sexual development is not easily amenable to experimentation in hydra. Therefore, the analysis of gene function and gene regulation requires the introduction of exogenous DNA in a large number of cells of the hydra polyps and the significant expression of reporter constructs in these cells. We present here the procedure whereby we coupled DNA injection into the gastric cavity to electroporation of the whole animal in order to efficiently transfect hydra polyps. We could detect GFP fluorescence in both endodermal and ectodermal cell layers of live animals and in epithelial as well as interstitial cell types of dissociated hydra. In addition, we could confirm GFP protein expression by showing colocalisation between GFP fluorescence and anti-GFP immunofluorescence. Finally, when a FLAG epitope was inserted in-frame with the GFP coding sequence, GFP fluorescence also colocalised with anti-FLAG immunofluorescence. This GFP expression in hydra cells was directed by various promoters, either homologous, like the hydra homeobox cnox-2 gene promoter, or heterologous, like the two nematode ribosomal protein S5 and L28 gene promoters, and the chicken beta-actin gene promoter. This strategy provides new tools for dissecting developmental molecular mechanisms in hydra; more specifically, the genetic regulations that take place in endodermal cells at the time budding or regeneration is initiated.

  7. Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs

    NASA Technical Reports Server (NTRS)

    Millwood, R. J.; Halfhill, M. D.; Harkins, D.; Russotti, R.; Stewart, C. N. Jr

    2003-01-01

    The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes.

  8. Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

    PubMed

    Shirzad, Hedayatollah; Esmaeili, Fariba; Bakhshalizadeh, Shabnam; Ebrahimie, Marzieh; Ebrahimie, Esmaeil

    2017-04-01

    Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP(+)). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP(+) into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP(+) cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation.

  9. Re-evaluation of the kinematics of Victoria Block using continuous GNSS data

    NASA Astrophysics Data System (ADS)

    Fernandes, R. M. S.; Miranda, J. M.; Delvaux, D.; Stamps, D. S.; Saria, E.

    2013-04-01

    The divergent boundary between the Somalia and Nubia plates is a complex tectonic domain where extensional processes are localized along narrow rift structures, isolating small blocks imbedded within the East African Rift. One of these tectonic units is the Victoria Block, which is the subject of this study. Here we process space-geodetic data for 37 permanent GNSS stations distributed along Nubia, Somalia and Victoria to (1) compute the motion of the three tectonic units in the ITRF2008 reference frame and (2) deduce the relative motion of Victoria with respect to its neighbouring plates. The Nubia Plate motion is computed from a set of 25 stations, the Somalia Plate motion from a set of 7 stations and the Victoria motion from a set of 5 stations. Although the number and distribution of the used stations is still not optimal, the good adjustment between observed and predicted motions confirms that Victoria acts as a rigid tectonic block. The instantaneous relative Euler poles for the Nubia-Victoria and Somalia-Victoria pairs are now evaluated as 10.66°N, 32.98E°, 0.120° Myr-1 and 8.02°S, 32.29°E, 0.159° Myr-1, respectively. The computation of the relative interplate velocities along Victoria's boundary is straightforward in most situations because the western and northeastern boundary segments correspond to well-developed rift basins, where extension is mostly normal to rift basin flanks and seismicity concentrates along narrow structures. This is particularly evident on the Western Branch between Victoria and Nubia. The southeastern limit of the Victoria Block is poorly defined, and geodetic data indicate that differential motion between Somalia and Victoria may be accommodated by a complex boundary area, which roughly encompasses the Masai Terrain. Geodetic observations of the Victoria-Somalia boundary along the Eastern Branch, particularly in the Manyara Rift, reveal highly oblique horizontal extension. In this region seismicity is sparse which suggests

  10. Health promotion in local churches in Victoria: an exploratory study.

    PubMed

    Ayton, Darshini; Manderson, Lenore; Smith, Ben J; Carey, Gemma

    2016-11-01

    Church-based health promotion has increasingly gained attention in strategies to address health disparities. In Australia, we have limited understanding of the role of local churches in health promotion and without this, how they might be involved in meaningful partnerships to tackle public health challenges. The objective of this qualitative study was to explore how churches are involved in health promotion in the state of Victoria. The research involved in-depth interviews with ministers from 30 churches in urban and rural Victoria, and case studies with 10 of these churches to enable further exploration. These case studies, conducted in 2010, included interviews with church staff, focus groups with volunteers, participant observation and document analysis. Analysis was iterative, utilising open, axial and thematic coding. Three different expressions of church - traditional, new modern and emerging - were identified and found to differentiate the levels and types of health promotion activity. Case studies illustrate the different expressions of how church mission influences health promotion activity. The traditional churches were involved particularly in disease screening and health education activities with their own, predominantly older congregation members. The new modern churches tended to have the material and human resources to be harnessed in health promotion activities involving congregation members and others. Emerging churches, in contrast, engaged in broad health-promoting activities, including disease prevention, lifestyle activities and socio-ecological approaches at a community level. These research findings highlight the opportunities and challenges of engaging with local churches in health promotion efforts and public health programmes to address health inequities.

  11. Offshore investigations on Wilkes land-Victoria land margin, Antarctica

    SciTech Connect

    Eittreim, S.L.

    1984-04-01

    In January 1984, the US Geological Survey research vessel S. P. Lee carried out investigations of the Antarctic continental margin in the Wilkes Land Victoria Land areas, using 24-channel and high-resolution seismic, sonobuoy refraction, gravity, magnetic, and bottom-sampling methods. This investigation augmented previous surveys of the Dumont d'Urville area by the French Petroleum Institute and explored new areas west and east to the boundary between the onshore Wilkes basin and the Victoria Land highlands. These surveys defined sediment thickness distribution and seismic stratigraphy in this frontier area. The tectonic style of the boundary between the East Antarctic craton and the younger crust of West Antarctica in the Ross Sea is revealed by one multichannel seismic line across this important boundary. The initial breakup of Antarctical from Australia occurred as a slowly spreading phase during the middle Cretaceous. According to Deep Sea Drilling Project results on the Tasman Rise, conditions of restricted circulation existed in the growing basin between the continents before the late Eocene. After the late Eocene, the major oceanic circulation pattern was established. Before that time, conditions were favorable for preservation of organic-carbon deposits on the sea floor. Among the questions to be addressed with this data are the following. How do apparent subsidence rates of this passive margin compare with others around the world. Does the onshore subglacial Wilkes basins to the Otway and Ceduna basins of Australia exists. What is the effect of the ice cap on the stratigraphy of this margin. Do the two major Tertiary ice advances have conspicuous seismic-stratigraphic signatures.

  12. PCBs in fish and their cestode parasites in Lake Victoria.

    PubMed

    Oluoch-Otiego, John; Oyoo-Okoth, Elijah; Kiptoo, Kipkorir Koross Godfrey; Chemoiwa, Emily J; Ngugi, Charles C; Simiyu, Gelas; Omutange, Elijah S; Ngure, Veronica; Opiyo, Mary A

    2016-08-01

    Polychlorinated biphenyls (PCBs) are classified as persistent organic pollutants (POPs) regulated by the Stockholm Convention (2001). Although their production and use was stopped almost three decades ago, PCBs are environmental persistent, toxic, and bioaccumulate in biota. We assessed the levels of 7 PCB congeners (IUPAC nos. 28, 52, 101, 118, 138, 153, and 180) in sediment and fish (Oreochromis niloticus, Lates niloticus, and Rastrineobola argentea) and evaluated the potential of cestode fish endoparasite (Monobothrioides sp., Proteocephalaus sp., and Ligula intestinalis) as biomonitors of PCBs in Lake Victoria, Kenya. The median concentration of Σ7PCBs in sediments and fish were 2.2-96.3 μg/kg dw and 300-3,000 μg/kg lw, respectively. At all the sampling sites, CB138, CB153, and CB180 were the dominant PCB congeners in sediment and fish samples. Compared to the muscle of the piscine host, Proteocephalaus sp. (infecting L. niloticus) biomagnified PCBs ×6-14 while Monobothrioides sp. (infecting O. niloticus) biomagnified PCBs ×4-8. Meanwhile, L. intestinalis (infecting R. argentea) biomagnified PCBs ×8-16 compared to the muscle of unparasitized fish. We demonstrate the occurrence of moderate to high levels of PCB in sediments and fish in Lake Victoria. We also provide evidence that fish parasites bioaccumulate higher levels of PCBs than their piscine hosts and therefore provide a promising biomonitor of PCBs. We urge further a long-term study to validate the use of the above cestode fish parasites as biomonitoring tools for PCBs.

  13. Impact of Land Use on Soil Respiration in Southwestern Victoria

    NASA Astrophysics Data System (ADS)

    Teodosio, B.; Daly, E.; Pauwels, V. R. N.

    2015-12-01

    Land use management is one of the key contributors to the global environmental change. Considerable changes in landscapes have been experienced in Southwestern Victoria, Australia in the past two decades. Eucalyptus globulus (blue gum) plantations have expanded, resulting in possible changes in the water and carbon balances of catchments. The shift from pastures to plantations could have a significant impact on the local carbon balance with possible effects on atmospheric CO2 concentration and vegetation productivity. We present preliminary measurements from a field study comparing soil respiration in a plantation and a pasture. Adjacent catchments in Southwestern Victoria, near Gatum, were used as study areas; the prominent difference between the two catchments is the land use, with one catchment being used as a pasture for livestock grazing and the other catchment being mainly planted with blue gums. The variability of soil respiration in the pasture is governed by differences in soil moisture and substrate content due to local features of the topography and livestock grazing. Soil respiration measurements in the plantation were taken on mounds, access tracks, and open spaces. Most observations on mounds had higher soil respiration possibly due to root and mycorrhizal respiration. The measurements in open spaces had comparable values with mound measurements; this might be due to a less limited radiation. The soil respiration between trees had lower values, possibly because of radiation limitation due to the canopy cover. These preliminary measurements allow us to compare soil respiration variability across catchments with different land uses. This is important to estimate CO2 fluxes from soil to the atmosphere in large areas and will be valuable in estimating gross primary production from measurements of net ecosystem exchange.

  14. Ghrelin receptor expression and colocalization with anterior pituitary hormones using a GHSR-GFP mouse line.

    PubMed

    Reichenbach, Alex; Steyn, Frederik J; Sleeman, Mark W; Andrews, Zane B

    2012-11-01

    Ghrelin is the endogenous ligand for the GH secretagogue receptor (GHSR) and robustly stimulates GH release from the anterior pituitary gland. Ghrelin also regulates the secretion of anterior pituitary hormones including TSH, LH, prolactin (PRL), and ACTH. However, the relative contribution of a direct action at the GHSR in the anterior pituitary gland vs. an indirect action at the GHSR in the hypothalamus remains undefined. We used a novel GHSR-enhanced green fluorescent protein (eGFP) reporter mouse to quantify GHSR coexpression with GH, TSH, LH, PRL, and ACTH anterior pituitary cells in males vs. females and in chow-fed or calorie-restricted (CR) mice. GHSR-eGFP-expressing cells were only observed in anterior pituitary. The number of GHSR-eGFP-expressing cells was higher in male compared with females, and CR did not affect the GHSR-eGFP cell number. Double staining revealed 77% of somatotrophs expressed GHSR-eGFP in both males and females. Nineteen percent and 12.6% of corticotrophs, 21% and 9% of lactotrophs, 18% and 19% of gonadotrophs, and 3% and 9% of males and females, respectively, expressed GHSR-eGFP. CR increased the number of TSH cells, but suppressed the number of lactotrophs and gonadotrophs, expressing GHSR-eGFP compared with controls. These studies support a robust stimulatory action of ghrelin via the GHSR on GH secretion and identify a previously unknown sexual dimorphism in the GHSR expression in the anterior pituitary. CR affects GHSR-eGFP expression on lactotrophs, gonadotrophs, and thyrotrophs, which may mediate reproductive function and energy metabolism during periods of negative energy balance. The low to moderate expression of GHSR-eGFP suggests that ghrelin plays a minor direct role on remaining anterior pituitary cells.

  15. Latrepirdine (Dimebon™) enhances autophagy and reduces intracellular GFP-Aβ42 levels in yeast

    PubMed Central

    Bharadwaj, Prashant R.; Verdile, Giuseppe; Barr, Renae K.; Gupta, Veer; Steele, John W.; Lachenmayer, M. Lenard; Yue, Zhenyu; Ehrlich, Michelle E.; Petsko, Gregory; Ju, Shulin; Ringe, Dagmar; Sankovich, Sonia E.; Caine, Joanne M.; Macreadie, Ian G.; Gandy, Sam; Martins, Ralph N.

    2012-01-01

    Latrepirdine (Dimebon™), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer’s disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of Aβ42 aggregates. Latrepirdine was shown to up-regulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro GFP tagged Aβ yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aβ42. GFP-Aβ42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aβ42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aβ42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aβ42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aβ42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aβ42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aβ42. PMID:22903131

  16. Fluorescent sperm in a transparent worm: validation of a GFP marker to study sexual selection

    PubMed Central

    2014-01-01

    Background Sexual selection has initially been thought to occur exclusively at the precopulatory stage in terms of contests among males and female mate choice, but research over the last four decades revealed that it often continues after copulation through sperm competition and cryptic female choice. However, studying these postcopulatory processes remains challenging because they occur internally and therefore are often difficult to observe. In the transparent free-living flatworm Macrostomum lignano, a recently established transgenic line that expresses green fluorescent protein (GFP) in all cell types, including sperm, offers a unique opportunity to non-invasively visualise and quantify the sperm of a GFP-expressing donor inside the reproductive tract of wild-type recipients in vivo. We here test several aspects of the reproductive performance of the transgenic individuals and the accuracy of the techniques involved in assessing the GFP-expressing worms and their sperm. We then show the usefulness of these methods in a study on sperm displacement. Results GFP-expressing worms do not differ from wild-type worms in terms of morphology, mating rate and reproductive success. In addition, we show that the GFP signal is reliably and unequivocally expressed by all GFP-expressing individuals observed under epifluorescence illumination. However, the intensity of the GFP signal emitted by sperm of GFP expressing donors can vary (which we show to be at least in part due to sperm ageing) and the GFP marker is inherited according to Mendel’s laws in most, but not all, of the individuals. Nevertheless, we argue these two issues can be addressed with an appropriate experimental design. Finally, we demonstrate the value of the GFP-techniques by comparing the number of GFP-expressing sperm in a wild-type recipient before and after mating with a competing sperm donor, providing clear experimental evidence for sperm displacement in M. lignano. This result suggests that sperm

  17. Denaturation studies reveal significant differences between GFP and blue fluorescent protein.

    PubMed

    Saeed, Ibtesam A; Ashraf, S Salman

    2009-10-01

    Green fluorescent protein (GFP) is an unusually stable fluorescent protein that belongs to a family of related auto-fluorescent proteins (AFPs). These AFPs have been generated from jellyfish GFP by mutating the amino acids in the chromophore or its vicinity. Variants that emit light in the blue region (Blue Fluorescent Protein, BFP), red region, or yellow region are readily available and are widely used in diverse applications. Previously, we had used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with SDS, urea, and heat. Surprisingly, we found that SDS, urea or heat, did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5, however, at pH 6.5, the protein lost all fluorescence within a very short period of time. These results suggested that GFP undergoes a structural/stability shift between pH 6.5 and 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat. In the present study, we wanted to explore whether the stability or structure of the closely related BFP is also pH dependent. As expected, we found heat-induced denaturation and renaturation of BFP to be pH dependent, very much like GFP. However, when exposed to other denaturants like urea/heat or SDS we found BFP to behave very differently than GFP. Unlike GFP, which at pH 8.5 and 7.5 is very resistant to SDS-induced denaturation, BFP readily lost about 20% of its fluorescence at pH 8.5 and about 60% fluorescence at pH 7.5. Also, our denaturation and renaturation studies show that under certain conditions, BFP is more stable than GFP, such that under conditions where GFP is completely denatured, BFP still retained significant fluorescence. Taken together, our preliminary results show that despite being very similar in both amino acid sequences and overall structures, there may be subtle and important structural/conformational differences between BFP and GFP.

  18. Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.

    PubMed

    Zhuang, Ran; Zhang, Yuan; Zhang, Rui; Song, Chaojun; Yang, Kun; Yang, Angang; Jin, Boquan

    2008-05-01

    GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.

  19. Modified Aequorin Shows Increased Bioluminescence Activity

    DTIC Science & Technology

    1993-08-18

    LW. Schultz, J.R. Deschamps, and KB. Ward. Preparation and Initial Characterization of Crystals of the Photoprotein Aequorin from Aequorea victoria ...C. Prasher, Virginia K Eckenrode, William W. Ward, Frank G. Prendergast, and Milton J. Cormier. Primary structure of the Aequorea victoria green...Prendergast, and William W. Ward. Chemical Structure of the Hexapeptide Chromophore of the Aequorea Green- Fluorescent Protein. Biochemistry 32: 1 212

  20. Modified Aequorin Shows Increased Bioluminescence Activity

    DTIC Science & Technology

    1993-08-18

    and Initial Characterization of Crystals of the Photoprotein Aequorin from Aequorea victoria . Proteins, Structure, & Genetics 15: 103-107. RELATED...Frank G. Prendergast, and Milton J. Cormier. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111 (2):229-233. PATENTS U.S...Hexapeptide Chromophore of the Aequorea Green- Fluorescent Protein. Biochemistry 32: 1212-1218. 1992 Dennis J. O’Kane, and Douglas C. Prasher

  1. Rapid detection of a gfp-marked Enterobacter aerogenes under anaerobic conditions by aerobic fluorescence recovery.

    PubMed

    Zhang, Chong; Xing, Xin-Hui; Lou, Kai

    2005-08-15

    A gfp- and kanamycin-resistance gene-containing plasmid pUCGK was successfully constructed and transformed into Enterobacter aerogenes to develop a rapid GFP-based method for quantifying the bacterial concentration under anaerobic conditions for production of biohydrogen. Since the use of GFP as a molecular reporter is restricted by its requirement for oxygen in the development of the fluorophore, fluorescence detection for the fluorescent E. aerogenes grown anaerobically for hydrogen production was performed by developing a method of aerobic fluorescence recovery (AFR) of the anaerobically expressed GFP. By using this AFR method, rapid and non-disruptive cell quantification of E. aerogenes by fluorescence density was achieved for analyzing the hydrogen production process.

  2. GFP-tagged E. coli shows bacterial distribution in mouse organs: pathogen tracking using fluorescence signal

    PubMed Central

    Park, Pil-Gu; Cho, Min-Hee; Rhie, Gi-eun; Jeong, Haeseul; Youn, Hyewon

    2012-01-01

    Purpose In vaccine efficacy evaluation, visualization of pathogens in whole organism at each time point would be able to reduce the consuming animals and provide the in vivo information within consistent background with identical organism. Materials and Methods Using IVIS spectrum whole live-animal imaging system, fluorescent intensity was optimized and visualized proportionately by concentrating Escherichia coli MC1061 strain which expresses GFP (E. coli-GFP) in BALB/C mice after injection. Results Local distribution of disseminated E. coli-GFP was traced in each organ by fluorescence. Detached organ showed more obvious fluorescent signal, and intestine showed strongest fluorescent signal. Conclusion This in vivo imaging method using GFP-tagged pathogen strain suggest quantified infected pathogens by fluorescence intensity in whole animals can provide the information about the localization and distribution after infection. PMID:23596581

  3. Structural basis for the fast maturation of Arthropoda green fluorescent protein.

    PubMed

    Evdokimov, Artem G; Pokross, Matthew E; Egorov, Nikolay S; Zaraisky, Andrey G; Yampolsky, Ilya V; Merzlyak, Ekaterina M; Shkoporov, Andrey N; Sander, Ian; Lukyanov, Konstantin A; Chudakov, Dmitriy M

    2006-10-01

    Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

  4. GFP plasmid-induced defects in Salmonella invasion depend on plasmid architecture, not protein expression.

    PubMed

    Clark, Leann; Martinez-Argudo, Isabel; Humphrey, Tom J; Jepson, Mark A

    2009-02-01

    We have investigated the impact of plasmids and GFP expression on invasion of cultured epithelial cells by Salmonella enterica Typhimurium strain SL1344. The invasiveness of SL1344 carrying plasmids derived from pBR322, encoding promoterless GFP or constitutively expressed rpsM-GFP, was compared under optimal growth conditions with that of SL1344(pBR322), unmodified SL1344 and a strain with chromosome-integrated rpsM-GFP. The strain carrying pBR322 exhibited normal invasion, but the presence of modified plasmids impaired invasiveness, and impairment was exacerbated by plasmid-encoded chloramphenicol resistance (CmR). Using a different antibiotic resistance marker, kanamycin (KmR), did not impair invasiveness. Despite the effect of plasmid-encoded CmR, the strain containing chromosomally encoded GFP, also carrying a CmR gene, was as invasive as the wild-type. To investigate the mechanism by which plasmid carriage decreases invasion, we monitored SPI-1 gene expression using prgH promoter activity as an index of SPI-1 activity. An SL1344 strain with a chromosome-integrated prgH::gfp reporter construct exhibited lower GFP expression during exponential phase when carrying plasmids incorporating CmR or gfp, mirroring invasion data. These data provide evidence that suppression of SPI-1 gene expression is a major factor in the loss of invasiveness associated with plasmid carriage. Our findings also indicate that some plasmids, especially those carrying CmR, should be used with caution, as virulence traits and gene expression may be affected by their presence. Integration of reporter proteins into the bacterial chromosome, however, appears to circumvent the adverse effects observed with plasmids.

  5. Fluorescence lifetime dynamics of eGFP in protein aggregates with expanded polyQ

    NASA Astrophysics Data System (ADS)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2009-02-01

    Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues (97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the process of polyQ protein aggregation per se.

  6. Sensitivity analysis of the GNSS derived Victoria plate motion

    NASA Astrophysics Data System (ADS)

    Apolinário, João; Fernandes, Rui; Bos, Machiel

    2014-05-01

    Fernandes et al. (2013) estimated the angular velocity of the Victoria tectonic block from geodetic data (GNSS derived velocities) only.. GNSS observations are sparse in this region and it is therefore of the utmost importance to use the available data (5 sites) in the most optimal way. Unfortunately, the existing time-series were/are affected by missing data and offsets. In addition, some time-series were close to the considered minimal threshold value to compute one reliable velocity solution: 2.5-3.0 years. In this research, we focus on the sensitivity of the derived angular velocity to changes in the data (longer data-span for some stations) by extending the used data-span: Fernandes et al. (2013) used data until September 2011. We also investigate the effect of adding other stations to the solution, which is now possible since more stations became available in the region. In addition, we study if the conventional power-law plus white noise model is indeed the best stochastic model. In this respect, we apply different noise models using HECTOR (Bos et al. (2013), which can use different noise models and estimate offsets and seasonal signals simultaneously. The seasonal signal estimation is also other important parameter, since the time-series are rather short or have large data spans at some stations, which implies that the seasonal signals still can have some effect on the estimated trends as shown by Blewitt and Lavellee (2002) and Bos et al. (2010). We also quantify the magnitude of such differences in the estimation of the secular velocity and their effect in the derived angular velocity. Concerning the offsets, we investigate how they can, detected and undetected, influence the estimated plate motion. The time of offsets has been determined by visual inspection of the time-series. The influence of undetected offsets has been done by adding small synthetic random walk signals that are too small to be detected visually but might have an effect on the

  7. Analysis of the water balance of Lake Victoria

    NASA Astrophysics Data System (ADS)

    Nossent, J.; de Brabanter, W.; Bauwens, W.

    2009-04-01

    Lake Victoria is situated within an elevated plateau in the western part of Africa's Great Rift Valley and lies within the territory of three countries: Tanzania, Uganda and Kenya. It is Africa's largest lake and the second widest fresh water lake in the world in terms of surface area. It is also the source of the longest branch of the River Nile, the White Nile. The lake's shallowness, limited river inflow, and large surface area relative to its volume make it vulnerable to climate changes and fluctuations of the water level. This affects the surrounding countries and their people a lot, especially in terms of their food supply and economy. The aim of this study was to get more information on the causes of these fluctuations by analysing the water balance of the lake for the period 1970-1974. It was based both on historical data and measurements and new calculations, and compared with previous studies (e.g. Suttcliffe and Parks, 1999). Precipitation and evaporation over the lake surface were calculated with the Thiessen Polygons method, using measurements from stations around the lake and on the islands. The total inflow of the lake is the sum of the contributions of twelve subbasins. One of these subcatchments, the Nzoia-catchment, was modeled with SWAT (Soil and Water Assessment Tool), a physically based, semi-distributed river basin simulator, as a contribution to the development of a water balance model for Lake Victoria. To calculate the outflow at the Owen Falls Dam in Jinja (Uganda), gauge heights of the lake were used in combination with the "Agreed Curve" (the relationship between water level and flow that was set by the policy makers). As the lake is assumed to be a system with a closed mass balance, the combination of the variations in the above mentioned components resulted in changes of the lake's storage, leading to fluctuations of the water level. For the period 1970-1974 the calculated mean monthly evaporation is 133 mm, with a standard deviation

  8. Trends and disparities in sepsis hospitalisations in Victoria, Australia.

    PubMed

    Ore, Timothy

    2015-12-14

    Objective The aim of the present study was to determine the clinical and epidemiological characteristics of patients with sepsis admitted to hospitals in Victoria, Australia, during the period 2004-14. The data include incidence, severity and mortality.Methods In all, 44 222 sepsis hospitalisations were identified between 2004-05 and 2013-14 from the Victorian Admitted Episodes Dataset. The dataset contains clinical and demographic information on all admissions to acute public and private hospitals. Using the International Classification of Diseases (10th Revision) Australian Modification codes, incidence rates, severity of disease and mortality were calculated.Results Sepsis hospitalisation rates per 10 000 population increased significantly (P < 0.01) over the period, from 6.9 (95% confidence interval (CI) 5.6-7.8) to 10.0 (95% CI 9.1-11.1), an annual growth rate of 3.8%. The age-standardised in-hospital death rates per 100 000 population grew significantly (P < 0.01) from 9.2 (95% CI 7.8-10.4) in 2004-05 to 13.0 (95% CI 11.7-14.6) in 2013-14, an annual growth rate of 3.1%. Among people under 45 years of age, the 0-4 years age group had the highest hospitalisation rate (3.0 per 10 000 population; 95% CI 2.7-3.4). Nearly half (46.2%) of all sepsis hospitalisations were among patients born overseas, with a rate of 14.5 per 10 000 population (95% CI 12.4-16.2) in that group compared with a rate of 5.9 per 10 000 population (95% CI 5.3-6.7) for patients born in Australia. The age-standardised sepsis hospitalisation rate was 2.6-fold greater in the lowest compared with highest socioeconomic areas (12.7 per 10 000 population (95% CI 11.2-13.8) vs 4.8 per 10 000 population (95% CI 4.1-5.7), respectively).Conclusion This paper shows a significant upward trend in both sepsis separation rates and in-hospital death rates over the period; unlike sepsis, in-hospital death rates from all diagnoses fell over the same period. The results can be used to stimulate review of

  9. Analysis of cytoskeleton dynamics and cell migration in drosophila ovaries using GFP-actin and E-cadherin-GFP fusion molecules

    NASA Astrophysics Data System (ADS)

    Verkhusha, Vladyslav V.; Tsukita, Shoichiro; Oda, Hiroki

    1999-06-01

    Coordination of cell migration and adhesion is essential for movement of tissues during morphogenesis. During Drosophila oogenesis so called border cells (BCs) break from an anterior epithelium of egg chamber, acquire a mesenchymal-like morphology, and migrate posteriorly between nurse cells to oocyte. The confocal microscopic observation of BCs has revealed well-developed forepart lamellipodium stained with Drosophila E-cadherin (DE-cadherin), PS2 integrin, cytoplasmic myosin and F-actin. To investigate mechanism of BC migration in vivo we have constructed a DE-cadherin-GFP and a GFP-actin fusion proteins and induced their expression BCs utilizing the UAS/GAL4 system. The DE-cadherin-GFP signal as well as immunostaining of PS2 integrin visualized a track of migrating BCs providing an evidence that adhesive molecules are pulled out and left behind on the surface of nurse cells. Our data suggest that two distinct adhesive systems, DE-cadherins and PS2 integrins simultaneously mediate the migration of BCs. Release of adhesive contacts in the tail region is a rate- limited event in BC migration. The spatial-temporal sequence of actin-based events visualized by the GFP-actin suggest a treadmilling model for actin behavior in BC lamellipodium. BC migration can be considered as simultaneous reiterating processes of lamellipodium extension and adhesive attachment, cytoskeletal contraction, and rear detachment.

  10. What do Victoria family physicians think about housecalls?

    PubMed Central

    Hammett, Tess

    2013-01-01

    Objective To determine the proportion of family physicians doing housecalls, the types of patients they think are appropriate to visit at home, whether physicians are satisfied with the number of housecalls they make, reasons family physicians list for not doing housecalls, and what they consider acceptable remuneration and travel time for housecalls. Design A 12-question paper survey was formulated specifically for this study and piloted by 6 family physicians in British Columbia. It was then mailed with a cover letter to 250 physicians' offices and faxed back anonymously. Setting Family physicians' private offices in Victoria, BC, between December 1 and 19, 2010. Participants A total of 250 randomly selected family physicians from a list of 552 physicians practising in Victoria on the College of Physicians and Surgeons of British Columbia website. Main outcome measures Proportion of physicians doing housecalls, reasons stated for not doing housecalls, and mean acceptable remuneration and travel time for a housecall. Results A total of 73 surveys (29.2%) were returned, 5 of which were not fully completed but were included for the questions that were answered. Sixty-four physicians (87.7%) did at least 1 housecall in the past year, 23 (31.5%) did housecalls at least once a month, and 12 (16.4%) did them at least once a week. Of 71 respondents, 64 physicians (90.1%) listed lack of time as a barrier to performing housecalls, 37 (52.1%) listed unsatisfactory remuneration, and 35 (49.3%) listed lengthy travel times. Most physicians indicated that appropriate remuneration for a housecall was either $142.21 (n = 30, 42.9%) or $108.41 (n = 26, 37.1%). Thirty-seven physicians (52.9%) noted that 20 minutes was an acceptable maximum 1-way travel time for a housecall, while 29 (41.4%) listed 10 minutes. Conclusion Several systemic factors, including lack of time, unsatisfactory remuneration, and large geographic catchment areas, make it difficult for urban family physicians

  11. Victorias energy efficiency and cogeneration project. Final report

    SciTech Connect

    1998-10-31

    This report describes a two-phase energy project currently contemplated for joint implementation at the Victorias Milling Company, a large sugar mill and refinery on the island of Negros in the Visayas region of the Philippines. The Energy Efficiency (EE) phase is expected to reduce of eliminate VMC`s fossil fuel consumption, which will have a direct and substantial impact on carbon emissions. Phase I is an EE project which involves the installation of equipment to reduce steam and electricity demand in the factories. Phase II, will involve retrofitting and increasing the capacity of the steam and power generation systems, and selling power to the grid. By increasing efficiency and output, the cogeneration project will allow the factory to use only bagasse sugar cane fiber waste as fuel for energy needs. The cogeneration project will also eliminate VMC`s electricity purchases and supply additional power for the island, which will offset generation capacity expansion on the island and the Visayas region.

  12. Establishing a sustainable childhood obesity monitoring system in regional Victoria.

    PubMed

    Crooks, Nicholas; Strugnell, Claudia; Bell, Colin; Allender, Steve

    2016-12-19

    Issue addressed: Childhood obesity poses a significant immediate and long-term burden to individuals, societies and health systems. Infrequent and inadequate monitoring has led to uncertainty about trends in childhood obesity prevalence in many countries. High-quality data, collected at regular intervals, over extended timeframes, with high response rates and timely feedback are essential to support prevention efforts. Our aim was to establish a sustainable childhood obesity monitoring system in regional Australia to collect accurate anthropometric and behavioural data, provide timely feedback to communities and build community engagement and capacity.Methods: All schools from six government regions of South-West Victoria were invited to participate. Passive (opt-out) consent was used to collect measured anthropometric and self-reported behavioural data from children in years 2, 4, and 6, aged 7-12 years.Results: We achieved a 70% school participation rate (n=46) and a 93% student response rate (n=2198) among government and independent schools. Results were reported within 10 weeks post data collection. Harnessing high levels of community engagement throughout the planning, data collection and reporting phases increased community capacity and data utility.Conclusions: The monitoring system achieved high response rates, community engagement and community capacity building, and delivered results back to the community in a timely manner.So what?: This system has the potential to provide sustainable monitoring of childhood obesity that is not dependent on external funding. The results of this monitoring will likely inform health promotion efforts in communities across the region.

  13. Forty Meters from Entry to Victoria Crater (Stereo)

    NASA Technical Reports Server (NTRS)

    2007-01-01

    [figure removed for brevity, see original site] Left-eye view of a color stereo pair for PIA09972 [figure removed for brevity, see original site] Right-eye view of a color stereo pair for PIA09972

    NASA's Mars Exploration Rover Opportunity used its navigation camera during the rover's 1,278th Martian day, or sol, (Aug. 28, 2007) to take the images combined into this stereo view. The rover was perched at the lip of Victoria Crater, which is about 800 meters (one-half mile) in diameter.

    After assessment of possible routes for Opportunity to descend into the crater, the rover team selected a site farther to the right along the rim. That selected entry point lies near the ripple of bright soil visible just outside the crater near the top center of this scene. The driving distance for Opportunity from the Sol 1,278 viewpoint to the selected entry point is about 40 meters (about 130 feet).

    This view combines a stereo pair and appears three-dimensional when seen through blue-red glasses. It is presented as a cylindrical-perspective projection with geometric seam correction.

  14. Mandatory bicycle helmet use: experience in Victoria, Australia.

    PubMed

    Vulcan, A P; Cameron, M H; Watson, W L

    1992-01-01

    On July 1, 1990, the legislation requiring wearing of an approved bicycle (safety) helmet by all pedal cyclists, unless exempted, came into effect in Victoria, Australia. The paper describes the more important activities which paved the way for this initiative and presents some preliminary information about the effect of the legislation on wearing rates and head injuries. Since 1980 there has been promotion of helmet use through bicycle education in schools, mass media publicity, support by professional organizations and community groups, bulk purchase schemes, and government rebates for helmet purchases. The Australian Standard for bicycle safety helmets has also been changed to meet community demands for lighter helmets with more provision for ventilation. There has been a steady increase in voluntary helmet use in Melbourne from 1983 to March 1990, as follows: 5% to 70% in primary school children; 2% to 20% in secondary students; and 27% to 40% in adults. In the period after the legislation, with relatively little enforcement, these three groups have shown substantial increases in helmet use rates, rising to 70-90% in most cases. Preliminary data show that the numbers of bicyclists with a head injury have dropped in the period since the legislation came into effect. The possible contributions to this reduction, of less bicycle use and lower risk of head injury in an accident, are discussed.

  15. Stratigraphie relations of australites in the Port Campbell Embayment, Victoria

    USGS Publications Warehouse

    Shoemakeri, E.M.; Ralph, Uhlherr H.

    1999-01-01

    In the Port Campbell Embayment of Victoria, australites have been found in situ in channel deposits of the Hanson Plain Sand of Pliocene and Pleistocene age. The large majority of the australites, however, occur as a lag deposit at the basal contact of the Sturgess Sand of late Pleistocene and Holocene age and are spatially correlated with ferruginous sandstone clasts that are derived from the Hanson Plain Sand. Some of the tektites are imbedded in or bonded to the ferruginous sandstone clasts, but most are found as individual tektite fragments. A few percent of the tektites have nearly perfectly preserved, complete aerodynamically shaped forms. The sandstone clasts and associated tektites have been reworked from the much older underlying Hanson Plain and have been locally concentrated in the lag deposit. Some tektites also occur at higher levels in the Sturgess Sand, almost invariably in association with stone flakes, exotic stones transported by the aborigines and, locally, with middens of mollusc shells. Circumstantial evidence indicates that the aborigines transported the tektites found in the upper part of the Sturgess, particularly at Stanhope Bay. As Port Campbell australites unequivocally occur in strata much older than the late Pleistocene and Holocene Sturgess, there is no longer any conflict between the apparent stratigraphie age of the tektites and the middle Pleistocene ages obtained by various Chronometrie methods. ?? Meteoritical Society, 1999.

  16. Injuries associated with fireworks in Victoria: an epidemiological overview

    PubMed Central

    Abdulwadud, O.; Ozanne-Smith, J.

    1998-01-01

    Objectives—To determine the epidemiological features of injuries associated with fireworks. Design—A retrospective study of reported cases. Subjects—Subjects were those who attended selected Victorian hospital emergency departments (n=17) and those admitted for firework related injuries (n=16). Results—The mean (SD) age of attenders at emergency department between January 1988 and June 1996, was 8.9 (6.2) years and most (88%) were under 18 years of age. Males accounted for 71% of the cases. The most common anatomical sites and types of injury were head (47%) and burns (88%), respectively. About 53% of the injuries were caused by firecrackers, the remainder by sparklers and penny bangers. Among those admitted to hospital between July 1987 and June 1996, the mean (SD) age was 22.9 (14.8) years and 50% were under 18 years of age. Males accounted for 87% of the cases. There was a significant difference in mean age between those admitted and not admitted to hospital, the former being significantly older. Conclusions—Although relatively rare, injuries from fireworks still occur in Victoria after legislative restrictions on their sale in 1985. Consequently, there is a potential risk for injuries among children, particularly from firecrackers. More enforcement of the regulations, education, and parental supervision are needed to prevent injuries from fireworks. PMID:9887417

  17. Remote sensing water observation for supporting Lake Victoria weed management.

    PubMed

    Cavalli, Rosa Maria; Laneve, Giovanni; Fusilli, Lorenzo; Pignatti, Stefano; Santini, Federico

    2009-05-01

    This paper aims to assess the suitability of remote sensing for enhancing the management of water body resources and for providing an inexpensive way to gather, on a wide area, weed infestation extent and optical parameter linked to the water body status. Remotely sensed satellite images and ancillary ground true data were used to produce land cover maps, trough classification techniques, and water compounds maps, applying radiative transfer models. The study proposed within the framework of the cooperation between Italian Foreign Affair Ministry (through the University of Rome) and Kenyan Authorities has been carried out on the Kenyan part of the Lake Victoria. This lake is one of the largest freshwater bodies of the world where, over the last few years environmental challenges and human impact have perturbed the ecological balance affecting the biodiversity. The objective of this research study is to define the thematic products, retrievable from satellite images, like weed abundance maps and water compound concentrations. These products, if provided with an appropriate time frequency, are useful to identify the preconditions for the occurrence of hazard events like abnormal macrophyte proliferation and to develop an up-to-date decision support system devoted to an apprised territory, environment and resource management.

  18. Spectral analysis of storm surge in Hong Kong Victoria Harbour

    NASA Astrophysics Data System (ADS)

    Tou, Stephen K. W.; Arumugam, K.

    Based on a linear model the dynamic characteristics of Victoria Harbour (Hong Kong) is obtained by means of spectral analysis of the storm surge hydrographs. The results show that the harbour is an ideal one which has a small gain factor and a flat response in the frequency range from 0 to 6 × 10 -5 Hz. The results also show that the power spectra possess the narrow band features which indicates that the periodic components associated with tidal motions are predominant over the random components. The power spectrum corresponding to a frequency of 2.3 × 10 -5 Hz is likely to be associated with the astronomical tides. The peaks in the power spectra at zero frequency suggest that the pumping mode of oscillations is dominant in a storm surge. This mode of oscillations represents the temporal variations in mean sea level. To demonstrate the full potential of the present model, more case studies should be conducted when surge as well as non-surge data are available.

  19. Mossbauer study of sediment cores from Victoria harbour, Hong Kong

    PubMed

    Tanner; Leong; Pan; Yu

    2000-12-01

    The concentrations of Fe and other abundant metals in 6 m-long sediment cores from four locations in the world's largest container port, Hong Kong, have been determined, in addition to physical characteristics and 210Pb activities. Fe is generally present at concentrations between 2% and 3% (depending on the particle size), similar to values found in granitic rocks. Its speciation was studied by room temperature Mossbauer spectroscopy. Two Fe(II) species and one Fe(III) species were found to be present in the cores. The relative proportions of Fe(II) and Fe(III) generally changed with the depth of sediment. Most noticeably, for the core taken from near the Star Ferry Pier at the Kowloon side of Victoria harbour, the proportion of Fe(II) was fairly constant down to 4.75 m, but then decreased with depth, so that near the core base (6 m depth), the iron was present almost exclusively as Fe(III). The colour of the core changed from olive grey to olive yellow between 5 and 6 m. According to the core chronology, this depth represents ca. 1900, before the ferry pier construction, when the harbour was unpolluted.

  20. Management of glioblastoma in Victoria, Australia (2006-2008).

    PubMed

    Gan, Hui K; Rosenthal, Mark A; Cher, Lawrence; Dally, Michael; Drummond, Katharine; Murphy, Michael; Thursfield, Vicky

    2015-09-01

    We describe the management of patients with newly diagnosed glioblastoma multiforme (GBM) in a population-based cohort and compare this to a previously studied cohort. We performed a retrospective cohort study of patients diagnosed with GBM from 2006-2008 in Victoria, Australia. Patients were identified from the population-based Victorian Cancer Registry and their treating doctors surveyed by questionnaire. Outcomes were then compared to a study of GBM patients who were diagnosed between 1998 and 2000 using an identical methodology. We reviewed 351 eligible patients. There were slightly more males (62%) and a minority had multifocal disease (13%). Total macroscopic resection, partial resection or biopsy only was performed in 32%, 37% and 24% of patients, respectively. The majority of patients were referred to a radiation oncologist and medical oncologist postoperatively. A total of 56% of patients were treated with postoperative radiotherapy with concurrent and sequential temozolomide and had a median survival of 14.4 months. This was significantly better than patients treated with postoperative radiotherapy alone in the current or earlier cohorts (2006-2008: median survival 6.2 months, p<0.0001 versus 1998-2000: 8.9 months, p<0.0001). This study demonstrates that postoperative chemoradiation has become the standard of care in this Victorian population with an associated improvement in median survival.

  1. Diversity of soil yeasts isolated from South Victoria Land, Antarctica

    USGS Publications Warehouse

    Connell, L.; Redman, R.; Craig, S.; Scorzetti, G.; Iszard, M.; Rodriguez, R.

    2008-01-01

    Unicellular fungi, commonly referred to as yeasts, were found to be components of the culturable soil fungal population in Taylor Valley, Mt. Discovery, Wright Valley, and two mountain peaks of South Victoria Land, Antarctica. Samples were taken from sites spanning a diversity of soil habitats that were not directly associated with vertebrate activity. A large proportion of yeasts isolated in this study were basidiomycetous species (89%), of which 43% may represent undescribed species, demonstrating that culturable yeasts remain incompletely described in these polar desert soils. Cryptococcus species represented the most often isolated genus (33%) followed by Leucosporidium (22%). Principle component analysis and multiple linear regression using stepwise selection was used to model the relation between abiotic variables (principle component 1 and principle component 2 scores) and yeast biodiversity (the number of species present at a given site). These analyses identified soil pH and electrical conductivity as significant predictors of yeast biodiversity. Species-specific PCR primers were designed to rapidly discriminate among the Dioszegia and Leucosporidium species collected in this study. ?? 2008 Springer Science+Business Media, LLC.

  2. GAD67-GFP+ Neurons in the Nucleus of Roller. II. Subthreshold and Firing Resonance Properties

    PubMed Central

    Berger, A. J.

    2011-01-01

    In the companion paper we show that GAD67-GFP+ (GFP+) inhibitory neurons located in the Nucleus of Roller of the mouse brain stem can be classified into two main groups (tonic and phasic) based on their firing patterns in responses to injected depolarizing current steps. In this study we examined the responses of GFP+ cells to fluctuating sinusoidal (“chirp”) current stimuli. Membrane impedance profiles in response to chirp stimulation showed that nearly all phasic cells exhibited subthreshold resonance, whereas the majority of tonic GFP+ cells were nonresonant. In general, subthreshold resonance was associated with a relatively fast passive membrane time constant and low input resistance. In response to suprathreshold chirp current stimulation at a holding potential just below spike threshold the majority of tonic GFP+ cells fired multiple action potentials per cycle at low input frequencies (<5 Hz) and either stopped firing or were not entrained by the chirp at higher input frequencies (= tonic low-pass cells). A smaller group of phasic GFP+ cells did not fire at low input frequency but were able to phase-lock 1:1 at intermediate chirp frequencies (= band-pass cells). Spike timing reliability was tested with repeated chirp stimuli and our results show that phasic cells were able to reliably fire when they phase-locked 1:1 over a relatively broad range of input frequencies. Most tonic low-pass cells showed low reliability and poor phase-locking ability. Computer modeling suggested that these different firing resonance properties among GFP+ cells are due to differences in passive and active membrane properties and spiking mechanisms. This heterogeneity of resonance properties might serve to selectively activate subgroups of interneurons. PMID:21047931

  3. Green fluorescence induced by EF-hand assembly in a split GFP system.

    PubMed

    Lindman, Stina; Johansson, Ida; Thulin, Eva; Linse, Sara

    2009-06-01

    The affinity between the 1-157 and 158-238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D(9k) to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1-157 of GFP are fused to residues 1-43 of calbindin, and in EF2-GFPC, residues 44-75 of calbindin are fused to residues 158-238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca(2+)-dependent.

  4. Distribution of CaMKIIα expression in the brain in vivo, studied by CaMKIIα-GFP mice

    PubMed Central

    Wang, Xinjun; Zhang, Chunzhao; Szábo, Gábor; Sun, Qian-Quan

    2013-01-01

    To facilitate the study of the CaMKIIα function in vivo, a CaMKIIα-GFP transgenic mouse line was generated. Here, our goal is to provide the first neuroanatomical characterization of GFP expression in the CNS of this line of mouse. Overall, CaMKIIα -GFP expression is strong and highly heterogeneous, with the dentate gyrus of the hippocampus as the most abundantly expressed region. In the hippocampus, around 70% of granule and pyramidal neurons expressed strong GFP. In the neocortex, presumed pyramidal neurons were GFP positive: around 32% of layer II/III and 35% of layer VI neurons expressed GFP, and a lower expression rate was found in other layers. In the thalamus and hypothalamus, strong GFP signals were detected in the neuropil. GFP-positive cells were also found in many other regions such as the spinal trigeminal nucleus, cerebellum and basal ganglia. We further compared the GFP expression with specific antibody staining for CaMKIIα and GABA. We found that GFP+ neurons were mostly positive for CaMKIIα-IR throughout the brain, with some exceptions throughout the brain, especially in the deeper layers of neocortex. GFP and GABA-IR marked distinct neuronal populations in most brain regions with the exception of granule cells in the olfactory bulb, purkinje cells in the cerebellar, and some layer I cells in neocortex. In conclusion, GFP expression in the CaMKIIα-GFP mice is similar to the endogenous expression of CaMKIIα protein, thus these mice can be used in in vivo and in vitro physiological studies in which visualization of CaMKIIα- neuronal populations is required. PMID:23632380

  5. E. coli RS2GFP Retention Mechanisms in Laboratory-Scale Fractured Rocks: A Statistical Model

    NASA Astrophysics Data System (ADS)

    Rodrigues, S. N.; Qu, J.; Dickson, S. E.

    2011-12-01

    With billions of gallons of groundwater being withdrawn every day in the US and Canada, it is imperative to understand the mechanisms which jeopardize this resource and the health of those who rely on it. Porous media aquifers have typically been considered to provide significant filtration of particulate matter (e.g. microorganisms), while the fractures in fractured rock aquifers and aquitards are considered to act as contaminant highways allowing a large fraction of pathogens to travel deep into an aquifer relatively quickly. Recent research results indicate that fractured rocks filter out more particulates than typically believed. The goal of the research presented here is to quantify the number of E. coli RS2GFP retained in a single, saturated, laboratory-scale fracture, and to relate the retention of E. coli RS2GFP to the aperture field characteristics and groundwater flow rate. To achieve this goal, physical experiments were conducted at the laboratory-scale to quantify the retention of E. coli RS2GFP through several single, saturated, dolomitic limestone fractures under a range of flow rates. These fractures were also cast with a transparent epoxy in order to visualize the transport mechanisms in the various different aperture fields. The E. coli RS2GFP is tagged with a green-fluorescent protein (GFP) that is used to obtain visualization data when excited by ultraviolet light. A series of experiments was conducted, each of which involved the release of a known number of E. coli RS2GFP at the upstream end of the fracture and measuring the effluent concentration profile. These experiments were conducted using both the natural rock and transparent cast of several different aperture fields, under a range of flow rates. The effects of different aperture field characteristics and flow rates on the retention of E. coli RS2GFP will be determined by conducting a statistical analysis of the retention data under different experimental conditions. The images captured

  6. In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice

    PubMed Central

    Takaishi, Shigeo; Shibata, Wataru; Tomita, Hiroyuki; Jin, Guangchun; Yang, Xiangdong; Ericksen, Russell; Dubeykovskaya, Zinaida; Asfaha, Samuel; Quante, Michael; Betz, Kelly S.; Shulkes, Arthur

    2011-01-01

    Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene. PMID:21051525

  7. Model system for plant cell biology: GFP imaging in living onion epidermal cells

    NASA Technical Reports Server (NTRS)

    Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

    1999-01-01

    The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

  8. In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice.

    PubMed

    Takaishi, Shigeo; Shibata, Wataru; Tomita, Hiroyuki; Jin, Guangchun; Yang, Xiangdong; Ericksen, Russell; Dubeykovskaya, Zinaida; Asfaha, Samuel; Quante, Michael; Betz, Kelly S; Shulkes, Arthur; Wang, Timothy C

    2011-02-01

    Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

  9. Potential of real-time measurement of GFP-fusion proteins.

    PubMed

    Jones, Jo J; Bridges, Angela M; Fosberry, Andrew P; Gardner, Sharmila; Lowers, Robert R; Newby, Rachel R; James, Philip J; Hall, Richard M; Jenkins, Owen

    2004-04-08

    Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 20l scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.

  10. A scalable strategy for high-throughput GFP tagging of endogenous human proteins

    PubMed Central

    Leonetti, Manuel D.; Sekine, Sayaka; Kamiyama, Daichi; Weissman, Jonathan S.; Huang, Bo

    2016-01-01

    A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. PMID:27274053

  11. Bistable isoelectric point photoswitching in green fluorescent proteins observed by dynamic immunoprobed isoelectric focusing.

    PubMed

    Hughes, Alex J; Tentori, Augusto M; Herr, Amy E

    2012-10-24

    We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the amino acid 222 E-to-G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF was employed to monitor dark (nonfluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3-4 formal charges distinguish the bright and dark populations of avGFP, as compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen-bond clusters within the GFP β-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events.

  12. Impact of cruise ship emissions in Victoria, BC, Canada

    NASA Astrophysics Data System (ADS)

    Poplawski, Karla; Setton, Eleanor; McEwen, Bryan; Hrebenyk, Dan; Graham, Mark; Keller, Peter

    2011-02-01

    Characterization of the effects of cruise ship emissions on local air quality is scarce. Our objective was to investigate community level concentrations of fine particulate matter (PM 2.5), nitrogen dioxide (NO 2) and sulphur dioxide (SO 2) associated with cruise ships in James Bay, Victoria, British Columbia (BC), Canada. Data obtained over four years (2005-2008) at the nearest air quality network site located 3.5 km from the study area, a CALPUFF modeling exercise (2007), and continuous measurements taken in the James Bay community over a three-month period during the 2009 cruise ship season were examined. Concentrations of PM 2.5 and nitrogen oxide (NO) were elevated on weekends with ships present with winds from the direction of the terminal to the monitoring station. SO 2 displayed the greatest impact from the presence of cruise ships in the area. Network data showed peaks in hourly SO 2 when ships were in port during all years. The CALPUFF modeling analysis found predicted 24-hour SO 2 levels to exceed World Health Organization (WHO) guidelines of 20 μg m -3 for approximately 3% of 24-hour periods, with a maximum 24-hour concentration in the community of 41 μg m -3; however, the CALPUFF model underestimated concentrations when predicted and measured concentrations were compared at the network site. Continuous monitoring at the location in the community predicted to experience highest SO 2 concentrations measured a maximum 24-hour concentration of 122 μg m -3 and 16% of 24-hour periods were above the WHO standard. The 10-minute concentrations of SO 2 reached up to 599 μg m -3 and exceeded the WHO 10-minute SO 2 guideline (500 μg m -3) for 0.03% of 10-minute periods. No exceedences of BC Provincial or Canadian guidelines or standards were observed.

  13. Evaluation of vehicle side airbag effectiveness in Victoria, Australia.

    PubMed

    D'Elia, Angelo; Newstead, Stuart; Scully, Jim

    2013-05-01

    Side airbag systems were first introduced into vehicles around 1995 to help protect occupants from injury in side impact crashes. International studies have shown that side airbags are effective in reducing the risk of death and injury, however, serious injuries can still occur even when side airbags deploy. The objective of this study was to use detailed injury information from insurance injury compensation claims data linked to Police reported crash data to determine the effectiveness of side airbags in reducing the risk of death or injury for occupants involved in side impact crashes in Victoria, Australia based on the specific body regions that side airbag systems are designed to protect. It was found that head and torso-protecting dual airbag systems designed to protect the head, neck, face, chest and abdomen are highly effective in reducing driver death or injury due to near side crashes. They were associated with a statistically significant reduction of 41.1% (25.9%, 53.2%) in the odds of death or injury across all body regions; and a 48.0% (28.0%, 62.4%) reduction in the odds of death or injury to the head, neck, face, chest and abdomen. The study did not find any evidence that torso-protecting airbags alone are effective in reducing death or injury. Analysis results indicate that head and torso-protecting side airbag systems in vehicles are a highly effective technology for reducing the risk of death or injury to vehicle occupants in near side crashes. The magnitude of the injury reduction benefits estimated indicate that fitment of this technology to all vehicles should be a high priority and will yield significant savings in overall road trauma.

  14. Making Fluorescent Streptococci and Enterococci for Live Imaging.

    PubMed

    Shabayek, Sarah; Spellerberg, Barbara

    2017-01-01

    Since the discovery of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, outstanding fluorescent labeling tools with numerous applications in vastly different areas of life sciences have been developed. To optimize GFP for diverse life science applications, a large variety of GFP derivatives with different environmental characteristics have been generated by mutagenesis. The enhanced green fluorescent protein (EGFP) is a well-known GFP derivative with highly increased fluorescence intensity compared to the GFP wild-type molecule. Further optimization strategies include numerous GFP derivatives with blue- and yellow-shifted fluorescence and increased pH-stability. The methods reported herein describe in detail the construction of customized fluorescent GFP reporter plasmids where the fluorescence gene is expressed under the control of a certain bacterial promoter of interest. Special attention is given to the GFP derivatives EGFP and Sirius. We explain how to generate EGFP/Sirius expressing streptococci and how to employ recombinantly labeled streptococci in different downstream fluorescent applications.

  15. Cocultures of GFP(+) -granule cells with GFP(-) -pyramidal cells and interneurons for the study of mossy fiber neurotransmission with paired recordings.

    PubMed

    Osorio, Beatriz; León, Uriel; Galván, Emilio J; Gutiérrez, Rafael

    2013-04-01

    Synaptic transmission of the granule cells (GCs) via their axons, the mossy fibers (MFs), is traditionally studied on acutely prepared or cultured slices. Usually, extracellular, bulk or minimal stimulation is used to evoke transmitter release from MF terminals, while recording from their postsynaptic target cells, the pyramidal cells and interneurons of CA3. However, the ideal method to assess MF neurotransmission, the simultaneous recording of a presynaptic GC and one of its target cells, is extremely difficult to achieve using slices. Alternatively, cultures of GCs establishing autapses have been developed, but in these, GCs do not contact their natural targets. We developed cocultures of GCs, dissociated from transgenic GFP(+) rats, with pyramidal cells and interneurons of CA3, dissociated from wild-type rats, and confirmed the expression of cell-specific markers by immunofluorescence. We conducted recordings of GFP(+) -GCs synaptically connected with their GFP(-) -target cells, and demonstrate that synaptic transmission and its plasticity have the signature of transmission of MF. Besides being strongly depressed by activation of mGluRs, high frequency activation of GC-to-pyramidal cells synapses undergo LTP, while GC-to-interneuron synapses undergo LTD. This coculture method allows a high reproducibility of recording connected pairs of identified cells, constituting a valuable tool to study MF transmission, as well as different combinations of identifiable pre- and postsynaptic cells.

  16. Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

    SciTech Connect

    Fujimura, Juri; E-mail: juri-f@nms.ac.jp; Ogawa, Rei; Mizuno, Hiroshi; Fukunaga, Yoshitaka; Suzuki, Hidenori

    2005-07-22

    Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.

  17. HU-GFP and DAPI co-localize on the Escherichia coli nucleoid.

    PubMed

    Wery, M; Woldringh, C L; Rouviere-Yaniv, J

    2001-02-01

    The heterodimeric HU protein, one of the most abundant DNA binding proteins, plays a pleiotropic role in bacteria. Among others, HU was shown to contribute to the maintenance of DNA superhelical density in Escherichia coli. By its properties HU shares some traits with histones and HMG proteins. More recently, its specific binding to DNA recombination and repair intermediates suggests that HU should be considered as a DNA damage sensor. For all these reasons, it will be of interest to follow the localization of HU within the living bacterial cells. To this end, we constructed HU-GFP fusion proteins and compared by microscopy the GFP green fluorescence with images of the nucleoid after DAPI staining. We show that DAPI and HU-GFP colocalize on the E. coli nucleoid. HU, therefore, can be considered as a natural tracer of DNA in the living bacterial cell.

  18. Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo.

    PubMed

    To, Tsz-Leung; Schepis, Antonino; Ruiz-González, Rubén; Zhang, Qiang; Yu, Dan; Dong, Zhiqiang; Coughlin, Shaun R; Shu, Xiaokun

    2016-07-21

    Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.

  19. Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays.

    PubMed

    Henriksen, Ulla; Fog, Jacob; Loechel, Frosty; Praestegaard, Morten

    2008-08-01

    Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFkappaB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca(2+) sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.

  20. Transformation of tobacco plants by Yali PPO-GFP fusion gene and observation of subcellular localization.

    PubMed

    Qi, Jing; Li, Gui-Qin; Dong, Zhen; Zhou, Wei

    2016-01-01

    To explore the subcellular localization of Polyphenol oxidase (PPO) from Pyrus bretschneideri, the 1779 bp cDNA of PPO gene excluding the termination codon TAA was cloned and fused with GFP to construct a binary vector pBI121-PPO-GFP. Then, the binary vector was transformed into Nicotiana tabacum by the tumefanciens-mediated method. Using confocal laser scanning microscopy, green fluorescent signals were localized in chloroplasts of the transformed Nicotiana tabacum cell, suggesting that the Polyphenol oxidase from Pyrus bretschneideri was a chloroplast protein.

  1. Reduction of Photo Bleaching and Long Term Archiving of Chemically Cleared GFP-Expressing Mouse Brains

    PubMed Central

    Becker, Klaus; Hahn, Christian Markus; Saghafi, Saiedeh; Jährling, Nina; Wanis, Martina; Dodt, Hans-Ulrich

    2014-01-01

    Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation. If required, high-power illumination or long exposure times can be applied with virtually no loss in signal quality and samples can be archived for years. PMID:25463047

  2. Understanding virtual water flows: A multiregion input-output case study of Victoria

    NASA Astrophysics Data System (ADS)

    Lenzen, Manfred

    2009-09-01

    This article explains and interprets virtual water flows from the well-established perspective of input-output analysis. Using a case study of the Australian state of Victoria, it demonstrates that input-output analysis can enumerate virtual water flows without systematic and unknown truncation errors, an issue which has been largely absent from the virtual water literature. Whereas a simplified flow analysis from a producer perspective would portray Victoria as a net virtual water importer, enumerating the water embodiments across the full supply chain using input-output analysis shows Victoria as a significant net virtual water exporter. This study has succeeded in informing government policy in Australia, which is an encouraging sign that input-output analysis will be able to contribute much value to other national and international applications.

  3. Stratigraphic architecture of bedrock reference section, Victoria Crater, Meridiani Planum, Mars

    USGS Publications Warehouse

    Edgar, Lauren A.; Grotzinger, John P.; Hayes, Alex G.; Rubin, David M.; Squyres, Steve W.; Bell, James F.; Herkenhoff, Ken E.

    2012-01-01

    The Mars Exploration Rover Opportunity has investigated bedrock outcrops exposed in several craters at Meridiani Planum, Mars, in an effort to better understand the role of surface processes in its geologic history. Opportunity has recently completed its observations of Victoria crater, which is 750 m in diameter and exposes cliffs up to ~15 m high. The plains surrounding Victoria crater are ~10 m higher in elevation than those surrounding the previously explored Endurance crater, indicating that the Victoria crater exposes a stratigraphically higher section than does the Endurance crater; however, Victoria strata overlap in elevation with the rocks exposed at the Erebus crater. Victoria crater has a well-developed geomorphic pattern of promontories and embayments that define the crater wall and that reveal thick bedsets (3–7m) of large-scale cross-bedding, interpreted as fossil eolian dunes. Opportunity was able to drive into the crater at Duck Bay, located on the western margin of Victoria crater. Data from the Microscopic Imager and Panoramic Camera reveal details about the structures, textures, and depositional and diagenetic events that influenced the Victoria bedrock. A lithostratigraphic subdivision of bedrock units was enabled by the presence of a light-toned band that lines much of the upper rim of the crater. In ascending order, three stratigraphic units are named Lyell, Smith, and Steno; Smith is the light-toned band. In the Reference Section exposed along the ingress path at Duck Bay, Smith is interpreted to represent a zone of diagenetic recrystallization; however, its upper contact also coincides with a primary erosional surface. Elsewhere in the crater the diagenetic band crosscuts the physical stratigraphy. Correlation with strata present at nearby promontory Cape Verde indicates that there is an erosional surface at the base of the cliff face that corresponds to the erosional contact below Steno. The erosional contact at the base of Cape Verde

  4. Monitoring the water balance of Lake Victoria, East Africa, from space

    NASA Astrophysics Data System (ADS)

    Swenson, Sean; Wahr, John

    2009-05-01

    SummaryUsing satellite gravimetric and altimetric data, we examine trends in water storage and lake levels of multiple lakes in the Great Rift Valley region of East Africa for the years 2003-2008. GRACE total water storage estimates reveal that water storage declined in much of East Africa, by as much as 60 {mm}/{year}, while altimetric data show that lake levels in some large lakes dropped by as much as 1-2 m. The largest declines occurred in Lake Victoria, the Earth's second largest freshwater body. Because the discharge from the outlet of Lake Victoria is used to generate hydroelectric power, the role of human management in the lake's decline has been questioned. By comparing catchment water storage trends to lake level trends, we confirm that climatic forcing explains only about 50decline. This analysis provides an independent means of assessing the relative impacts of climate and human management on the water balance of Lake Victoria that does not depend on observations of dam discharge, which may not be publically available. In the second part of the study, the individual components of the lake water balance are estimated. Satellite estimates of changes in lake level, precipitation, and evaporation are used with observed lake discharge to develop a parameterization for estimating subsurface inflows due to changes in groundwater storage estimated from satellite gravimetry. At seasonal timescales, this approach provides closure to Lake Victoria's water balance to within 17 {mm}/{month}. The third part of this study uses the water balance of a downstream water body, Lake Kyoga, to estimate the outflow from Lake Victoria remotely. Because Lake Kyoga is roughly 20 times smaller in area than Lake Victoria, its water balance is strongly influenced by inflow from Lake Victoria. Lake Kyoga has been shown to act as a linear reservoir, where its outflow is proportional to the height of the lake. This model can be used with satellite altimetric lake levels to estimate a

  5. Assessing Human Impacts on the Water Balance of Lake Victoria Using GRACE and Altimeters.

    NASA Astrophysics Data System (ADS)

    Swenson, S. C.; Wahr, J. M.

    2008-12-01

    GRACE has provided global estimates of vertically integrated water storage anomalies at monthly intervals with a useful spatial resolution of 300-500 km. Water levels of large lakes are now routinely monitored by satellite altimeters. By combining these datasets, it may be possible to separate the effects of climate and human management on lake levels. During the period 2002-2006, the levels of Lake Victoria dropped about two meters. Water storage changes of Lake Victoria and other large lakes in the East African Rift Valley are compared, and the relative impacts of climate and human management are shown to be of similar magnitude.

  6. Chemodenitrification in the cryoecosystem of Lake Vida, Victoria Valley, Antarctica.

    PubMed

    Ostrom, N E; Gandhi, H; Trubl, G; Murray, A E

    2016-11-01

    Lake Vida, in the Victoria Valley of East Antarctica, is frozen, yet harbors liquid brine (~20% salt, >6 times seawater) intercalated in the ice below 16 m. The brine has been isolated from the surface for several thousand years. The brine conditions (permanently dark, -13.4 °C, lack of O2 , and pH of 6.2) and geochemistry are highly unusual. For example, nitrous oxide (N2 O) is present at a concentration among the highest reported for an aquatic environment. Only a minor (17) O anomaly was observed in N2 O, indicating that this gas was predominantly formed in the lake. In contrast, the (17) O anomaly in nitrate (NO3-) in Lake Vida brine indicates that approximately half or more of the NO3- present is derived from atmospheric deposition. Lake Vida brine was incubated in the presence of (15) N-enriched substrates for 40 days. We did not detect microbial nitrification, dissimilatory reduction of NO3- to ammonium (NH4+), anaerobic ammonium oxidation, or denitrification of N2 O under the conditions tested. In the presence of (15) N-enriched nitrite (NO2-), both N2 and N2 O exhibited substantial (15) N enrichments; however, isotopic enrichment declined with time, which is unexpected. Additions of (15) N-NO2- alone and in the presence of HgCl2 and ZnCl2 to aged brine at -13 °C resulted in linear increases in the δ(15) N of N2 O with time. As HgCl2 and ZnCl2 are effective biocides, we interpret N2 O production in the aged brine to be the result of chemodenitrification. With this understanding, we interpret our results from the field incubations as the result of chemodenitrification stimulated by the addition of (15) N-enriched NO2- and ZnCl2 and determined rates of N2 O and N2 production of 4.11-41.18 and 0.55-1.75 nmol L(-1)  day(-1) , respectively. If these rates are representative of natural production, the current concentration of N2 O in Lake Vida could have been reached between 6 and 465 years. Thus, chemodenitrification alone is sufficient to explain the

  7. Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish

    PubMed Central

    Ota, Satoshi; Taimatsu, Kiyohito; Yanagi, Kanoko; Namiki, Tomohiro; Ohga, Rie; Higashijima, Shin-ichi; Kawahara, Atsuo

    2016-01-01

    The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes. PMID:27725766

  8. Automated, high-throughput platform for protein solubility screening using a split-GFP system.

    PubMed

    Listwan, Pawel; Terwilliger, Thomas C; Waldo, Geoffrey S

    2009-03-01

    Overproduction of soluble and stable proteins for functional and structural studies is a major bottleneck for structural genomics programs and traditional biochemistry laboratories. Many high-payoff proteins that are important in various biological processes are "difficult to handle" as protein reagents in their native form. We have recently made several advances in enabling biochemical technologies for improving protein stability (http://www.lanl.gov/projects/gfp/), allowing stratagems for efficient protein domain trapping, solubility-improving mutations, and finding protein folding partners. In particular split-GFP protein tags are a very powerful tool for detection of stable protein domains. Soluble, stable proteins tagged with the 15 amino acid GFP fragment (amino acids 216-228) can be detected in vivo and in vitro using the engineered GFP 1-10 "detector" fragment (amino acids 1-215). If the small tag is accessible, the detector fragment spontaneously binds resulting in fluorescence. Here, we describe our current and on-going efforts to move this process from the bench (manual sample manipulation) to an automated, high-throughput, liquid-handling platform. We discuss optimization and validation of bacterial culture growth, lysis protocols, protein extraction, and assays of soluble and insoluble protein in multiple 96 well plate format. The optimized liquid-handling protocol can be used for rapid determination of the optimal, compact domains from single ORFS, collections of ORFS, or cDNA libraries.

  9. Protection against Autoimmune Diabetes by Silkworm-Produced GFP-Tagged CTB-Insulin Fusion Protein

    PubMed Central

    Meng, Qiaohong; Wang, Wenfeng; Shi, Xiaowen; Jin, Yongfeng; Zhang, Yaozhou

    2011-01-01

    In animals, oral administration of the cholera toxin B (CTB) subunit conjugated to the autoantigen insulin enhances the specific immune-unresponsive state. This is called oral tolerance and is capable of suppressing autoimmune type 1 diabetes (T1D). However, the process by which the CTB-insulin (CTB-INS) protein works as a therapy for T1D in vivo remains unclear. Here, we successfully expressed a green fluorescent protein- (GFP-) tagged CTB-Ins (CTB-Ins-GFP) fusion protein in silkworms in a pentameric form that retained the native ability to activate the mechanism. Oral administration of the CTB-Ins-GFP protein induced special tolerance, delayed the development of diabetic symptoms, and suppressed T1D onset in nonobese diabetic (NOD) mice. Moreover, it increased the numbers of CD4+CD25+Foxp3+ T regulatory (Treg) cells in peripheral lymph tissues and affected the biological activity of spleen cells. This study demonstrated that the CTB-Ins-GFP protein produced in silkworms acted as an oral protein vaccine, inducing immunological tolerance involving CD4+CD25+Foxp3+ Treg cells in treating T1D. PMID:21765853

  10. Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum

    PubMed Central

    Ewe, Daniela; Río Bártulos, Carolina; Kroth, Peter G.; Gruber, Ansgar

    2016-01-01

    An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns. PMID:27635322

  11. Visualization of coral host-pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

    NASA Astrophysics Data System (ADS)

    Pollock, F. Joseph; Krediet, Cory J.; Garren, Melissa; Stocker, Roman; Winn, Karina; Wilson, Bryan; Huete-Stauffer, Carla; Willis, Bette L.; Bourne, David G.

    2015-06-01

    The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host-pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain's virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen's interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.

  12. In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.

    PubMed

    Lark, Arianna R; Kitamoto, Toshihiro; Martin, Jean-René

    2016-01-08

    Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca(2+)-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and - with the addition of its cofactor coelenterazine - emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca(2+)-transients and Ca(2+)-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca(2+)-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

  13. Colonization of spinach (Spinacia oleracea L.) by GFP-tagged verticillium dahliae.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The soilborne fungus, Verticillium dahliae, causes wilt in a wide range of hosts, including spinach (Spinacia oleracea L.). The interaction between a green fluorescent protein (GFP)-tagged V. dahliae strain and spinach was studied by confocal laser scanning microscopy. The roots of spinach seedlings...

  14. A mechanistically relevant cytotoxicity assay based on the detection of cellular GFP.

    PubMed

    Halter, Michael; Almeida, Jamie L; Tona, Alessandro; Cole, Kenneth D; Plant, Anne L; Elliott, John T

    2009-08-01

    Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.

  15. Non-invasive imaging of transgenic GFP expression in neonatal mouse brain

    NASA Astrophysics Data System (ADS)

    Ho, Gideon; Zhang, Chunyan; Zhuo, Lang

    2007-02-01

    Glial fibrillary acidic protein (GFAP) is a traditional biomarker for astrocytes of the central nervous system. In this study, non-invasive in vivo imaging of GFAP-GFP (green fluorescent protein) expression in the brain of neonatal transgenic mice is used as a novel method to investigate the relationship between the expression of the transgene at 0, 2, 4, 6 and 8 hr post-treatment in mice subjected to a single administration of 12 mg/kg of neurotoxin 1-methyl-4(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH 3-MPTP). The GFP elevation was found to peak at 6 hr and lasted to at least 8 hr after the toxin treatment. Histological examination of fixed brain sections using immunohistochemistry (IHC) shows an increase in GFP and GFAP signal from the substantia nigra pars compacta (SNpc) and the hippocampus. The results have provided quantitative fluorescence and qualitative histological evidence for the activation of the GFAP-GFP transgene in astrocytes following neurotoxin 2'-CH 3-MPTP administration, suggesting that the model described here could be used to study neuronal degeneration such as Parkinson's disease and in general, developmental neurotoxicity in live animals.

  16. Structural Analysis of the Victoria Quadrangle (H2) of Mercury based on NASA MESSENGER Data

    NASA Astrophysics Data System (ADS)

    Galluzzi, Valentina

    2015-04-01

    Objective of this thesis is the mapping and structural analysis of the H2 quadrangle, “Victoria”, and a reconnaissance study of the geometry and kinematics of lobate scarps on Mercury. To this end, I produced a 1:3,000,000 geologic map of the area using the images provided by the NASA spacecraft MESSENGER, which has been orbiting the planet since March, 2011. The geologic map shows the distribution of smooth plains, intermediate plains, intercrater plains units and a classification of crater materials based on an empirical distinction among three stages of degradation. Structural mapping shows that the H2 quadrangle is dominated by N-S faults (here grouped into the Victoria system) to the east and NE-SW faults (Larrocha system) to the west, with the secondary existence of NW-SE-trending faults (Carnegie system) in the north-western area of the quadrangle. A systematic analysis of these systems has led to the following results. 1) The Victoria system is characterized by a main array of faults located along Victoria Rupes - Endeavour Rupes - Antoniadi Dorsum. The segmentation of this array into three different sectors changes from north to south and is spatially linked to the presence of three volcanic vents located at the boundaries between each sector and at the northern end of the Victoria Rupes sector, suggesting that volcanism and faulting are interrelated. 2) The main array of Carnegie system is kinematically linked and antithetical to the Victoria system. Both systems have arguably controlled the growth of a longitudinal, fault-free, crustal and gravimetric bulge in the central area of the Victoria quadrangle, which is interpreted as a regional contractional pop-up. 3) The Larrocha system is interrupted against the central bulge and thus is probably older than the Victoria and Carnegie systems. Buffered crater counting performed on the Victoria system confirms the young relative age of its fault segments with respect to the map units. The faults of the

  17. Sorting of GFP Tagged NtSyr1, an ABA Related Syntaxin

    PubMed Central

    Gigante, Massimiliano; De Domenico, Stefania; Piro, Gabriella; Dalessandro, Giuseppe

    2006-01-01

    Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers. NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane. In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane. PMID:19521480

  18. Interplay of Promoter Usage and Intragenic CpG Content: Impact on GFP Reporter Gene Expression.

    PubMed

    Krinner, Simone; Heitzer, Asli; Asbach, Benedikt; Wagner, Ralf

    2015-12-01

    Successful therapeutic protein production in vitro and in vivo requires efficient and long-term transgene expression supported by optimized vector and transgene cis-regulatory sequence elements. This study provides a comparative analysis of CpG-rich, highly expressed, versus CpG-depleted, poorly expressed green fluorescent protein (GFP) reporter transgenes, transcribed by various promoters in two different cell systems. Long-term GFP expression from a defined locus in stable Chinese hamster ovary cells was clearly influenced by the combination of transgene CpG content and promoter usage, as shown by differential silencing effects on selection pressure removal among the cytomegalovirus (CMV) promoter and elongation factor (EF)-1α promoter. Whereas a high intragenic CpG content promoted local DNA methylation, CpG depletion rather accelerated transgene loss and increased the local chromatin density. On lentiviral transfer of various expression modules into epigenetically sensitive P19 embryonic pluripotent carcinoma cells, CMV promoter usage led to rapid gene silencing irrespective of the intragenic CpG content. In contrast, EF-1α promoter-controlled constructs showed delayed silencing activity and high-level transgene expression, in particular when the CpG-rich GFP reporter was used. Notably, GFP silencing in P19 cells could be prevented completely by the bidirectional, dual divergently transcribed A2UCOE (ubiquitously acting chromatin-opening element derived from the human HNRPA2B1-CBX3 locus) promoter. Because the level of GFP expression by the A2UCOE promoter was entirely unaffected by the intragenic CpG level, we suggest that A2UCOE can overcome chromatin compaction resulting from intragenic CpG depletion due to its ascribed chromatin-opening abilities. Our analyses provide insights into the interplay of the intragenic CpG content with promoter sequences and regulatory sequence elements, thus contributing toward the design of therapeutic transgene expression

  19. Regulation of rhodopsin-eGFP distribution in transgenic xenopus rod outer segments by light.

    PubMed

    Haeri, Mohammad; Calvert, Peter D; Solessio, Eduardo; Pugh, Edward N; Knox, Barry E

    2013-01-01

    The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial variation of fluorescence intensity in cells expressing a fluorescently tagged rhodopsin transgene. There was a light synchronized fluctuation in intensity, with higher intensity in disks formed at night and lower intensity for those formed during the day. This fluctuation was absent in constant light or dark conditions. There was also a slow modulation of the overall expression level that was not synchronized with the lighting cycle or between cells in the same retina. The axial variations of other membrane-associated fluorescent proteins, eGFP-containing two geranylgeranyl acceptor sites and eGFP fused to the transmembrane domain of syntaxin, were greatly reduced or not detectable, respectively. In acutely light-adapted rods, an arrestin-eGFP fusion protein also exhibited axial variation. Both the light-sensitive Rho-eGFP and arrestin-eGFP banding were in phase with the previously characterized birefringence banding (Kaplan, Invest. Ophthalmol. Vis. Sci. 21, 395-402 1981). In contrast, endogenous rhodopsin did not exhibit such axial variation. Thus, there is an axial inhomogeneity in membrane composition or structure, detectable by the rhodopsin transgene density distribution and regulated by the light cycle, implying a light-regulated step for disk assembly in the OS. The impact of these results on the use of chimeric proteins with rhodopsin fused to fluorescent proteins at the carboxyl terminus is discussed.

  20. Preferential and Bidirectional Labeling of the Rubrospinal Tract with Adenovirus-GFP for Monitoring Normal and Injured Axons

    PubMed Central

    Wang, Xiaofei; Smith, George M.

    2011-01-01

    Abstract The rodent rubrospinal tract (RST) has been studied extensively to investigate regeneration and remodeling of central nervous system (CNS) axons. Currently no retrograde tracers can specifically label rubrospinal axons and neurons (RSNs). The RST can be anterogradely labeled by injecting tracers into the red nucleus (RN), but accurately locating the RN is a technical challenge. Here we developed a recombinant adenovirus carrying a green fluorescent protein reporter gene (Adv-GFP) which can preferentially, intensely, and bi-directionally label the RST. When Adv-GFP was injected into the second lumbar spinal cord, the GFP was specifically transported throughout the entire RST, with peak labeling seen at 2 weeks post-injection. When Adv-GFP was injected directly into the RN, GFP was anterogradely transported throughout the RST. Following spinal cord injury (SCI), injection of Adv-GFP resulted in visualization of GFP in transected, spared, or sprouted RST axons bi-directionally. Thus Adv-GFP could be used as a novel tool for monitoring and evaluating strategies designed to maximize RST axonal regeneration and remodeling following SCI. PMID:21299337

  1. Determination of the in vivo redox potential using roGFP and fluorescence spectra obtained from one-wavelength excitation

    NASA Astrophysics Data System (ADS)

    Wierer, S.; Elgass, K.; Bieker, S.; Zentgraf, U.; Meixner, A. J.; Schleifenbaum, F.

    2011-02-01

    The analysis of molecular processes in living (plant) cells such as signal transduction, DNA replication, carbon metabolism and senescence has been revolutionized by the use of green fluorescent protein (GFP) and its variants as specific cellular markers. Many cell biological processes are accompanied by changes in the intracellular redox potential. To monitor the redox potential, a redox-sensitive mutant of GFP (roGFP) was created, which shows changes in its optical properties in response to changes in the redox state of its surrounding medium. For a quantitative analysis in living systems, it is essential to know the optical properties of roGFP in vitro. Therefore, we applied spectrally resolved fluorescence spectroscopy on purified roGFP exposed to different redox potentials to determine shifts in both the absorption and the emission spectra of roGFP. Based on these in vitro findings, we introduce a new approach using one-wavelength excitation to use roGFP for the in vivo analysis of cell biological processes. We demonstrate the ability this technique by investigating chloroplast-located Grx1-roGFP2 expressing Arabidopsis thaliana cells as example for dynamically moving intracellular compartments. This is not possible with the two-wavelength excitation technique established so far, which hampers a quantitative analysis of highly mobile samples due to the time delay between the two measurements and the consequential displacement of the investigated area.

  2. Functional expression and cellular localization of cercosporin-resistance proteins fused with the GFP in Cercospora nicotianae.

    PubMed

    Chung, Kuang-Ren; Ehrenshaft, Marilyn; Daub, Margaret E

    2002-06-01

    The Cercospora nicotianae pdx1 and crg1 genes were previously identified as genes required for resistance to the singlet oxygen ((1)O(2))-generating toxin cercosporin. The pdx1 gene has subsequently been shown to be required for pyridoxine biosynthesis, but both the precise biochemical function of the PDX1 protein and the function of the CRG1 protein remain undefined, as both sequences lack defined enzymatic domains or cofactor-binding sites. The gfp gene encoding green fluorescent protein was translationally fused with pdx1 and crg1. Transformation of these constructs into strains mutant in these respective genes resulted in green-fluorescent transformants complemented for the mutant phenotype. Microscopic studies revealed that in transformants transformed with gfp alone, fluorescence was distributed evenly throughout the cytoplasm and excluded from the vacuoles. Expression of PDX1::GFP either under the constitutive Aspergillus nidulans gpdA promoter or its own native promoter was visualized as distinct fluorescent circular structures in the cytoplasm, suggesting that PDX1::GFP was probably localized in the intracellular vesicles. Expression of CRG1 fused with GFP at either its N- or C-terminus resulted in low green fluorescence, compared with that of GFP alone or PDX1::GFP. The green fluorescence of either of the CRG1::GFP fusion proteins was barely observable in transformants and was generally seen as a few scattered regions of fluorescence in the hyphae. Southern blot analysis indicated multiple copies of the constructs were integrated into the fungal genome. Northern analysis revealed that pdx1:: gfp and crg1:: gfp were each expressed as an intact transcriptional unit. Cell fractionation followed by immunoblotting against a GFP antibody showed that GFP alone and PDX1::GFP were detected exclusively in the cytoplasmic fraction. The two CRG1::GFP proteins were barely detected in the cytoplasmic fraction and not at all from the membrane fraction, a result

  3. Equity Indicators: Measures of Socio-Economic Status at Victoria University.

    ERIC Educational Resources Information Center

    Sinclair, Genevieve; Doughney, James; Palermo, Josephine

    After a review of relevant literature on socioeconomic status (SES) and the ways in which is used for higher education institutional research and policy, a detailed data analysis of Victoria University (VU), Australia student data was undertaken. Between 10,000 and 15,000 domestic student addresses were geocoded to Australian Bureau of Statistics…

  4. A Survey of People with Intellectual Disabilities Living in Residential Aged Care Facilities in Victoria

    ERIC Educational Resources Information Center

    Bigby, C.; Webber, R.; Bowers, B.; McKenzie-Green, B.

    2008-01-01

    Background: Australia's national ageing policy recognises that people ageing with intellectual disability (ID) require particular attention, yet there is no policy framework concerning this population. This study describes the distribution and characteristics of people with ID in residential aged care in Victoria, provides insights into the…

  5. The Killing of the Workers' Educational Association of Victoria: A Myth Challenged

    ERIC Educational Resources Information Center

    Dadswell, Gordon

    2004-01-01

    On 21 March 1941, the Council of the Workers' Educational Association of Victoria, Australia, (the Association) voted the organisation out of existence. The demise was in no way contemplated, and there was no practical reason why the Council acted in the way it did. This paper is the story of the destruction of a successful adult education…

  6. Connections '96. Proceedings of a Faculty Conference (2nd, Victoria, British Columbia, Canada, May 1996).

    ERIC Educational Resources Information Center

    Dayton-Sakari, Mary, Ed.; Miller, Carole S., Ed.; Liedtke, Werner, Ed.

    This proceedings contains 19 papers presented at the second annual faculty conference at the University of Victoria (British Columbia, Canada). Papers cover a wide variety of disciplines, including preschool education, classroom communication, mathematics instruction, theater, attention deficit disorders, distance learning by rural home schoolers,…

  7. Increase in Meningococcal Serogroup W Disease, Victoria, Australia, 2013–2015

    PubMed Central

    Stevens, Kerrie; Sohail, Asma; Franklin, Lucinda J.; Bond, Katherine A.; Brahmi, Aicha; Romanes, Finn; Ong, Katherine S.

    2016-01-01

    In Victoria, Australia, invasive meningococcal disease caused by Neisseria meningitidis serogroup W increased from 4% of all cases in 2013 to 30% in 2015. This increase resulted largely from strains similar to those in the serogroup W sequence type 11 clonal complex, previously described in the United Kingdom and South America. PMID:27648521

  8. Visy Cares Hub and Victoria University: Making the Door of a University Open to the Community

    ERIC Educational Resources Information Center

    Broadbent, Robyn

    2011-01-01

    In 1999, a group of men embarked on a remarkable project that resulted in building a two million dollar youth centre in one of Melbourne's most disadvantaged communities. From the outset, Victoria University (VU) was a keen partner in the project. This project had key synergies with the current experiences of the University--a dual sector higher…

  9. Perception of Aquaculture Education to Support Further Growth of Aquaculture Industry in Victoria, Australia

    ERIC Educational Resources Information Center

    Awal, Sadiqul; Christie, Andrew; Watson, Matthew; Hannadige, Asanka G. T.

    2012-01-01

    Purpose: The central aim of this study was to determine the perception of aquaculture educational provisions in the state of Victoria, and whether they are sufficient to ultimately support further growth of the industry. Design/methodology/approach: Questionnaires were formulated and distributed to participants in a variety of ways, including via…

  10. Employment Shifts in the TAFE Workforce in Victoria, 1993-98. Working Paper.

    ERIC Educational Resources Information Center

    Shah, Chandra

    Data on the work force in Technical and Further Education (TAFE) institutes in Victoria, Australia, for 1993-1998 reveal a number of structural changes. First, the number of women staff increased from 46% to 53%, although men still constitute 54% of the teaching staff. As full-time staff employment dropped an average of 1.1% annually, part-time…

  11. Vulnerability to Bushfires in Rural Australia: A Case Study from East Gippsland, Victoria

    ERIC Educational Resources Information Center

    Whittaker, Joshua; Handmer, John; Mercer, David

    2012-01-01

    This paper investigates the nature and causes of vulnerability to bushfires in the Wulgulmerang district of East Gippsland, Victoria, in south-eastern Australia. In 2003 bushfires devastated the small population of this isolated farming district, destroying homes, agricultural assets and public infrastructure. The fires also adversely affected the…

  12. History and timing of human impact on Lake Victoria, East Africa.

    PubMed Central

    Verschuren, Dirk; Johnson, Thomas C; Kling, Hedy J; Edgington, David N; Leavitt, Peter R; Brown, Erik T; Talbot, Michael R; Hecky, Robert E

    2002-01-01

    Lake Victoria, the largest tropical lake in the world, suffers from severe eutrophication and the probable extinction of up to half of its 500+ species of endemic cichlid fishes. The continuing degradation of Lake Victoria's ecological functions has serious long-term consequences for the ecosystem services it provides, and may threaten social welfare in the countries bordering its shores. Evaluation of recent ecological changes in the context of aquatic food-web alterations, catchment disturbance and natural ecosystem variability has been hampered by the scarcity of historical monitoring data. Here, we present high-resolution palaeolimnological data, which show that increases in phytoplankton production developed from the 1930s onwards, which parallels human-population growth and agricultural activity in the Lake Victoria drainage basin. Dominance of bloom-forming cyanobacteria since the late 1980s coincided with a relative decline in diatom growth, which can be attributed to the seasonal depletion of dissolved silica resulting from 50 years of enhanced diatom growth and burial. Eutrophication-induced loss of deep-water oxygen started in the early 1960s, and may have contributed to the 1980s collapse of indigenous fish stocks by eliminating suitable habitat for certain deep-water cichlids. Conservation of Lake Victoria as a functioning ecosystem is contingent upon large-scale implementation of improved land-use practices. PMID:11839198

  13. From Aspiration to Destination: Understanding the Decisions of University Applicants in Regional Victoria

    ERIC Educational Resources Information Center

    Harvey, Andrew; Burnheim, Catherine; Joschko, Lucie; Luckman, Michael

    2012-01-01

    This paper examines the choices and destinations of prospective university students from three regional areas in Victoria. The study is based on information collected for tertiary applicants in the Gippsland, Bendigo and Mildura areas, all of which host a local university campus. Using application and enrollment data, we examine the choices that…

  14. 75 FR 9114 - FM Table of Allotments, Markham, Ganado, and Victoria, Texas

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-01

    ... COMMISSION 47 CFR Part 73 FM Table of Allotments, Markham, Ganado, and Victoria, Texas AGENCY: Federal... counterproposal filed by Fort Bend Broadcasting Company, licensee of Station KHTZ(FM), Ganado, Texas, to upgrade Station KHTZ(FM) from Channel 284C2 to Channel 235C and to modify its license accordingly....

  15. History and timing of human impact on Lake Victoria, East Africa.

    PubMed

    Verschuren, Dirk; Johnson, Thomas C; Kling, Hedy J; Edgington, David N; Leavitt, Peter R; Brown, Erik T; Talbot, Michael R; Hecky, Robert E

    2002-02-07

    Lake Victoria, the largest tropical lake in the world, suffers from severe eutrophication and the probable extinction of up to half of its 500+ species of endemic cichlid fishes. The continuing degradation of Lake Victoria's ecological functions has serious long-term consequences for the ecosystem services it provides, and may threaten social welfare in the countries bordering its shores. Evaluation of recent ecological changes in the context of aquatic food-web alterations, catchment disturbance and natural ecosystem variability has been hampered by the scarcity of historical monitoring data. Here, we present high-resolution palaeolimnological data, which show that increases in phytoplankton production developed from the 1930s onwards, which parallels human-population growth and agricultural activity in the Lake Victoria drainage basin. Dominance of bloom-forming cyanobacteria since the late 1980s coincided with a relative decline in diatom growth, which can be attributed to the seasonal depletion of dissolved silica resulting from 50 years of enhanced diatom growth and burial. Eutrophication-induced loss of deep-water oxygen started in the early 1960s, and may have contributed to the 1980s collapse of indigenous fish stocks by eliminating suitable habitat for certain deep-water cichlids. Conservation of Lake Victoria as a functioning ecosystem is contingent upon large-scale implementation of improved land-use practices.

  16. Compatibility of Ugandan Schistosoma mansoni isolates with Biomphalaria snail species from Lake Albert and Lake Victoria.

    PubMed

    Adriko, Moses; Standley, Claire J; Tinkitina, Benjamin; Mwesigwa, Gerald; Kristensen, Thomas K; Stothard, J Russell; Kabatereine, Narcis B

    2013-11-01

    In order to investigate the capacity of being intermediate host for Schistosoma mansoni, the Ugandan F1 generation of Biomphalaria snail species that were laboratory-bred from parent populations originally collected from either Lake Victoria or Lake Albert was challenged with sympatric and non-sympatric S. mansoni isolates. After a prepatent period of 20 days, a daily 10-hourly snail shedding for cercariae was done to determine the infection rate, cercarial production per hour and survival period of infected snails. The study suggests that when parasite strains from a different geographical origin is used for infection, survival of infected snails increase, leading to an increased transmission potential. Although earlier literature had indicated that the Lake Victoria Biomphalaria sudanica is refractory to S. mansoni, we showed that all Ugandan Biomphalaria spp., including B. sudanica from all locations, were highly susceptible to the S. mansoni isolates. Thus if B. choanomphala, which is an efficient intermediate host in Lake Victoria, is given an opportunity to occupy Lake Albert, it will most likely be compatible with the Albertine S. mansoni parasites. Equally, if B. stanleyi, currently restricted to Lake Albert invades Lake Victoria, it is likely to act as an efficient intermediate host. Future work should concentrate on intraspecific population-level differences in compatibility.

  17. What Do the Public Search for on the Catalogue of the State Library of Victoria?

    ERIC Educational Resources Information Center

    Waller, Vivienne

    2009-01-01

    This study examines what the public search for in the catalogue of the State Library of Victoria (SLV). As well as indicating the type of content being accessed, this gives an indication of what catalogue users expect of the State Library collection. A content analysis was undertaken of a random, stratified sample of 4,000 search queries typed in…

  18. Establishing an Online Community of Inquiry at the Distance Education Centre, Victoria

    ERIC Educational Resources Information Center

    Jackson, Luke C.; Jackson, Alun C.; Chambers, Dianne

    2013-01-01

    This pilot intervention focused on three courses that were redesigned to utilize the online environment to establish an online community of inquiry (CoI). The setting for this research study was the Distance Education Centre, Victoria (DECV), an Australian co-educational school with approximately 3000 students who, for a variety of reasons, are…

  19. Connections '98. Proceedings of a Faculty Conference (4th, Victoria, British Columbia, Canada, May 1998).

    ERIC Educational Resources Information Center

    Gibbons, Sandra L., Ed.; Anderson, John O., Ed.

    This proceedings contains 13 papers from the 1998 annual Faculty of Education conference at the University of Victoria, British Columbia (Canada). The papers are: (1) "Struggling with Re-Presentation, Voice, and Self in Narrative Research" (Marla Arvay); (2) "Women's Soccer in Canada: A Slow Road to Equity" (Meredith Bogle,…

  20. Connections '99. Proceedings of a Faculty Conference (5th, Victoria, British Columbia, Canada, May 1999).

    ERIC Educational Resources Information Center

    Gibbons, Sandra L., Ed.; Liedtke, Werner W., Ed.

    This proceedings contains 13 papers from the 1999 annual conference of the Faculty of Education, University of Victoria (British Columbia). The papers are: (1) "Sacred and the Profane in Advertising Art" (Bill Zuk, Robert Dalton); (2) "Finding the Fund$ in Fun Run: Evaluating the Effectiveness and Efficiency of Physical Activity…

  1. Connections '97. Proceedings of a Faculty Conference (3rd, Victoria, British Columbia, Canada, May 1997).

    ERIC Educational Resources Information Center

    Liedtke, Werner, Ed.

    This proceedings contains 17 papers presented at the third annual faculty conference at the University of Victoria (British Columbia, Canada). Papers cover a wide variety of disciplines and topics, including student teaching, athletics, researcher-teacher collaboration, hands-on science instruction, violence prevention, youth violence, counseling,…

  2. Testing the Marine Hypothesis for The Opportunity Landing Site at Victoria Crater

    NASA Astrophysics Data System (ADS)

    Parker, T. J.

    2006-12-01

    Hypothesis Summary: 1. Meridiani Planum is a marine sulfate platform deposit, analogous to terrestrial carbonate platforms but with sulfate mineralogy, laid down during one or multiple marine transgressions over the landing site region. 2. Outcrop laminations, ripples, and larger, dune-scale bedforms are subaqueous in origin, produced by tidal currents. Aqueous deposits may be interbedded with crossbedded aeolian deposits of same composition derived from subjacent water-lain deposits during lowstands. At the scale of observations made by Opportunity, the marine hypothesis differs from the consensus, sabkha model, in two relatively minor ways (but with important differences in the inferred paleoenvironments). 3. Blueberry and cobble "lag" on top of outcrop is a lag, but indicates erosion of perhaps many meters, even tens of meters or more of outcrop material from the region by water. Wind erosion has been very limited over geologic time (perhaps less than a meter locally). 4. Remarkably-flat, horizontal geomorphic surface of outcrop was produced by shallow standing water locally, controlling both deposition and erosion of the sulfate outcrop material to within a few meters of sea level. 5. Craters in the landing site exhibit a continuum of degradation states, with Endurance and Victoria typifying the best preserved craters visited (or to be visited) by Opportunity, and Erebus and Terra Nova representing the most degraded craters visited that are larger than 100 meters across. Terra Nova is similar to numerous kilometer-scale "rock ring" craters in the Meridiani Planum landing site. 6. "Serrated" rim at Victoria is similar to, but fresher in expression, rim morphology at Erebus Crater, and may indicate water pouring over crater rim during tidal or storm surges in water level across the region. Predictions to be tested at Victoria Crater: 1. The crater's ejecta and raised rim have been destroyed by tidal currents in shallow standing water. There is no Endurance

  3. Advances in fluorescent protein technology.

    PubMed

    Shaner, Nathan C; Patterson, George H; Davidson, Michael W

    2007-12-15

    Current fluorescent protein (FP) development strategies are focused on fine-tuning the photophysical properties of blue to yellow variants derived from the Aequorea victoria jellyfish green fluorescent protein (GFP) and on the development of monomeric FPs from other organisms that emit in the yellow-orange to far-red regions of the visible light spectrum. Progress toward these goals has been substantial, and near-infrared emitting FPs may loom over the horizon. The latest efforts in jellyfish variants have resulted in new and improved monomeric BFP, CFP, GFP and YFP variants, and the relentless search for a bright, monomeric and fast-maturing red FP has yielded a host of excellent candidates, although none is yet optimal for all applications. Meanwhile, photoactivatable FPs are emerging as a powerful class of probes for intracellular dynamics and, unexpectedly, as useful tools for the development of superresolution microscopy applications.

  4. Photoacid Behaviour in a Fluorinated Green Fluorescent Protein Chromophore: Ultrafast Formation of Anion and Zwitterion States.(†).

    PubMed

    Laptenok, S P; Conyard, J; Page, P C Bulman; Chan, Y; You, M; Jaffrey, S R; Meech, S R

    2016-01-01

    The photophysics of the chromophore of the green fluorescent protein in Aequorea victoria (avGFP) are dominated by an excited state proton transfer reaction. In contrast the photophysics of the same chromophore in solution are dominated by radiationless decay, and photoacid behaviour is not observed. Here we show that modification of the pKa of the chromophore by fluorination leads to an excited state proton transfer on an extremely fast (50 fs) time scale. Such a fast rate suggests a barrierless proton transfer and the existence of a pre-formed acceptor site in the aqueous solution, which is supported by solvent and deuterium isotope effects. In addition, at lower pH, photochemical formation of the elusive zwitterion of the GFP chromophore is observed by means of an equally fast excited state proton transfer from the cation. The significance of these results for understanding and modifying the properties of fluorescent proteins are discussed.

  5. Role of Coherent Low-Frequency Motion in Excited-State Proton Transfer of Green Fluorescent Protein Studied by Time-Resolved Impulsive Stimulated Raman Spectroscopy.

    PubMed

    Fujisawa, Tomotsumi; Kuramochi, Hikaru; Hosoi, Haruko; Takeuchi, Satoshi; Tahara, Tahei

    2016-03-30

    Green fluorescent protein (GFP) from jellyfish Aequorea victoria, an essential bioimaging tool, luminesces via excited-state proton transfer (ESPT) in which the phenolic proton of the p-hydroxybenzylideneimidazolinone chromophore is transferred to Glu222 through a hydrogen-bond network. In this process, the ESPT mediated by the low-frequency motion of the chromophore has been proposed. We address this issue using femtosecond time-resolved impulsive stimulated Raman spectroscopy. After coherently exciting low-frequency modes (<300 cm(-1)) in the excited state of GFP, we examined the excited-state structural evolution and the ESPT dynamics within the dephasing time of the low-frequency vibration. A clear anharmonic vibrational coupling is found between one high-frequency mode of the chromophore (phenolic CH bend) and a low-frequency mode at ∼104 cm(-1). However, the data show that this low-frequency motion does not substantially affect the ESPT dynamics.

  6. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed Central

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-01-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit. PMID:9776758

  7. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-11-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.

  8. Pleistocene desiccation in East Africa bottlenecked but did not extirpate the adaptive radiation of Lake Victoria haplochromine cichlid fishes.

    PubMed

    Elmer, Kathryn R; Reggio, Chiara; Wirth, Thierry; Verheyen, Erik; Salzburger, Walter; Meyer, Axel

    2009-08-11

    The Great Lakes region of East Africa, including Lake Victoria, is the center of diversity of the mega-diverse cichlid fishes (Perciformes: Teleostei). Paleolimnological evidence indicates dramatic desiccation of this lake ca. 18,000-15,000 years ago. Consequently, the hundreds of extant endemic haplochromine species in the lake must have either evolved since then or refugia must have existed, within that lake basin or elsewhere, from which Lake Victoria was recolonized. We studied the population history of the Lake Victoria region superflock (LVRS) of haplochromine cichlids based on nuclear genetic analysis (12 microsatellite loci from 400 haplochomines) of populations from Lake Kivu, Lake Victoria, and the connected and surrounding rivers and lakes. Population genetic analyses confirmed that Lake Kivu haplochromines colonized Lake Victoria. Coalescent analyses show a 30- to 50-fold decline in the haplochromine populations of Lake Victoria, Lake Kivu, and the region ca. 18,000-15,000 years ago. We suggest that this coincides with drastic climatic and geological changes in the late Pleistocene. The most recent common ancestor of the Lake Victoria region haplochromines was estimated to have existed about 4.5 million years ago, which corresponds to the first radiation of cichlids in Lake Tanganyika and the origin of the tribe Haplochrominii. This relatively old evolutionary origin may explain the high levels of polymorphism still found in modern haplochromines. This degree of polymorphism might have acted as a "genetic reservoir" that permitted the explosive radiation of hundreds of haplochromines and their array of contemporary adaptive morphologies.

  9. Framework for Integrated Water, Energy, and Environmental Resources Assessment, Planning, and Management: Lake Victoria Case Study

    NASA Astrophysics Data System (ADS)

    Georgakakos, A.; Yao, H.; Tidwell, A.

    2006-12-01

    This article describes a recent assessment study for Lake Victoria in East Africa. The study includes three interlinked components. The first pertains to Lake Victoria regulation and includes climate and hydrologic forecasting (seasonal, inter-annual, and centennial), and outflow regulation (water resources planning). The second pertains to energy system planning, and the third to environmental and socio-economic impact assessments. These components converge at the operational level where water, energy, and environmental management are harmonized through the use of a decision support system, the Lake Victoria Decision Support Tool. Some of the broad study findings are summarized below: (1)Lake Victoria is entering a new era in which sustainable water resources management is tightly linked to and can only be achieved by proactive energy planning. To meet this new challenge, and maximize the benefit of the decision support system, water and energy sector decisions must be institutionally coordinated. (2)Climate and hydrologic forecasts of sufficient skill are critical for effective water resources and energy planning and management. More specifically, extensive assessments with several GCMs and climate scenarios indicate that Lake Victoria will most likely be adversely impacted by climate change with potentially serious local and regional consequences. Furthermore, seasonal and inter-annual forecasts are very important in meeting medium term water and energy demands and minimizing the costs of thermal energy generation. (3)Wetland ecological and socio-economic benefits are comparable to power sector benefits, underscoring the need for more comprehensive evaluations of non-power water uses and integrative water, energy, and environmental planning and management. (4)Integrated forecast-decision systems are excellent means to bring to bear and make practically available advances in various water-related disciplines for the benefit of managers and policy makers.

  10. Regional nitrogen budget of the Lake Victoria Basin, East Africa: syntheses, uncertainties and perspectives

    NASA Astrophysics Data System (ADS)

    Zhou, Minghua; Brandt, Patric; Pelster, David; Rufino, Mariana C.; Robinson, Timothy; Butterbach-Bahl, Klaus

    2014-10-01

    Using the net anthropogenic nitrogen input (NANI) approach we estimated the N budget for the Lake Victoria Basin in East Africa. The NANI of the basin ranged from 887 to 3008 kg N km-2 yr-1 (mean: 1827 kg N km-2 yr-1) for the period 1995-2000. The net nitrogen release at basin level is due primarily to livestock and human consumption of feed and foods, contributing between 69% and 85%. Atmospheric oxidized N deposition contributed approximately 14% to the NANI of the Lake Victoria Basin, while either synthetic N fertilizer imports or biological N fixations only contributed less than 6% to the regional NANI. Due to the low N imports of feed and food products (<20 kg N km-2 yr-1), nitrogen release to the watershed must be derived from the mining of soil N stocks. The fraction of riverine N export to Lake Victoria accounted for 16%, which is much lower than for watersheds located in Europe and USA (25%). A significant reduction of the uncertainty of our N budget estimate for Lake Victoria Basin would be possible if better data on livestock systems and riverine N export were available. Our study indicates that at present soil N mining is the main source of nitrogen in the Lake Victoria Basin. Thus, sustainable N management requires increasing agricultural N inputs to guarantee food security and rehabilitation and protection of soils to minimize environmental costs. Moreover, to reduce N pollution of the lake, improving management of human and animal wastes needs to be carefully considered in future.

  11. Transformation of Escherichia coli K-12 with a high-copy plasmid encoding the green fluorescent protein reduces growth: implications for predictive microbiology.

    PubMed

    Oscar, T P; Dulal, K; Boucaud, D

    2006-02-01

    The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (mumax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40 degrees C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40 degrees C, mumax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.

  12. Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer.

    PubMed

    Schmid, J A; Birbach, A; Hofer-Warbinek, R; Pengg, M; Burner, U; Furtmüller, P G; Binder, B R; de Martin, R

    2000-06-02

    We investigated the dynamics of nuclear transcription factor kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IkappaBalpha similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappaB-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature-dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 x 10(6) m(-1), although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant.

  13. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore

    SciTech Connect

    Huang, Hao; Suslov, Nikolai B.; Li, Nan-Sheng; Shelke, Sandip A.; Evans, Molly E.; Koldobskaya, Yelena; Rice, Phoebe A.; Piccirilli, Joseph A.

    2014-08-21

    Spinach is an in vitro–selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

  14. A synthetic GFP-like chromophore undergoes base-catalyzed autoxidation into acylimine red form.

    PubMed

    Ivashkin, Pavel E; Lukyanov, Konstantin A; Lukyanov, Sergey; Yampolsky, Ilia V

    2011-04-15

    Fluorescent proteins are widely used in modern experimental biology, but much controversy exists regarding details of maturation of different types of their chromophores. Here we studied possible mechanisms of DsRed-type red chromophore formation using synthetic biomimetic GFP-like chromophores, bearing an acylamino substituent, corresponding to an amino acid residue at position 65. We have shown these model compounds to readily react with molecular oxygen to produce a highly unstable DsRed-like acylimine, isolated in the form of stable derivatives. Under the same aerobic conditions an unusual red-shifted imide chromophore--a product of 4-electron oxidation of Gly65 residue--is formed. Our data showed that GFP chromophore is prone to autoxidation at position 65 Cα by its chemical nature with basic conditions being the only key factor required.

  15. Application of fluorescence spectroscopy and multispectral imaging for non-invasive estimation of GFP transfection efficiency

    NASA Astrophysics Data System (ADS)

    Tamošiūnas, M.; Jakovels, D.; Lihačovs, A.; Kilikevičius, A.; Baltušnikas, J.; Kadikis, R.; Šatkauskas, S.

    2014-10-01

    Electroporation and ultrasound induced sonoporation has been showed to induce plasmid DNA transfection to the mice tibialis cranialis muscle. It offers new prospects for gene therapy and cancer treatment. However, numerous experimental data are still needed to deliver the plausible explanation of the mechanisms governing DNA electro- or sono-transfection, as well as to provide the updates on transfection protocols for transfection efficiency increase. In this study we aimed to apply non-invasive optical diagnostic methods for the real time evaluation of GFP transfection levels at the reduced costs for experimental apparatus and animal consumption. Our experimental set-up allowed monitoring of GFP levels in live mice tibialis cranialis muscle and provided the parameters for DNA transfection efficiency determination.

  16. Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

    SciTech Connect

    Didier, P.; Weiss, E.; Sibler, A.-P.; Philibert, P.; Martineau, P.; Bigot, J.-Y.; Guidoni, L.

    2008-02-22

    Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the same scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.

  17. IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

    NASA Astrophysics Data System (ADS)

    Awazu, Kunio; Kinpara, Takeshi; Tamiya, Eiichi

    2002-05-01

    A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm 2. FEL irradiation at a wavelength of 5.75 and 6.1 μm, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 μm. The maximum transfer efficiency was about 0.5%.

  18. Formation of Schiff-base for photoreaction mechanism of red shift of GFP spectra.

    PubMed

    Koseki, Jun; Kita, Yukiumi; Tachikawa, Masanori

    2010-04-01

    We have proposed the formation of Schiff-base between R96 and chromophore (CRO) to elucidate the reaction mechanism for the irreversible red shift of green fluorescent protein (GFP) spectra under the absence of oxygen. The difference between absorption energies of reactant and product for our GFP models with CIS(D)/6-31G* level is 0.21eV, which is in reasonable agreement with the corresponding experimental value of 0.25eV. We have suggested the irreversible photoreaction mechanism, where the CRO excited from ground (S(0)) state to first excited singlet (S(1)) state immediately turns to the first excited triplet (T(1)) state, and the nucleophilic addition reaction occurs on the T(1) state.

  19. Evaluation of a GFP Report Gene Construct for Environmental Arsenic Detection

    SciTech Connect

    Roberto, F.F.; Barnes, J.M.; Bruhn, D.F.

    2002-03-28

    Detection of arsenic and other heavy metal contaminants in the environment is critical to ensuring safe drinking water and effective cleanup of historic activities that have led to widespread contamination of soil and groundwater. Biosensors have the potential to significantly reduce the costs associated with site characterization and long term environmental monitoring. By exploiting the highly selective and sensitive natural mechanisms by which bacteria and other living organisms respond to heavy metals, and fusing transcriptionally active components of these mechanisms to reporter genes, such as B-galactosidase, bacterial luciferase (lux), or green fluorescent protein (GFP) from marine jellyfish, it is possible to produce inexpensive, yet effective biosensors. This article describes the response to submicrogram quantities of arsenite and arsenate of a whole cell arsenic biosensor utilizing a GFP reporter gene.

  20. Therapeutic targeting of tumors with imageable GFP-expressing Salmonella typhimurium auxotrophic mutants

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.; Hayashi, Katsuhiro; Zhao, Ming

    2008-02-01

    Tumor targeting Salmonella typhimurium has been developed. These bacteria were mutagenized and a strain auxotrophic for leucine and arguine was selected. This strain was also engineered to express GFP. This train, termed A1, could target prostate tumors in nude mouse models and inhibit their growth. A1 was passaged through a tumor and re-isolated and termed A1-R. A1-R had greater antitumor efficacy and could cure breast, prostate, pancreatic, and lung tumors in nude mouse models.

  1. The hESC line Envy expresses high levels of GFP in all differentiated progeny.

    PubMed

    Costa, Magdaline; Dottori, Mirella; Ng, Elizabeth; Hawes, Susan M; Sourris, Koula; Jamshidi, Pegah; Pera, Martin F; Elefanty, Andrew G; Stanley, Edouard G

    2005-04-01

    Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.

  2. A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag.

    PubMed

    Slack, J M; Dougherty, E M; Lawrence, S D

    2001-09-01

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74-GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74-GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74-GFP chimeras in recombinant baculoviruses. When C-terminal region S580-F645 was deleted from p74, p74-GFP chimera localization became non-specific and chimeras became soluble. p74 region S580-F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.

  3. A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.

    PubMed

    Jailani, A Abdul Kader; Solanki, Vikas; Roy, Anirban; Sivasudha, T; Mandal, Bikash

    2017-03-02

    A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits.

  4. Development and validation of four Leishmania species constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies.

    PubMed

    Patel, Asha Parbhu; Deacon, Andrew; Getti, Giulia

    2014-04-01

    Green fluorescent protein (GFP)-parasite transfectants have been widely used as a tool for studying disease pathogenesis in several protozoan models and their application in drug screening assays has increased rapidly. In the past decade, the expression of GFP has been established in several Leishmania species, mostly for in vitro studies. The current work reports generation of four transgenic parasites constitutively expressing GFP (Leishmania mexicana, Leishmania aethiopica, Leishmania tropica and Leishmania major) and their validation as a representative model of infection. This is the first report where stable expression of GFP has been achieved in L. aethiopica and L. tropica. Integration of GFP was accomplished through homologous recombination of the expression construct, pRib1.2αNEOαGFP downstream of the 18S rRNA promoter in all species. A homogeneous and high level expression of GFP was detected in both the promastigote and the intracellular amastigote stages. All transgenic species showed the same growth pattern, ability to infect mammalian host cells and sensitivity to reference drugs as their wild type counterparts. All four transgenic Leishmania are confirmed as models for in vitro and possibly in vivo infections and represent an ideal tool for medium throughput testing of compound libraries.

  5. Low-Cost Synthesis of Smart Biocompatible Graphene Oxide Reduced Species by Means of GFP.

    PubMed

    Masullo, Tiziana; Armata, Nerina; Pendolino, Flavio; Colombo, Paolo; Lo Celso, Fabrizio; Mazzola, Salvatore; Cuttitta, Angela

    2016-02-01

    The aim of this work is focused on the engineering of biocompatible complex systems composed of an inorganic and bio part. Graphene oxide (GO) and/or graphite oxide (GtO) were taken into account as potential substrates to the linkage of the protein such as Anemonia sulcata recombinant green fluorescent protein (rAsGFP). The complex system is obtained through a reduction process between GO/GtO and rAsGFP archiving an environmentally friendly biosynthesis. Spectroscopic measurements support the formation of reduced species. In particular, photoluminescence shows a change in the activity of the protein when a bond is formed, highlighted by a loss of the maximum emission signal of rAsGFP and a redshift of the maximum absorption peak of the GO/GtO species. Moreover, the hemolysis assay reveals a lower value in the presence of less oxidized graphene species providing evidence for a biocompatible material. This singular aspect can be approached as a promising method for circulating pharmaceutical preparations via intravenous administration in the field of drug delivery.

  6. Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein

    PubMed Central

    Senejani, Alireza G.; Gogarten, J. Peter

    2007-01-01

    Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1), is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn2+ and not Mg2+ metal cations for activity. PMID:17389927

  7. GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

    PubMed

    Santillan, Beatriz A; Moye, Christopher; Mittelman, David; Wilson, John H

    2014-01-01

    Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

  8. GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

    PubMed Central

    Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

    2014-01-01

    Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

  9. Mechanistic Insights into Reversible Photoactivation in Proteins of the GFP Family

    PubMed Central

    Gayda, Susan; Nienhaus, Karin; Nienhaus, G. Ulrich

    2012-01-01

    Light-controlled modification of the fluorescence emission properties of proteins of the GFP family is of crucial importance for many imaging applications including superresolution microscopy. Here, we have studied the reversibly photoswitchable fluorescent protein mIrisGFP using optical spectroscopy. By analyzing the pH dependence of isomerization and protonation equilibria and the isomerization kinetics, we have obtained insight into the coupling of the chromophore to the surrounding protein moiety and a better understanding of the photoswitching mechanism. A different acid-base environment of the chromophore’s protonating group in its two isomeric forms, which can be inferred from the x-ray structures of IrisFP, is key to the photoswitching function and ensures that isomerization and protonation are correlated. Amino acids near the chromophore, especially Glu212, rearrange upon isomerization, and Glu212 protonation modulates the chromophore pKa. In mIrisGFP, the cis chromophore protonates in two steps, with pKcis of 5.3 and 6, which is much lower than pKtrans (>10). Based on these results, we have put forward a mechanistic scheme that explains how the combination of isomeric and acid-base properties of the chromophore in its protein environment can produce negative and positive photoswitching modes. PMID:23260054

  10. Differentiation potential and GFP labeling of sheep bone marrow-derived mesenchymal stem cells.

    PubMed

    Czernik, Marta; Fidanza, Antonella; Sardi, Martina; Galli, Cesare; Brunetti, Dario; Malatesta, Daniela; Della Salda, Leonardo; Matsukawa, Kazutsugu; Ptak, Grazyna E; Loi, Pasqualino

    2013-01-01

    Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow-derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34- and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre-clinical sheep model to test the efficiency and safety of cell replacement therapy.

  11. Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein.

    PubMed

    Senejani, Alireza G; Gogarten, J Peter

    2007-02-16

    Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1), is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn(2+) and not Mg(2+) metal cations for activity.

  12. Clonal and territorial development of the pancreas as revealed by eGFP-labelled mouse chimeras.

    PubMed

    Eberhard, Daniel; Jockusch, Harald

    2010-10-01

    The clonal structure of the pancreas was analysed in neonatal and adult mouse chimeras in which one partner displayed cell patches expressing green fluorescent protein (eGFP). Coherent growth during pancreatic histogenesis was suggested by the presence of large eGFP-labelled acinar clusters rather than a scattered distribution of individual labelled acinar cells. The adult chimeric pancreas contained monophenotypic acini, whereas surprisingly 5% of acini in neonates were polyclonal. Monophenotypic acini presumably arose by coherent expansion leading to large 3D patches and may not be monoclonal. Islets of Langerhans were oligoclonal at both ages investigated. The proportion of eGFP positive cells within islets did not correlate with that of the surrounding acinar tissue indicating clonal independence of islets from their neighbourhood. The patterns observed argue against a secondary contribution of blood-borne progenitor/stem cells to the acinar compartment during tissue turnover. The different clonal origins of acini and islets are integrated into a model of pancreatic histogenesis.

  13. The structure of a GFP-based antibody (fluorobody) to TLH, a toxin from Vibrio parahaemolyticus.

    PubMed

    Chen, Yaoguang; Huang, Xiaocheng; Wang, Rongzhi; Wang, Shihua; Shi, Ning

    2015-07-01

    A fluorobody is a manmade hybrid molecule that is composed of green fluorescent protein (GFP) and a fragment of antibody, which combines the affinity and specificity of an antibody with the visibility of a GFP. It is able to provide a real-time indication of binding while avoiding the use of tags and secondary binding reagents. Here, the expression, purification and crystal structure of a recombinant fluorobody for TLH (thermolabile haemolysin), a toxin from the lethal food-borne disease bacterium Vibrio parahaemolyticus, are presented. This is the first structure of a fluorobody to be reported. Crystals belonging to space group P4(3)2(1)2, with unit-cell parameters a = b = 63.35, c = 125.90 Å, were obtained by vapour diffusion in hanging drops and the structure was refined to an Rfree of 16.7% at 1.5 Å resolution. The structure shows a CDR loop of the antibody on the GFP scaffold.

  14. Selection of IgE-binding aptameric green fluorescent protein (Ap-GFP) by the ribosome display (RD) platform

    SciTech Connect

    Chen, S.-S. Yang Yongmin; Barankiewicz, Teresa J.

    2008-09-26

    GFP-C{kappa} fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 {mu}g of the dual site aptameric GFP library exhibiting a diversity of 7.5 x 10{sup 11} was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand.

  15. 75 FR 71467 - Exelon Nuclear Texas Holdings, LLC, Early Site Permit Application for the Victoria County Station...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-23

    ... From the Federal Register Online via the Government Publishing Office NUCLEAR REGULATORY COMMISSION Exelon Nuclear Texas Holdings, LLC, Early Site Permit Application for the Victoria County Station... Contention Preparation AGENCY: Nuclear Regulatory Commission (NRC or the Commission). ACTION: Notice...

  16. Draft Genome Sequences of 15 Staphylococcus aureus Isolates Recovered from Raw Milk and Associated Milk Filters from Victoria, Australia

    PubMed Central

    McMillan, Kate; Allnutt, Theodore R.

    2017-01-01

    ABSTRACT This study describes draft whole genomes of 15 Staphylococcus aureus isolates from dairy farms located in Victoria, Australia. Two novel sequence types (ST3183 and ST3184) were identified among these isolates. PMID:28082499

  17. 78 FR 60826 - Foreign-Trade Zone 155-Calhoun/Victoria Counties, Texas; Authorization of Production Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-10-02

    ... Production Activity; Caterpillar, Inc. (Excavator and Frame Assembly Production); Victoria, Texas On May 29... proposed production activity to the Foreign-Trade Zones (FTZ) Board on behalf of Caterpillar, Inc.,...

  18. The Cambrian Ross Orogeny in northern Victoria Land (Antarctica) and New Zealand: A synthesis

    USGS Publications Warehouse

    Federico, L.; Capponi, G.; Crispini, L.; Bradshaw, J.D.

    2007-01-01

    In the Cambrian, the paleo-Pacific margin of the Gondwana supercontinent included East Antarctica, Australia, Tasmania and New Zealand and was affected by themajor Ross-Delamerian Orogeny. In Antarctica, evidence suggests that this resulted from oblique subduction and that in northern Victoria Land it was accompanied by the opening and subsequent closure of a back-arc basin. Comparison of the type and timing of sedimentary, magmatic and metamorphic events in areas noted above shows strong similarities between northern Victoria Land and New Zealand. In both regions Middle Cambrian volcanites are interpreted as arc/back-arc assemblages produced by west-directed subduction; sediments interbedded with the volcanites show provenance both from the arc and from the Gondwana margin and therefore place the basin close to the continent. Back-arc closure in the Late Cambrian was likely accomplished through a second subduction system

  19. Strain and displacement measurements for the June 9, 1980 Victoria, Mexico Earthquake

    NASA Astrophysics Data System (ADS)

    Darby, D.; Nyland, E.; Suarez, F.; Chavez, D.; Gonzalez, J.

    A microgeodetic network 22 km south east of Est. Guadalupe Victoria, Baja California Norte, installed in late May 1980, has been resurveyed in an experiment that started 12 hours after the June 9, 1980 Victoria earthquake, which had an epicenter at 10 km depth about 12 km from the network. The resurvey was complete by June 13. Both the initial observations and the resurvey were done with HP3800 distance meter equipment. Some angular control was provided with a Wild T3 theodolite. The network underwent a compressive strain of 7 ± 3 micro strain essentially parallel the Cerro Prieto fault about the time of the earthquake. Strains of this size are associated with simple dislocation models of earthquakes of this magnitude. Its direction appears to be anomalous however. This may indicate compression related to soil liquefaction processes or strain near the end of the slip plane.

  20. Environmental impacts of cage culture in Lake Victoria: the case of Shirati Bay-Sota, Tanzania.

    PubMed

    Kashindye, Benedicto Boniphace; Nsinda, P; Kayanda, R; Ngupula, G W; Mashafi, C A; Ezekiel, C N

    2015-01-01

    The experimental cage culture was conducted at Shirati bay, Lake Victoria from February to August 2013, to investigate the impacts of the small scale cage culture on the environment. Three locations along the cages, at the intermediate and one in the offshore (control) were sampled for water quality parameters, phytoplankton and macro invertebrates. A notable increase in nutrient concentration was observed after the set of cages among the stations. However DO, pH, and water transparency showed no major changes and was within the recommended ranges. Cyanophytes an indicator of inorganic pollution dominated before and after the set of cages, an increase in phytoplankton numerical abundance was observed after stocking of fish in cages. In addition there was an increase in the invertebrate community especially bivalves and gastropods. In conclusion we found no consistent environmental change caused by cage culture, and therefore it can be allowed in Lake Victoria, Tanzania part, with close monitoring of its impacts.

  1. A fresh insight into transmission of schistosomiasis: a misleading tale of Biomphalaria in Lake Victoria.

    PubMed

    Standley, Claire J; Wade, Christopher M; Stothard, J Russell

    2011-01-01

    Lake Victoria is a known hot-spot for Schistosoma mansoni, which utilises freshwater snails of the genus Biomphalaria as intermediate hosts. Different species of Biomphalaria are associated with varying parasite compatibility, affecting local transmission. It is thought that two species, B. choanomphala and B. sudanica, inhabit Lake Victoria; despite their biomedical importance, the taxonomy of these species has not been thoroughly examined. This study combined analysis of morphological and molecular variables; the results demonstrated that molecular groupings were not consistent with morphological divisions. Habitat significantly predicted morphotype, suggesting that the different Lake Victorian forms of Biomphalaria are ecophentoypes of one species. The nomenclature should be revised accordingly; the names B. choanomphala choanomphala and B. c. sudanica are proposed. From a public health perspective, these findings can be utilised by policy-makers for better understanding of exposure risk, resulting in more effective and efficient control initiatives.

  2. [Occurrence and seasonal changes of antibiotics in the Victoria Harbour and the Pearl River, South China].

    PubMed

    Xu, Wei-hai; Zhang, Gan; Zou, Shi-chun; Li, Xiang-dong; Liu, Yu-chun

    2006-12-01

    The occurrence and seasonal changes of antibiotics in the Victoria Harbour and the Pearl River were studied using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring (MRM). The results showed that the concentrations of antibiotics were mainly below the limit of quantification (LOQ) in the ambient marine water of Victoria Harbour. However, except for amoxicillin, all the target antibiotics were detected in the Pearl River during high (summer) and low (early spring) water seasons with the concentrations ranging from 13 to 69 ng x L(-1) and from 70 to 489 ng x L(-1), respectively. The concentrations of antibiotics in the Pearl River are all higher than that in America and other western developed countries, no matter in spring or summer. The concentrations of antibiotics showed sensitive daily changes in the high water season.

  3. VLUIS, a land use data product for Victoria, Australia, covering 2006 to 2013.

    PubMed

    Morse-McNabb, Elizabeth; Sheffield, Kathryn; Clark, Rob; Lewis, Hayden; Robson, Susan; Cherry, Don; Williams, Steve

    2015-11-24

    Land Use Information is a key dataset required to enable an understanding of the changing nature of our landscapes and the associated influences on natural resources and regional communities. The Victorian Land Use Information System (VLUIS) data product has been created within the State Government of Victoria to support land use assessments. The project began in 2007 using stakeholder engagement to establish product requirements such as format, classification, frequency and spatial resolution. Its genesis is significantly different to traditional methods, incorporating data from a range of jurisdictions to develop land use information designed for regular on-going creation and consistency. Covering the entire landmass of Victoria, the dataset separately describes land tenure, land use and land cover. These variables are co-registered to a common spatial base (cadastral parcels) across the state for the period 2006 to 2013; biennially for land tenure and land use, and annually for land cover. Data is produced as a spatial GIS feature class.

  4. VLUIS, a land use data product for Victoria, Australia, covering 2006 to 2013

    PubMed Central

    Morse-McNabb, Elizabeth; Sheffield, Kathryn; Clark, Rob; Lewis, Hayden; Robson, Susan; Cherry, Don; Williams, Steve

    2015-01-01

    Land Use Information is a key dataset required to enable an understanding of the changing nature of our landscapes and the associated influences on natural resources and regional communities. The Victorian Land Use Information System (VLUIS) data product has been created within the State Government of Victoria to support land use assessments. The project began in 2007 using stakeholder engagement to establish product requirements such as format, classification, frequency and spatial resolution. Its genesis is significantly different to traditional methods, incorporating data from a range of jurisdictions to develop land use information designed for regular on-going creation and consistency. Covering the entire landmass of Victoria, the dataset separately describes land tenure, land use and land cover. These variables are co-registered to a common spatial base (cadastral parcels) across the state for the period 2006 to 2013; biennially for land tenure and land use, and annually for land cover. Data is produced as a spatial GIS feature class. PMID:26602150

  5. A Fresh Insight into Transmission of Schistosomiasis: A Misleading Tale of Biomphalaria in Lake Victoria

    PubMed Central

    Standley, Claire J.; Wade, Christopher M.; Stothard, J. Russell

    2011-01-01

    Lake Victoria is a known hot-spot for Schistosoma mansoni, which utilises freshwater snails of the genus Biomphalaria as intermediate hosts. Different species of Biomphalaria are associated with varying parasite compatibility, affecting local transmission. It is thought that two species, B. choanomphala and B. sudanica, inhabit Lake Victoria; despite their biomedical importance, the taxonomy of these species has not been thoroughly examined. This study combined analysis of morphological and molecular variables; the results demonstrated that molecular groupings were not consistent with morphological divisions. Habitat significantly predicted morphotype, suggesting that the different Lake Victorian forms of Biomphalaria are ecophentoypes of one species. The nomenclature should be revised accordingly; the names B. choanomphala choanomphala and B. c. sudanica are proposed. From a public health perspective, these findings can be utilised by policy-makers for better understanding of exposure risk, resulting in more effective and efficient control initiatives. PMID:22046308

  6. New fossil teeth of Theropithecus oswaldi (Cercopithecoidea) from the Early Pleistocene at Cueva Victoria (SE Spain).

    PubMed

    Ferràndez-Cañadell, Carles; Ribot, Francesc; Gibert, Lluís

    2014-09-01

    The presence of Theropithecus oswaldi in Europe was first reported in 1995 from the Early Pleistocene site of Cueva Victoria (SE Spain), showing the dispersal of this genus above 30° north latitude and into Europe. Later claims of the presence in Italy of Theropithecus in the Early Pleistocene, based on vertebral remains, are controversial. Here we report four additional teeth of T. oswaldi from Cueva Victoria. These and the previously described tooth correspond to a minimum of two individuals. The presence of T. oswaldi in North Africa and SE Iberia during the Early Pleistocene suggests a possible faunal dispersal from Africa into Europe through the Straits of Gibraltar, which would have acted as a filter bridge.

  7. The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants.

    PubMed

    Lacorte, Cristiano; Ribeiro, Simone G; Lohuis, Dick; Goldbach, Rob; Prins, Marcel

    2007-09-01

    Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner. To evaluate this potential, the N protein was fused to the Aequorea victoria green fluorescent protein (GFP), either at the amino or carboxy terminus, and expressed in plants from binary vectors in Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens and evaluated after 4 days, revealing an intense GFP fluorescence under UV light. Microscopic analysis revealed that upon expression of the GFP:N fusion a small number of large aggregates were formed, whereas N:GFP expression led to a large number of smaller aggregates scattered throughout the cytoplasm. A simple purification method was tested, based on centrifugation and filtration, yielding a gross extract that contained large amounts of N:GFP aggregates, as confirmed by GFP fluorescence and Western blot analysis. These results show that the homopolymerization properties of the N protein can be used as a fast and simple way to purify large amounts of proteins from plants.

  8. [The green fluorescent protein that glows in bioscience].

    PubMed

    Millán, María Inés; Becú-Villalobos, Damasia

    2009-01-01

    Green fluorescent protein (GFP) is a protein produced by the jellyfish Aequorea victoria, that emits bioluminescence in the green zone of the visible spectrum. The GFP gene has been cloned and is used in molecular biology as a marker. The three researchers that participated independently in elucidating the structure and function of this and its related proteins, Drs. Shimomura, Chalfie and Tsien were awarded the Nobel Prize in Chemistry 2008. Dr. Shimomura discovered and studied the properties of GFP. Using molecular biological techniques, Chalfie succeeded in introducing the GFP gene into the DNA of the small, almost transparent worm C. elegans, and initiated an era in which GFP would be used as a glowing marker for cellular biology. Finally, Dr.Tsien found precisely how GFP's structure produces the observed green fluorescence, and succeeded in modifying the structure to generate molecules that emit light at slightly different wavelengths, which gave tags of different colors. Fluorescent proteins are very versatile and are being used in many areas, such as microbiology, biotechnology, physiology, environmental engineering, development, etc. They can, for example, illuminate growing cancer tumours; show the development of Alzheimer's disease, or detect arsenic traces in water. Finding the key to how a marine organism produces light unexpectedly ended up providing researchers with a powerful array of tools with which to visualize cell biology in action.

  9. Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors

    PubMed Central

    Akimoto, Masayuki; Cheng, Hong; Zhu, Dongxiao; Brzezinski, Joseph A.; Khanna, Ritu; Filippova, Elena; Oh, Edwin C. T.; Jing, Yuezhou; Linares, Jose-Luis; Brooks, Matthew; Zareparsi, Sepideh; Mears, Alan J.; Hero, Alfred; Glaser, Tom; Swaroop, Anand

    2006-01-01

    The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl−/− retinas, the GFP+ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP+ photoreceptors from wild-type and Nrl−/− retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies. PMID:16505381

  10. The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages

    PubMed Central

    Guglielmi, Luca; Patzel, Eva; Sel, Saadettin; Auffarth, Gerd U.; Carl, Matthias; Poggi, Lucia

    2017-01-01

    Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP) transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC) containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers. PMID:28107513

  11. Imaging the static dielectric constant in vitro and in living cells by a bioconjugable GFP chromophore analog.

    PubMed

    Signore, Giovanni; Abbandonato, Gerardo; Storti, Barbara; Stöckl, Martin; Subramaniam, Vinod; Bizzarri, Ranieri

    2013-02-28

    A fluorescent probe structurally similar to the GFP chromophore is demonstrated to report the local static dielectric constant. This probe can be chemically functionalized for selective targeting at the intracellular level.

  12. B chromosomes have a functional effect on female sex determination in Lake Victoria cichlid fishes.

    PubMed

    Yoshida, Kohta; Terai, Yohey; Mizoiri, Shinji; Aibara, Mitsuto; Nishihara, Hidenori; Watanabe, Masakatsu; Kuroiwa, Asato; Hirai, Hirohisa; Hirai, Yuriko; Matsuda, Yoichi; Okada, Norihiro

    2011-08-01

    The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

  13. Late Pleistocene Desiccation of Lake Victoria and Rapid Evolution of Cichlid Fishes

    NASA Astrophysics Data System (ADS)

    Johnson, Thomas C.; Scholz, Christopher A.; Talbot, Michael R.; Kelts, Kerry; Ricketts, R. D.; Ngobi, Gideon; Beuning, Kristina; Ssemmanda, Immacculate; McGill, J. W.

    1996-08-01

    Lake Victoria is the largest lake in Africa and harbors more than 300 endemic species of haplochromine cichlid fish. Seismic reflection profiles and piston cores show that the lake not only was at a low stand but dried up completely during the Late Pleistocene, before 12,400 carbon-14 years before the present. These results imply that the rate of speciation of cichlid fish in this tropical lake has been extremely rapid.

  14. Satellite-Based Assessment of the spatial extent of Aquatic Vegetation in Lake Victoria

    NASA Astrophysics Data System (ADS)

    Clark, W.; Aligeti, N.; Jeyaprakash, T.; Martins, M.; Stodghill, J.; Winstanley, H.

    2011-12-01

    Lake Victoria in Africa is the second largest freshwater lake in the world and is known for its abundance of aquatic wildlife. In particular over 200 different fish species are caught and sold by local fisherman. The lake is a major contributor to the local economy as a corridor of transportation, source of drinking water, and source of hydropower. However, the invasion of aquatic vegetation such as water hyacinth in the lake has disrupted each of these markets. Aquatic vegetation now covers a substantial area of the coastline blocking waterways, disrupting hydropower, hindering the collection of drinking water and decreasing the profitability of fishing. The vegetation serves as a habitat for disease carrying mosquitoes as well as snakes and snails that spread the parasitic disease bilharzia. The current control measures of invasive aquatic vegetation rely on biological, chemical and mechanical control. The objective of this study was to utilize remote sensing to map aquatic vegetation within Lake Victoria from 2000 to 2011. MODIS, Landsat 4-5TM, and Landsat 7-ETM imagery was employed to perform change detections in vegetation and identify the extent of aquatic vegetation throughout the years. The efficiency of containment efforts were evaluated and ideal time for application of such efforts were suggested. A methodology for aquatic vegetation surveillance was created. The results of this project were presented as a workshop to the Lake Victoria Fisheries Organization, SERVIR, and other partner organizations. The workshop provided instruction into the use of NASA and other satellite derived products. Time series animations of the spatial extent of aquatic vegetation within the lake were created. By identifying seasons of decreased aquatic vegetation, ideal times to employ control efforts were identified. SERVIR will subsequently utilize the methodologies and mapping results of this study to develop operational aquatic vegetation surveillance for Lake Victoria.

  15. Leuconidae (Crustacea: Cumacea) from the collections of the Museum Victoria, Australia.

    PubMed

    Gerken, Sarah

    2016-01-21

    The collections of the Museum Victoria have yielded six new leuconid species in five genera from Australian waters: Austroleucon adiazetos n. sp., A. dolosolevis n. sp., Eudorellopsis mykteros n. sp., Kontiloleucon australiensis n. gen., n. sp., Leucon (Alytoleucon) dolichorhinos n. sp., Ommatoleucon megalopos n. sp. as well as the new genus Kontiloleucon. Leucon (Leucon) echinolophotos n. sp. is a new species from off Enderby Land, Antarctica. Keys to the Australian leuconid genera and species are included.

  16. Urban eutrophication and its spurring conditions in the Murchison Bay of Lake Victoria.

    PubMed

    Kabenge, Martin; Wang, Hongtao; Li, Fengting

    2016-01-01

    The efficiency of Lake Victoria in providing its ecosystem services to riparian states, both immediate and along the Nile river basin, is strongly related to its water quality. Over the past few decades, eutrophication has increased in the lake arising from increased inflow of nutrients. This study was carried out in the Murchison Bay area of Lake Victoria with the aims of assessing the progress of eutrophication nutrient enrichment into the lake between 1990 and 2014. Using Landsat satellite floating algae index (FAI) products and data from laboratory analysis of water samples, the study revealed that floating algae reoccurred periodically with coverage varying between 1 and 18 km(2). The findings also indicated that the range of nitrate-nitrogen concentrations increased greatly with maximum concentrations recorded at 31.2 mg l(-1) in 2007 from 0.084 mg l(-1) in 1990. The soluble reactive phosphorus concentration range showed a maximum of 1.45 mg l(-1) in 2007 from 0.043 mg l(-1) in 1990. The chlorophyll levels increased from an average of 17 μg l(-1) in 1992 by threefold in 1996 but had however declined and halved in intensity by 2011. The eutrophication that has occurred in Lake Victoria over the past decades has been due to pollution from industrial, residential, and agricultural areas within the catchment.

  17. Parameterization of the inherent optical properties of Murchison Bay, Lake Victoria.

    PubMed

    Okullo, Willy; Ssenyonga, Taddeo; Hamre, Børge; Frette, Oyvind; Sørensen, K; Stamnes, Jakob J; Steigen, Andreas; Stamnes, Knut

    2007-12-20

    Lake Victoria, Africa's largest freshwater lake, suffers greatly from negative changes in biomass of species of fish and also from severe eutrophication. The continuing deterioration of Lake Victoria's ecological functions has great long-term consequences for the ecosystem benefits it provides to the countries bordering its shores. However, knowledge about temporal and spatial variations of optical properties and how they relate to lake constituents is important for a number of reasons such as remote sensing, modeling of underwater light fields, and long-term monitoring of lake waters. Based on statistical analysis of data from optical measurements taken during half a year of weekly cruises in Murchison Bay, Lake Victoria, we present a three-component model for the absorption and a two-component model for the scattering of light in the UV and the visible regions of the solar spectrum along with tests of their ranges of validity. The three-component input to the model for absorption is the chlorophyll-a (Chl-a), total suspended materials concentrations, and yellow substance absorption, while the two-component input to the model for scattering is the Chl-a concentration and total suspended materials.

  18. Parameterization of the inherent optical properties of Murchison Bay, Lake Victoria

    NASA Astrophysics Data System (ADS)

    Okullo, Willy; Ssenyonga, Taddeo; Hamre, Børge; Frette, Øyvind; Sørensen, K.; Stamnes, Jakob J.; Steigen, Andreas; Stamnes, Knut

    2007-12-01

    Lake Victoria, Africa's largest freshwater lake, suffers greatly from negative changes in biomass of species of fish and also from severe eutrophication. The continuing deterioration of Lake Victoria's ecological functions has great long-term consequences for the ecosystem benefits it provides to the countries bordering its shores. However, knowledge about temporal and spatial variations of optical properties and how they relate to lake constituents is important for a number of reasons such as remote sensing, modeling of underwater light fields, and long-term monitoring of lake waters. Based on statistical analysis of data from optical measurements taken during half a year of weekly cruises in Murchison Bay, Lake Victoria, we present a three-component model for the absorption and a two-component model for the scattering of light in the UV and the visible regions of the solar spectrum along with tests of their ranges of validity. The three-component input to the model for absorption is the chlorophyll-a (Chl-a), total suspended materials concentrations, and yellow substance absorption, while the two-component input to the model for scattering is the Chl-a concentration and total suspended materials.

  19. Mapping dominant annual land cover from 2009 to 2013 across Victoria, Australia using satellite imagery

    PubMed Central

    Sheffield, Kathryn; Morse-McNabb, Elizabeth; Clark, Rob; Robson, Susan; Lewis, Hayden

    2015-01-01

    There is a demand for regularly updated, broad-scale, accurate land cover information in Victoria from multiple stakeholders. This paper documents the methods used to generate an annual dominant land cover (DLC) map for Victoria, Australia from 2009 to 2013. Vegetation phenology parameters derived from an annual time series of the Moderate Resolution Imaging Spectroradiometer Vegetation Indices 16-day 250 m (MOD13Q1) product were used to generate annual DLC maps, using a three-tiered hierarchical classification scheme. Classification accuracy at the broadest (primary) class level was over 91% for all years, while it ranged from 72 to 81% at the secondary class level. The most detailed class level (tertiary) had accuracy levels ranging from 61 to 68%. The approach used was able to accommodate variable climatic conditions, which had substantial impacts on vegetation growth patterns and agricultural production across the state between both regions and years. The production of an annual dataset with complete spatial coverage for Victoria provides a reliable base data set with an accuracy that is fit-for-purpose for many applications. PMID:26602009

  20. Genetic structure of pelagic and littoral cichlid fishes from Lake Victoria.

    PubMed

    Takeda, Miyuki; Kusumi, Junko; Mizoiri, Shinji; Aibara, Mitsuto; Mzighani, Semvua Isa; Sato, Tetsu; Terai, Yohey; Okada, Norihiro; Tachida, Hidenori

    2013-01-01

    The approximately 700 species of cichlids found in Lake Victoria in East Africa are thought to have evolved over a short period of time, and they represent one of the largest known examples of adaptive radiation. To understand the processes that are driving this spectacular radiation, we must determine the present genetic structure of these species and elucidate how this structure relates to the ecological conditions that caused their adaptation. We analyzed the genetic structure of two pelagic and seven littoral species sampled from the southeast area of Lake Victoria using sequences from the mtDNA control region and 12 microsatellite loci as markers. Using a Bayesian model-based clustering method to analyze the microsatellite data, we separated these nine species into four groups: one group composed of pelagic species and another three groups composed mainly of rocky-shore species. Furthermore, we found significant levels of genetic variation between species within each group at both marker loci using analysis of molecular variance (AMOVA), although the nine species often shared mtDNA haplotypes. We also found significant levels of genetic variation between populations within species. These results suggest that initial groupings, some of which appear to have been related to habitat differences, as well as divergence between species within groups took place among the cichlid species of Lake Victoria.

  1. A reinterpretation of geomorphological evidence for Glacial Lake Victoria, McMurdo Dry Valleys, Antarctica

    NASA Astrophysics Data System (ADS)

    McGowan, Hamish A.; Neil, David T.; Speirs, Johanna C.

    2014-03-01

    The largely snow and ice free McMurdo Dry Valleys of Antarctica are one of the coldest and driest locations on Earth. It has been proposed that during the Last Glacial Maximum (LGM) to the early Holocene large lakes up to 200 m deep and 100 km2 in area occupied these valleys. We present the first topographic survey of features reported to be shorelines from one such lake, Glacial Lake Victoria, in Victoria Valley. In combination with the analysis of laser altimetry data obtained from the NASA Airborne Topographic Mapper system and cosmogenic dating of granite boulders we show that the features previously thought to be shorelines are not horizontally or linearly continuous. Rather, we conclude that they are scars from ancient slope mass movement deposits. 10Be cosmogenic dating indicates that their formation is on timescales of at least 160 ka before present and not 20 ka as the LGM mega-lake hypothesis suggests. We conclude that the geomorphic features believed to be shorelines and which underpin the LGM mega-lake hypothesis in Victoria Valley are mass movement deposits and not lake shorelines. Our results support an emerging body of literature unable to find evidence to verify the McMurdo Dry Valleys LGM mega-lake hypothesis. Accordingly we suggest caution in invoking such significant landscape features in discussions of the environmental past of this unique region until such time as further research provides an unequivocal history of the region's geomorphic past.

  2. Mapping dominant annual land cover from 2009 to 2013 across Victoria, Australia using satellite imagery.

    PubMed

    Sheffield, Kathryn; Morse-McNabb, Elizabeth; Clark, Rob; Robson, Susan; Lewis, Hayden

    2015-11-24

    There is a demand for regularly updated, broad-scale, accurate land cover information in Victoria from multiple stakeholders. This paper documents the methods used to generate an annual dominant land cover (DLC) map for Victoria, Australia from 2009 to 2013. Vegetation phenology parameters derived from an annual time series of the Moderate Resolution Imaging Spectroradiometer Vegetation Indices 16-day 250 m (MOD13Q1) product were used to generate annual DLC maps, using a three-tiered hierarchical classification scheme. Classification accuracy at the broadest (primary) class level was over 91% for all years, while it ranged from 72 to 81% at the secondary class level. The most detailed class level (tertiary) had accuracy levels ranging from 61 to 68%. The approach used was able to accommodate variable climatic conditions, which had substantial impacts on vegetation growth patterns and agricultural production across the state between both regions and years. The production of an annual dataset with complete spatial coverage for Victoria provides a reliable base data set with an accuracy that is fit-for-purpose for many applications.

  3. Pervasive, tholeiitic refertilisation and heterogeneous metasomatism in Northern Victoria Land lithospheric mantle (Antarctica)

    NASA Astrophysics Data System (ADS)

    Pelorosso, Beatrice; Bonadiman, Costanza; Coltorti, Massimo; Faccini, Barbara; Melchiorre, Massimiliano; Ntaflos, Theodoros; Gregoire, Michel

    2016-04-01

    The petrology of peridotite xenoliths in the Cenozoic volcanics from Greene Point (Northern Victoria Land, Antarctica) provides new constraints on the characterisation of the lithospheric mantle beneath the West Antarctic Rift. Based on mineral major and trace element models, this mantle domain is proposed to represent a residuum after 10% and 20% partial melting. Moreover, melting models and isotopic results for Sr and Nd systematics highlight the substantial contribution of tholeiitic melts percolating through peridotites. Close correlation with trace element contents in clinopyroxene phenocrysts from Ferrar and Karoo tholeiites allows us to ascribe this refertilisation event to the Jurassic. This asthenospheric melt was also able to transfer a garnet signature to the Northern Victoria Land mantle segment. The rare presence of glass and secondary phases indicate that Greene Point xenoliths were heterogeneously affected by alkaline metasomatism, probably related to the West Antarctic Rift System opening; this has also been widely observed in other Northern Victoria Land localities (i.e., Baker Rocks). Temperature and fO2 were calculated (950 °C; Δlog fO2 (QFM), - 1.70 to - 0.39) at a fixed pressure of 15 kbar, confirming the tendency of the anhydrous Greene Point xenolith population to have higher equilibration temperatures and comparable redox conditions, compared to the nearby amphibole-bearing peridotites from Baker Rocks.

  4. Development of GFP fusions for examination of the effects of the space environment on gene expression in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Mancinelli, R.; Fahlen, T.

    The goal of the In situ Space Gene Expression on Nano-satillites (ISGEN) program is to be ready to fly technology that can support a fully automated experiment to quantify changes in model organisms in situ in low earth orbit in a free flyer platform in less than two years. A straightforward gene expression assay that meets the ISGEN flight objective for testing flight hardware as well as return data regarding the effects of microgravity on gene expression has been developed. Escherichia coli K-12, a bacterium that exhibits changes in its growth pattern when flown in micro-gravity on the Space Shuttle, was used. The scientific objective of this work is to determine if there is a discernable change in metabolic and stress pathway gene expression due to growth in the space environment. To that end, we have linked the green fluorescent protein (GFP) reporter gfp to phoP, a gene that responds to extracellular Mg2+ levels, and pykF, a gene involved in the glycolytic pathway that responds to changes in intracellular pyruvate. These genes respond to the metabolic needs of the cell and may be altered in the micro-gravity environment. E. coli cells containing a plasmid encoding the phoP-gfp-mut3 reporter construct were grown with or without MgSO_4. The effect of the added MgSO_4 is the repression of the expression of GFP. This is the expected result if GFP expression were under the control of a magnesium-regulated promoter such as phoP. Consistent with the negative feedback loop, we observe repression of GFP production in cells containing our pykF-gfp plasmid construct, when grown in the presence of excess glucose. Thus, the pykF-gfp fusion functions as a glucose sensor.

  5. No adverse effects of transgenic maize on population dynamics of endophytic Bacillus subtilis strain B916-gfp.

    PubMed

    Sun, Chongsi; Geng, Lili; Wang, Meiling; Shao, Gaoxiang; Liu, Yongfeng; Shu, Changlong; Zhang, Jie

    2017-02-01

    Endophytic bacterial communities play a key role in promoting plant growth and combating plant diseases. However, little is known about their population dynamics in plant tissues and bulk soil, especially in transgenic crops. This study investigated the colonization of transgenic maize harboring the Bacillus thuringiensis (Bt) cry1Ah gene by Bacillus subtilis strain B916-gfp present in plant tissues and soil. Bt and nontransgenic maize were inoculated with B916-gfp by seed soaking, or root irrigation under both laboratory greenhouse and field conditions. During the growing season, B916-gfp colonized transgenic as well as nontransgenic plants by both inoculation methods. No differences were observed in B916-gfp population size between transgenic and nontransgenic plants, except at one or two time points in the roots and stems that did not persist over the examination period. Furthermore, planting transgenic maize did not affect the number of B916-gfp in bulk soil in either laboratory or field trials. These results indicate that transgenic modification of maize with the cry1Ah gene has no influence on colonization by the endophytic bacteria B916-gfp present in the plant and in bulk soil.

  6. Isolation, plant colonization potential, and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp. Ph6-gfp.

    PubMed

    Sun, Kai; Liu, Juan; Gao, Yanzheng; Jin, Li; Gu, Yujun; Wang, Wanqing

    2014-06-26

    This investigation provides a novel method of endophyte-aided removal of polycyclic aromatic hydrocarbons (PAHs) from plant bodies. A phenanthrene-degrading endophytic bacterium Pseudomonas sp. Ph6 was isolated from clover (Trifolium pratense L.) grown in a PAH-contaminated site. After being marked with the GFP gene, the colonization and distribution of strain Ph6-gfp was directly visualized in plant roots, stems, and leaves for the first time. After ryegrass (Lolium multiflorum Lam.) roots inoculation, strain Ph6-gfp actively and internally colonized plant roots and transferred vertically to the shoots. Ph6-gfp had a natural capacity to cope with phenanthrene in vitro and in planta. Ph6-gfp degraded 81.1% of phenanthrene (50 mg · L(-1)) in a culture solution within 15 days. The inoculation of plants with Ph6-gfp reduced the risks associated with plant phenanthrene contamination based on observations of decreased concentration, accumulation, and translocation factors of phenanthrene in ryegrass. Our results will have important ramifications in the assessment of the environmental risks of PAHs and in finding ways to circumvent plant PAH contamination.

  7. Isolation, plant colonization potential, and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp. Ph6-gfp

    NASA Astrophysics Data System (ADS)

    Sun, Kai; Liu, Juan; Gao, Yanzheng; Jin, Li; Gu, Yujun; Wang, Wanqing

    2014-06-01

    This investigation provides a novel method of endophyte-aided removal of polycyclic aromatic hydrocarbons (PAHs) from plant bodies. A phenanthrene-degrading endophytic bacterium Pseudomonas sp. Ph6 was isolated from clover (Trifolium pratense L.) grown in a PAH-contaminated site. After being marked with the GFP gene, the colonization and distribution of strain Ph6-gfp was directly visualized in plant roots, stems, and leaves for the first time. After ryegrass (Lolium multiflorum Lam.) roots inoculation, strain Ph6-gfp actively and internally colonized plant roots and transferred vertically to the shoots. Ph6-gfp had a natural capacity to cope with phenanthrene in vitro and in planta. Ph6-gfp degraded 81.1% of phenanthrene (50 mg.L-1) in a culture solution within 15 days. The inoculation of plants with Ph6-gfp reduced the risks associated with plant phenanthrene contamination based on observations of decreased concentration, accumulation, and translocation factors of phenanthrene in ryegrass. Our results will have important ramifications in the assessment of the environmental risks of PAHs and in finding ways to circumvent plant PAH contamination.

  8. Matrix metalloproteinase 2 fused to GFP, expressed in E. coli, successfully tracked MMP-2 distribution in vivo.

    PubMed

    Azevedo, A; Prado, A F; Issa, J P M; Gerlach, R F

    2016-08-01

    Matrix Metalloproteinases (MMPs) participate in many physiological and pathological processes. One major limitation to a better understanding of the role MMPs play in these processes is the lack of well-characterized chimeric proteins and characterization of their fluorescence. The specialized literature has reported on few constructs bearing MMPs fused to the sequence of the green fluorescent protein (GFP), but none of the described constructs have been intended for expression in bacteria or for purification and use in vivo. This work has tested a recombinant reporter protein containing the MMP-2 catalytic domain fused to GFP in terms of purification efficiency, degradation of substrates in solution and in zymograms, kinetic activity, GFP fluorescence, and GFP fluorescence in whole animals after injection of the purified and lyophilized fluorescent protein. This work has also characterized rhMMP-2 (recombinant human MMP-2) and inactive clones and used them as negative controls in experiments employing catMMP-2/GFP and rhMMP-2. To our knowledge, this is the first study that has fully characterized a chimeric protein with the MMP-2 catalytic domain fused to GFP, that has efficiently purified such protein from bacteria in a single-step, and that has obtained an adequate chimeric protein for injection in animals and tracking of MMP-2 fate and activity in vivo.

  9. A complex adenovirus vector that delivers FASL-GFP with combined prostate-specific and tetracycline-regulated expression.

    PubMed

    Rubinchik, S; Wang, D; Yu, H; Fan, F; Luo, M; Norris, J S; Dong, J Y

    2001-11-01

    Cell-type-restricted transgene expression delivered by adenovirus vectors is highly desirable for gene therapy of cancer, as it can limit cytotoxic gene expression to tumor cells. However, many tumor- and tissue-specific promoters are weaker than the constitutively active promoters and are thus less effective. To combine cell-type specificity with high-level regulated transgene expression, we have developed a complex adenoviral vector. We have placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a mouse Tnfsf6 (encoding FASL)-GFP fusion gene under the control of the tetracycline responsive promoter. We have incorporated both expression cassettes into a single construct. We show that FASL-GFP expression from this vector is essentially restricted to prostate cancer cells, in which it can be regulated by doxycycline. Higher levels of prostate-specific FASL-GFP expression were generated by this approach than by driving the FASL-GFP expression directly with ARR2PB. More FASL-GFP expression correlated with greater induction of apoptosis in prostate cancer LNCaP cells. Mouse studies confirmed that systemic delivery of both the prostate-specific and the prostate-specific/tet-regulated vectors was well tolerated at doses that were lethal for FASL-GFP vector with CMV promoter. This strategy should be able to improve the safety and efficacy of cancer gene therapy using other cytotoxic genes as well.

  10. GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

    PubMed Central

    Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

    2008-01-01

    Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195

  11. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

    PubMed

    Crispo, M; Vilariño, M; dos Santos-Neto, P C; Núñez-Olivera, R; Cuadro, F; Barrera, N; Mulet, A P; Nguyen, T H; Anegón, I; Menchaca, A

    2015-02-01

    Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

  12. Characterization and Dynamics of Aggresome Formation by a Cytosolic Gfp-Chimera✪

    PubMed Central

    García-Mata, Rafael; Bebök, Zsuzsa; Sorscher, Eric J.; Sztul, Elizabeth S.

    1999-01-01

    Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883–1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481–490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end–directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins. PMID:10491388

  13. Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene

    PubMed Central

    Stefaniuk, Marzena; Gualda, Emilio J.; Pawlowska, Monika; Legutko, Diana; Matryba, Paweł; Koza, Paulina; Konopka, Witold; Owczarek, Dorota; Wawrzyniak, Marcin; Loza-Alvarez, Pablo; Kaczmarek, Leszek

    2016-01-01

    Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy. PMID:27312902

  14. Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization

    PubMed Central

    Ozawa, Akihiko; Brunori, Gloria; Mercatelli, Daniela; Wu, Jinhua; Cippitelli, Andrea; Zou, Bende; Xie, Xinmin (Simon); Williams, Melissa; Zaveri, Nurulain T.; Low, Sarah; Scherrer, Grégory; Kieffer, Brigitte L.

    2015-01-01

    The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [35S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native

  15. Analysis of cargo transport by IFT and GFP imaging of IFT in Chlamydomonas.

    PubMed

    Diener, Dennis

    2009-01-01

    Chlamydomonas reinhardtii is the organism in which intraflagellar transport (IFT) was first visualized and in which the composition of IFT particles was originally elucidated. As the universality of IFT among ciliated/flagellated cells was uncovered, the diversity of organisms used to study IFT has grown. Still, because of the ease of isolation of flagella from Chlamydomonas and the battery of temperature-sensitive mutants affecting IFT proteins and motors, this unicellular alga remains the principal model for biochemical studies of IFT motors and cargo; furthermore, the long, exposed flagella of this cell are ideally suited for observing IFT in real time with GFP-tagged components of IFT.

  16. Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

    PubMed Central

    van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.

    2016-01-01

    ABSTRACT Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition

  17. Engineering FRET constructs using CFP and YFP.

    PubMed

    Shimozono, Satoshi; Miyawaki, Atsushi

    2008-01-01

    Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Here we discuss how to engineer constructs for FRET between the cyan- and yellow-emitting variants of green fluorescent protein (GFP) from Aequorea victoria (CFP and YFP, respectively). Throughout this chapter, we stress the fact that FRET is highly sensitive to the relative orientation and distance between the donor and the acceptor. The chapter consists of two parts. First, we discuss FRET-based indicators encoded by single genes, which were developed in our laboratory. In this approach, a number of different constructs can be made for a comparative assessment of their FRET efficiencies. For example, the length and sequence of the linker between the fluorescent protein and the host protein should be optimized for each specific application. In the second part, we describe the use of long and flexible linkers for engineering FRET constructs, including an introduction to a general and efficient tool for making successful fusion proteins with long and flexible linkers. When CFP and YFP are fused through floppy linkers to two protein domains that interact with each other, the two fluorescent proteins will associate due to the weak dimerization propensity of Aequorea GFP, which results in moderate FRET. This approach has become even more powerful due to the construction of a new pair of fluorescent proteins for FRET: CyPet and YPet.

  18. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

    PubMed Central

    Pletnev, Vladimir Z.; Pletneva, Nadya V.; Lukyanov, Konstantin A.; Souslova, Ekaterina A.; Fradkov, Arkady F.; Chudakov, Dmitry M.; Chepurnykh, Tatyana; Yampolsky, Ilia V.; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-01-01

    A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 Cβ atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis. PMID:23999308

  19. Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

    PubMed

    Pletnev, Vladimir Z; Pletneva, Nadya V; Lukyanov, Konstantin A; Souslova, Ekaterina A; Fradkov, Arkady F; Chudakov, Dmitry M; Chepurnykh, Tatyana; Yampolsky, Ilia V; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-09-01

    A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C(β) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.

  20. The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.

    PubMed

    Rakosy-Tican, Elena; Aurori, Cristian M; Dijkstra, Camelia; Thieme, Ramona; Aurori, Adriana; Davey, Michael R

    2007-05-01

    Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

  1. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  2. Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

    PubMed

    Sivakumar, Krishnakumar; Mukherjee, Manisha; Cheng, Hsin-I; Zhang, Yingdan; Ji, Lianghui; Cao, Bin

    2015-03-01

    Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.

  3. The fluorescent photobleaching properties of GFP expressed in human lung cancer cells

    NASA Astrophysics Data System (ADS)

    Jin, Ying; Xing, Da

    2003-12-01

    The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the dicistronic expression vector (pEGFP-C1) was used to transfected into human lung cancer cell line (ASTC-a-1) and a positive clone which stably expressed GFP in high level was obtained. After more than three months' passengers, the cells were also remained the strong fluorescence under fluorescent microscope. The results showed that the green fluorescent protein expressed in tumor cells was also photobleached under intense irradiation (approximately 488 nm) and the degree of photobleaching varied with the difference of the intensity of the excitation. Using different interdiction parcel (None, ND4, ND8, ND16), there were significant differences in photobleaching among the different excitation. The photobleaching was also affected by the time length of excitation, and the intensity of fluorescence was obviously decreased along with the increasing of excitation time, especially to stronger excitation.

  4. Establishment of enhancer detection lines expressing GFP in the gut of the ascidian Ciona intestinalis.

    PubMed

    Yoshida, Reiko; Sasakura, Yasunori

    2012-01-01

    The gut is a tubular, endodermal organ for digesting food and absorbing nutrients. In this study, we characterized eight enhancer detection lines that express green fluorescent protein (GFP) in the whole or part of the digestive tube of the ascidian Ciona intestinalis. Three enhancer detection lines for the pyloric gland, a structure associated with the digestive tube, were also analyzed. These lines are valuable markers for analyzing the mechanisms of development of the gut. Based on the GFP expression of the enhancer detection lines together with morphological characteristics, the digestive tube of Ciona can be subdivided into at least 10 compartments in which different genetic cascades operate. Causal insertion sites of the enhancer detection lines were identified, and the expression pattern of the genes near the insertion sites were characterized by means of whole-mount in situ hybridization. We have characterized four and two genes that were specifically or strongly expressed in the digestive tube and pyloric gland, respectively. The present data provide the basic information and useful resources for studying gut formation in Ciona.

  5. Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Ojala, Kirsi; Michel, Patrik O; Vuento, Matti; Oker-Blom, Christian

    2002-12-01

    Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

  6. Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

    NASA Technical Reports Server (NTRS)

    Granger, C. L.; Cyr, R. J.

    2000-01-01

    Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

  7. Overlap of Doxycycline Fluorescence with that of the Redox-Sensitive Intracellular Reporter roGFP.

    PubMed

    Khader, Heba; Solodushko, Victor; Al-Mehdi, Abu Bakr; Audia, Jonathon; Fouty, Brian

    2014-03-01

    Tetracycline-inducible systems allow for either suppression or induction of transgene expression to facilitate studies of cell physiology. Doxycycline is a preferred inducer for these gene expression systems due to its membrane permeability; however, the heterocyclic structure of doxycycline exhibits fluorogenic properties that can potentially bias measurement of other fluorochromes. Thus the simultaneous use of tetracycline-inducible systems and fluorescent proteins as reporter genes or as intracellular biosensors may lead to potentially confounding results. Herein, using cells which co-express the ratiometric redox sensitive intracellular reporter, roGFP, and a tetracycline-inducible reporter plasmid encoding the reporter gene, mCherry, as a model system, we describe the overlapping intracellular fluorescent signals between doxycycline and commonly used intracellular fluorescent probes. In our cells, the addition of doxycycline to cells caused a dose- and time-dependent increase in cell fluorescence with 405 nm excitation which overlapped with that of the oxidized configuration of roGFP. Incubating cells in concentrations of doxycycline less than 1 μg/mL and removing doxycycline from the media 60 min before performing experiments eliminated fluorescence interference while still maintaining maximal reporter transgene activation.

  8. Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning

    PubMed Central

    Drobizhev, M.; Callis, P. R.; Nifosì, R.; Wicks, G.; Stoltzfus, C.; Barnett, L.; Hughes, T. E.; Sullivan, P.; Rebane, A.

    2015-01-01

    The majority of protein functions are governed by their internal local electrostatics. Quantitative information about these interactions can shed light on how proteins work and allow for improving/altering their performance. Green fluorescent protein (GFP) and its mutation variants provide unique optical windows for interrogation of internal electric fields, thanks to the intrinsic fluorophore group formed inside them. Here we use an all-optical method, based on the independent measurements of transition frequency and one- and two-photon absorption cross sections in a number of GFP mutants to evaluate these internal electric fields. Two physical models based on the quadratic Stark effect, either with or without taking into account structural (bond-length) changes of the chromophore in varying field, allow us to separately evaluate the long-range and the total effective (short- and long-range) fields. Both types of the field quantitatively agree with the results of independent molecular dynamic simulations, justifying our method of measurement. PMID:26286372

  9. Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

    PubMed

    Maximova, S; Miller, C; Antúnez de Mayolo, G; Pishak, S; Young, A; Guiltinan, M J

    2003-06-01

    We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.

  10. SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP.

    PubMed

    Melnik, Bogdan S; Povarnitsyna, Tatiana V; Glukhov, Anatoly S; Melnik, Tatyana N; Uversky, Vladimir N; Sarma, Ramaswamy H

    2012-01-01

    Abstract The most attractive and methodologically convenient way to enhance protein stability is via the introduction of disulphide bond(s). However, the effect of the artificially introduced SS-bond on protein stability is often quite unpredictable. This raises the question of how to choose the protein sites in an intelligent manner, so that the 'fastening' of these sites by the SS-bond(s) would provide maximal protein stability. We hypothesize that the successful design of a stabilizing SS-bond requires finding highly mobile protein regions. Using GFP as an illustrative example, we demonstrate that the knowledge of the peculiarities of the intramolecular hydrophobic interactions, combined with the understanding of the local intrinsic disorder propensities (that can be evaluated by various disorder predictors, e.g., PONDRFIT), is sufficient to find the candidate sites for the introduction of stabilizing SS-bridge(s). In fact, our analysis revealed that the insertion of the engineered SS-bridge between two highly flexible regions of GFP noticeably increased the conformational stability of this protein toward the thermal and chemical unfolding. Therefore, our study represents a novel approach for the rational design of stabilizing disulphide bridges in proteins.

  11. Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP.

    PubMed

    Tsutsui, Motomu; Hasegawa, Hitoki; Adachi, Koichi; Miyata, Maiko; Huang, Peng; Ishiguro, Naoki; Hamaguchi, Michinari; Iwamoto, Takashi

    2008-08-08

    Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.

  12. High prevalence of non-synonymous substitutions in mtDNA of cichlid fishes from Lake Victoria.

    PubMed

    Shirai, Kazumasa; Inomata, Nobuyuki; Mizoiri, Shinji; Aibara, Mitsuto; Terai, Yohey; Okada, Norihiro; Tachida, Hidenori

    2014-12-01

    When a population size is reduced, genetic drift may fix slightly deleterious mutations, and an increase in nonsynonymous substitution is expected. It has been suggested that past aridity has seriously affected and decreased the populations of cichlid fishes in Lake Victoria, while geographical studies have shown that the water levels in Lake Tanganyika and Lake Malawi have remained fairly constant. The comparably stable environments in the latter two lakes might have kept the populations of cichlid fishes large enough to remove slightly deleterious mutations. The difference in the stability of cichlid fish population sizes between Lake Victoria and the Lakes Tanganyika and Malawi is expected to have caused differences in the nonsynonymous/synonymous ratio, ω (=dN/dS), of the evolutionary rate. Here, we estimated ω and compared it between the cichlids of the three lakes for 13 mitochondrial protein-coding genes using maximum likelihood methods. We found that the lineages of the cichlids in Lake Victoria had a significantly higher ω for several mitochondrial loci. Moreover, positive selection was indicated for several codons in the mtDNA of the Lake Victoria cichlid lineage. Our results indicate that both adaptive and slightly deleterious molecular evolution has taken place in the Lake Victoria cichlids' mtDNA genes, whose nonsynonymous sites are generally conserved.

  13. Population-structure and genetic diversity in a haplochromine cichlid fish [corrected] of a satellite lake of Lake Victoria.

    PubMed

    Abila, Romulus; Barluenga, Marta; Engelken, Johannes; Meyer, Axel; Salzburger, Walter

    2004-09-01

    The approximately 500 species of the cichlid fish species flock of Lake Victoria, East Africa, have evolved in a record-setting 100,000 years and represent one of the largest adaptive radiations. We examined the population structure of the endangered cichlid species Xystichromis phytophagus from Lake Kanyaboli, a satellite lake to Lake Victoria in the Kenyan Yala wetlands. Two sets of molecular markers were analysed--sequences of the mitochondrial control region as well as six microsatellite loci--and revealed surprisingly high levels of genetic variability in this species. Mitochondrial DNA sequences failed to detect population structuring among the three sample populations. A model-based population assignment test based on microsatellite data revealed that the three populations most probably aggregate into a larger panmictic population. However, values of population pairwise FST indicated moderate levels of genetic differentiation for one population. Eleven distinct mitochondrial haplotypes were found among 205 specimens of X. phytophagus, a relatively high number compared to the total number of 54 haplotypes that were recovered from hundreds of specimens of the entire cichlid species flock of Lake Victoria. Most of the X. phytophagus mitochondrial DNA haplotypes were absent from the main Lake Victoria, corroborating the putative importance of satellite lakes as refugia for haplochromine cichlids that went extinct from the main lake in the last decades and possibly during the Late Pleistocene desiccation of Lake Victoria.

  14. Active inclusion bodies of acid phosphatase PhoC: aggregation induced by GFP fusion and activities modulated by linker flexibility

    PubMed Central

    2013-01-01

    Background Biologically active inclusion bodies (IBs) have gained much attention in recent years. Fusion with IB-inducing partner has been shown to be an efficient strategy for generating active IBs. To make full use of the advantages of active IBs, one of the key issues will be to improve the activity yield of IBs when expressed in cells, which would need more choices on IB-inducing fusion partners and approaches for engineering IBs. Green fluorescent protein (GFP) has been reported to aggregate when overexpressed, but GFP fusion has not been considered as an IB-inducing approach for these fusion proteins so far. In addition, the role of linker in fusion proteins has been shown to be important for protein characteristics, yet impact of linker on active IBs has never been reported. Results Here we report that by fusing GFP and acid phosphatase PhoC via a linker region, the resultant PhoC-GFPs were expressed largely as IBs. These IBs show high levels of specific fluorescence and specific PhoC activities (phosphatase and phosphotransferase), and can account for up to over 80% of the total PhoC activities in the cells. We further demonstrated that the aggregation of GFP moiety in the fusion protein plays an essential role in the formation of PhoC-GFP IBs. In addition, PhoC-GFP IBs with linkers of different flexibility were found to exhibit different levels of activities and ratios in the cells, suggesting that the linker region can be utilized to manipulate the characteristics of active IBs. Conclusions Our results show that active IBs of PhoC can be generated by GFP fusion, demonstrating for the first time the potential of GFP fusion to induce active IB formation of another soluble protein. We also show that the linker sequence in PhoC-GFP fusion proteins plays an important role on the regulation of IB characteristics, providing an alternative and important approach for engineering of active IBs with the goal of obtaining high activity yield of IBs. PMID:23497261

  15. Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue.

    PubMed

    Nobusue, Hiroyuki; Endo, Tsuyoshi; Kano, Koichiro

    2008-06-01

    We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro.

  16. Glacial geomorphology of the Victoria Valley System, Ross Sea Region, Antarctica

    NASA Astrophysics Data System (ADS)

    Bockheim, James G.; McLeod, Malcolm

    2013-07-01

    During the 2011-2012 austral summer, we had the opportunity to verify a surficial geology map prepared nearly 50 years ago for the Victoria Valley system (VVS), the largest of the McMurdo Dry Valleys. We used high-resolution landsat images and a digital elevation model to identify landforms and prepare detailed maps of each of the five valleys in the VVS, including lateral and end moraines, rock glaciers, gelifluction sheets, gravel ripples, and hummocky and ice-cored drifts. Our mapping suggests that the Bull drift is less extensive than previously thought, attains a maximum elevation of ~ 750 m in Balham and Barwick Valleys and the upper Bull Pass region, and does not occur in McKelvey Valley. We found Insel drift to 850 m elevation in eastern McKelvey Valley and upper Bull Pass and were able to trace Insel drift down Bull Pass where it becomes Peleus drift in Wright Valley. The Victoria Lower Glacier likely responded to grounding of ice in the Ross Embayment and was out-of-phase with alpine glaciers elsewhere in the VVS. We amplified and quantified Calkin's relative chronology and provide here our multiple-parameter relative chronology for the McMurdo Dry Valleys that is based on surface-boulder weathering, soil weathering, salt stage, degree of development of the desert pavement, and form of patterned ground. Except for Victoria Lower Valley, we correlate Packard drift with Taylor II drift (ca., 120 ka), Vida drift with Taylor III drift (ca., 300 ka), Bull drift with Taylor IVb drift (2.7-3.5 Ma, and Insel drift with Peleus drift (> 3.7 Ma, < 5.4 Ma). The lack of a strong correlation between soil salt stage and depth of visible salts with elevation leads us to question whether a high-level lake (ca., 200 m deep) existed in the VVS during the early Holocene.

  17. Metals in Nile perch (Lates niloticus) and suspended particulate matter from Lake Victoria, Tanzania.

    PubMed

    Machiwa, John F

    2005-01-01

    A study was conducted to assess the levels of pollutant metals in suspended particulate matter and Nile perch from Lake Victoria. The metals in particulate matter were determined to ascertain their concentrations at the base of the food chain. Nile perch samples were collected in September 2003 from five major fish processing factories at the shores of Lake Victoria in Mwanza and Musoma. The concentrations of total Hg, Pb, Cd, and Cu were generally low in particulate matter and in most locations were close to or below their limits of detection. The concentrations of Zn were high in suspended particulate matter, the highest being 219.4 +/- 153.0 microg L(-1) found in particulate matter from Nungwe Bay in the southern part of Lake Victoria. Nile perch generally contained low levels of heavy metals; the range for Pb was <0.01-0.08 microg g(-1) ww, Cd was <0.001-0.04 microg g(-1) ww, Cu was 0.01-0.97 microg g(-1) ww, and Zn was <0.01-18.94 microg g(-1) ww. The concentration of total mercury ranged between 31.0 and 684.2 ng g(-1) ww; generally, it was below the Food and Agriculture Organization of the United Nations/World Health Organization (1000 ng total Hg g(-1) ww for piscivorous fish species) maximum allowable level. Indeed, all Nile perch samples that weighed less than 10 kg had less than 200 ng total Hg g(-1) ww and therefore are safe for regular consumption by at-risk groups such as children and pregnant women. Levels of mercury and other heavy metals in Nile perch at present is, therefore, not a severe environmental issue; however, urgent regulatory measures should be taken to minimize metal input into the lake to maintain the current levels in the fish.

  18. Irrigator responses to groundwater resource management in northern Victoria, southeastern Australia

    NASA Astrophysics Data System (ADS)

    Gill, Bruce C.; Webb, John; Wilkinson, Roger; Cherry, Don

    2014-10-01

    In northern Victoria, farmers are the biggest users of groundwater and therefore the main stakeholders in plans that seek to sustainably manage the resource. Interviews with 30 irrigation farmers in two study areas, analysed using qualitative social research methods, showed that the overwhelming majority of groundwater users agreed with the need for groundwater management and thought that the current plans had achieved sustainable resource use. The farmers also expressed a strong need for clear technical explanations for management decisions, in particular easily understood water level data. The social licence to implement the management plans arose through effective consultation with the community during plan development. Several additional factors combined to gain acceptance for the plans: good data on groundwater usage and aquifer levels is available; irrigation farmers had been exposed to usage restrictions since the late 1990s; an ‘adaptive’ management approach is in use which allowed refinements to be readily incorporated and fortuitously, plan development coincided with the 1998-2009 drought, when declines in groundwater levels reinforced the usefulness of the plans. The imposition of a nation-wide water use reduction plan in 2012 had relatively little impact in Victoria because of the early implementation of effective groundwater management plans. However, economic difficulties that reduce groundwater users’ capacity to pay groundwater management charges mean that the future of the plans in Victoria is not assured. Nevertheless, the high level of trust that exists between Victorian irrigation farmers and the management agencies suggests that the continued use of a consultative approach will continue to produce workable outcomes. Lessons from the Victorian experience may be difficult to apply in other areas of groundwater use in Australia and overseas, where there may be a quite different history of development and culture of groundwater management.

  19. Stable isotope paleoecology of Late Pleistocene Middle Stone Age humans from the Lake Victoria basin, Kenya.

    PubMed

    Garrett, Nicole D; Fox, David L; McNulty, Kieran P; Faith, J Tyler; Peppe, Daniel J; Van Plantinga, Alex; Tryon, Christian A

    2015-05-01

    Paleoanthropologists have long argued that environmental pressures played a key role in human evolution. However, our understanding of how these pressures mediated the behavioral and biological diversity of early modern humans and their migration patterns within and out of Africa is limited by a lack of archaeological evidence associated with detailed paleoenvironmental data. Here, we present the first stable isotopic data from paleosols and fauna associated with Middle Stone Age (MSA) sites in East Africa. Late Pleistocene (∼100-45 ka, thousands of years ago) sediments on Rusinga and Mfangano Islands in eastern Lake Victoria (Kenya) preserve a taxonomically diverse, non-analog faunal community associated with MSA artifacts. We analyzed the stable carbon and oxygen isotope composition of paleosol carbonate and organic matter and fossil mammalian tooth enamel, including the first analyses for several extinct bovids such as Rusingoryx atopocranion, Damaliscus hypsodon, and an unnamed impala species. Both paleosol carbonate and organic matter data suggest that local habitats associated with human activities were primarily riverine woodland ecosystems. However, mammalian tooth enamel data indicate that most large-bodied mammals consumed a predominantly C4 diet, suggesting an extensive C4 grassland surrounding these riverine woodlands in the region at the time. These data are consistent with other lines of paleoenvironmental evidence that imply a substantially reduced Lake Victoria at this time, and demonstrate that C4 grasslands were significantly expanded into equatorial Africa compared with their present distribution, which could have facilitated dispersal of human populations and other biotic communities. Our results indicate that early populations of Homo sapiens from the Lake Victoria region exploited locally wooded and well-watered habitats within a larger grassland ecosystem.

  20. Malaria vectors in Lake Victoria and adjacent habitats in western Kenya.

    PubMed

    Minakawa, Noboru; Dida, Gabriel O; Sonye, George O; Futami, Kyoko; Njenga, Sammy M

    2012-01-01

    The prevalence of malaria among the residents of the Lake Victoria basin remains high. The environment associated with the lake may maintain a high number of malaria vectors. Lake habitats including water hyacinths have been suspected to be the source of vectors. This study investigated whether malaria vectors breed in the lake habitats and adjacent backwater pools. Anopheline larvae were collected within the littoral zone of the lake and adjacent pools located along approximately 24.3 km of the lakeshore in western Kenya, and their breeding sites characterized. Three primary vector species, Anopheles arabiensis, Anopheles gambiae s.s. and Anopheles funestus s.s., and three potential vectors, were found in the lake habitats. Unexpectedly, An. arabiensis was the most dominant vector species in the lake sampling sites. Its habitats were uncovered or covered with short grass. A potential secondary malaria vector, Anopheles rivulorum, dominated the water hyacinths in the lake. Most breeding sites in the lake were limited to areas that were surrounded by tall emergent plants, including trees, and those not exposed to waves. Nearly half of adjacent habitats were lagoons that were separated from the lake by sand bars. Lagoons contained a variety of microhabitats. Anopheles arabiensis dominated open habitats, whereas An. funestus s.s. was found mainly in vegetated habitats in lagoons. The current study confirmed that several breeding sites are associated with Lake Victoria. Given that Lake Victoria is the second largest lake in the world, the lake related habitats must be extensive; therefore, making targeted vector control difficult. Further exploration is necessary to estimate the effects of lake associated habitats on malaria transmission so as to inform a rational decision-making process for vector control.

  1. Population genetic structure of Anopheles gambiae mosquitoes on Lake Victoria islands, west Kenya

    PubMed Central

    Chen, Hong; Minakawa, Noboru; Beier, John; Yan, Guiyun

    2004-01-01

    Background Understanding the genetic structure of island Anopheles gambiae populations is important for the current tactics in mosquito control and for the proposed strategy using genetically-modified mosquitoes (GMM). Genetically-isolated mosquito populations on islands are a potential site for testing GMM. The objective of this study was to determine the genetic structure of A. gambiae populations on the islands in Lake Victoria, western Kenya. Methods The genetic diversity and the population genetic structures of 13 A. gambiae populations from five islands on Lake Victoria and six villages from the surrounding mainland area in the Suba District were examined using six microsatellite markers. The distance range of sampling sites varied between 2.5 and 35.1 km. Results A similar level of genetic diversity between island mosquito populations and adjacent mainland populations was found. The average number of alleles per locus was 7.3 for the island populations and 6.8 for the mainland populations. The average observed heterozygosity was 0.32 and 0.28 for the island and mainland populations, respectively. A low but statistically significant genetic structure was detected among the island populations (FST = 0.019) and between the island and mainland populations (FST = 0.003). A total of 12 private alleles were found, and nine of them were from the island populations. Conclusion A level of genetic differentiation between the island and mainland populations was found. Large extent of gene flow between the island and mainland mosquito populations may result from wind- or human-assisted dispersal. Should the islands on Lake Victoria be used as a trial site for the release program of GMM, mosquito dispersal between the islands and between the island and the mainland should be vigorously monitored. PMID:15581429

  2. Amplification of extreme precipitation response to climate change over Lake Victoria

    NASA Astrophysics Data System (ADS)

    Thiery, Wim; Davin, Edouard; Seneviratne, Sonia; Bedka, Kristopher; van Lipzig, Nicole

    2015-04-01

    Casualties among fishermen operating on Lake Victoria are estimated to amount up to several thousand per year, leading to the dubious distinction of "world's most lethal lake". Most of the casualties are caused by severe thunderstorms occurring at night, when surface winds converge over the lake and trigger deep convection of air masses moistened by the lake. With the climate change induced raise in troposphere temperatures, the frequency and intensity of these extremes are likely to increase. However, up to now only very little is known about the processes underlying this nighttime convection, and how it will be affected by climate change. We examine the impact of climate change on hazardous thunderstorms over Lake Victoria by conducting a set of regional climate model simulations which resolve individual lakes and explicitly compute lake temperatures. The regional climate model COSMO-CLM² is used to dynamically downscale a CORDEX-Africa projection (COSMO-CLM/MPI-ESM-LR) under RCP8.5 to 7 km grid spacing for the periods 1981-2010 and 2071-2100. Based on these high resolution simulations, we project that the increase in extreme precipitation is amplified over Lake Victoria compared to surrounding land area, consistent with projections from the (courser-scale) CORDEX-Africa ensemble. Moreover, the strongest extremes are found to follow the Clausius-Clapeyron scaling over the lake surface only. Finally, we investigate controls on the occurrence of this extreme precipitation in the present-day climate using satellite observations and a dynamical reanalysis downscaling, and detect a strong relationship with antecedent daytime land thunderstorms. Besides supplying moisture, these storms also modify mesoscale circulation in favor of strong over-lake convection the following night. Extending this analysis will make it possible to attribute the projected lake amplification effect to changes in the controlling factors.

  3. Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

    NASA Technical Reports Server (NTRS)

    Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

    1998-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

  4. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    NASA Technical Reports Server (NTRS)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  5. Measurements of Ca²⁺ concentration with recombinant targeted luminescent probes.

    PubMed

    Ottolini, Denis; Calì, Tito; Brini, Marisa

    2013-01-01

    In the last two decades the study of Ca(2+) homeostasis in living cells has been enhanced by the explosive development of genetically encoded Ca(2+)-indicators. The cloning of the Ca(2+)-sensitive photoprotein aequorin and of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been enormously advantageous. As polypeptides, aequorin and GFP allow their endogenous production in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localize them within the cell by including defined targeting signals in the amino acid sequence. These two proteins have been extensively engineered to obtain several recombinant probes for different biological parameters, among which Ca(2+) concentration reporters are probably the most relevant. The GFP-based Ca(2+) probes and aequorin are widely employed in the study of intracellular Ca(2+) homeostasis. The new generation of bioluminescent probes that couple the Ca(2+) sensitivity of aequorin to GFP fluorescence emission allows real-time measurements of subcellular Ca(2+) changes in single cell imaging experiments and the video-imaging of Ca(2+) concentrations changes in live transgenic animals that express GFP-aequorin bifunctional probes.

  6. Ultra-Fast Excited State Dynamics in Green Fluorescent Protein: Multiple States and Proton Transfer

    NASA Astrophysics Data System (ADS)

    Chattoraj, Mita; King, Brett A.; Bublitz, Gerold U.; Boxer, Steven G.

    1996-08-01

    The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.

  7. Pesticide residues and heavy metals in Lake Victoria Nile perch, Lates niloticus, belly flap oil.

    PubMed

    Ogwok, P; Muyonga, J H; Sserunjogi, M L

    2009-05-01

    Oil was extracted from the belly flaps of varied sizes of Nile perch caught from Lake Victoria (Uganda). The oil was analyzed for pesticide residues and heavy metals. Total residual concentration of dichlorodiphenyltrichloroethane, endosulfan, hexachlorocyclohexane, hexachlorobenzene, heptachlor, chlordane, endrin, aldrin and chlorofenvinphos increased significantly (p < 0.05) with fish size. Mercury and lead were detected in most samples while arsenic and cadmium were below detection limits. Nile perch may, therefore, accumulate significant amount of chemical contaminants. Levels of contaminants in Nile perch oil were, in general, within limits considered acceptable by the stringent German Food Law for human consumption.

  8. Shape of allied health: an environmental scan of 27 allied health professions in Victoria.

    PubMed

    Nancarrow, Susan A; Young, Gretchen; O'Callaghan, Katy; Jenkins, Mathew; Philip, Kathleen; Barlow, Kegan

    2016-08-11

    Objective In 2015, the Victorian Department of Health and Human Services commissioned the Victorian Allied Health Workforce Research Program to provide data on allied health professions in the Victorian public, private and not-for-profit sectors. Herein we present a snapshot of the demographic profiles and distribution of these professions in Victoria and discuss the workforce implications.Methods The program commenced with an environmental scan of 27 allied health professions in Victoria. This substantial scoping exercise identified existing data, resources and contexts for each profession to guide future data collection and research. Each environmental scan reviewed existing data relating to the 27 professions, augmented by an online questionnaire sent to the professional bodies representing each discipline.Results Workforce data were patchy but, based on the evidence available, the allied health professions in Victoria vary greatly in size (ranging from just 17 child life therapists to 6288 psychologists), are predominantly female (83% of professions are more than 50% female) and half the professions report that 30% of their workforce is aged under 30 years. New training programs have increased workforce inflows to many professions, but there is little understanding of attrition rates. Professions reported a lack of senior positions in the public sector and a concomitant lack of senior specialised staff available to support more junior staff. Increasing numbers of allied health graduates are being employed directly in private practice because of a lack of growth in new positions in the public sector and changing funding models. Smaller professions reported that their members are more likely to be professionally isolated within an allied health team or larger organisations. Uneven rural-urban workforce distribution was evident across most professions.Conclusions Workforce planning for allied health is extremely complex because of the lack of data, fragmented

  9. Gambling revenues as a public administration issue: electronic gaming machines in Victoria.

    PubMed

    Pickernell, David; Keast, Robyn; Brown, Kerry; Yousefpour, Nina; Miller, Chris

    2013-12-01

    Gambling activities and the revenues derived have been seen as a way to increase economic development in deprived areas. There are also, however, concerns about the effects of gambling in general and electronic gaming machines (EGMs) in particular, on the resources available to the localities in which they are situated. This paper focuses on the factors that determine the extent and spending of community benefit-related EGM-generated resources within Victoria, Australia, focusing in particular on the relationships between EGM activity and socio-economic and social capital indicators, and how this relates to the community benefit resources generated by gaming.

  10. Climate Change Impacts on Water Resources in the Lake Victoria Basin

    NASA Astrophysics Data System (ADS)

    Tidwell, A. C.; Georgakakos, A. P.; Yao, H.

    2007-12-01

    The success of water resources planning and management decisions is predicated on decision maker access to reliable and conclusive information, including climate forecasts. This is becoming increasingly critical as climate changes are predicted to take place over the same time horizons used in water and energy resources planning. The results of climate scenario analysis for Lake Victoria in East Africa are presented and used in a detailed climate change impact assessment for water resources. This region is shown to be particularly vulnerable to climate changes through projected impacts on net basin supplies, lake levels, lake releases, and energy generation.

  11. A review of mercury in Lake Victoria, East Africa: implications for human and ecosystem health.

    PubMed

    Campbell, Linda; Dixon, D G; Hecky, R E

    2003-01-01

    Lake Victoria, East Africa, has been the site of many recent studies measuring mercury (Hg) concentrations in water, fish, sediment, soil, and humans. Most of these studies were motivated by concerns about Hg contamination from processing of gold ore on the southern shores. Total Hg (THg) concentrations in fish were usually below permissible World Health Organization (WHO) concentrations and international marketing limits and do not threaten the lucrative export industry. Nile perch 3-10 kg and most >10 kg had THg concentrations above the WHO threshold concentrations for at-risk groups (200 ng/g). Elevated THg concentrations in large Nile perch are not of major concern because Nile perch are rarely consumed by the people living on Lake Victoria and very large Nile perch are becoming increasingly rare in catches. Water THg concentrations were below Canadian drinking water guidelines but were elevated relative to those in the northern Great Lakes. Sediment and soil THg concentrations were within inter-national guidelines and are comparable to those in northern latitudes but are lower than those in the Amazon basin. Biomass burning and soil erosion are estimated to be the major sources of THg for the lake and probably constitute a larger source of THg than gold mining in Tanzania.THg concentrations in urine and hair from human volunteers indicate that while gold miners and frequent skin-bleaching cream users are at risk of inorganic mercury poisoning, the rest of the population, including fishermen, is not. Human exposure assessments demonstrated that fish consumption and soil geophagy constitute major sources of THg for humans, but the total estimated daily intake of THg was below the Health Canada tolerable daily intake (TDI) limits. The use of beauty creams containing high inorganic Hg concentrations, however, caused the estimated THg exposure to exceed the TDI. The high THg content in the hair of regular cream users supports this assessment. The nutritional

  12. VICTORIA: A mechanistic model of radionuclide behavior in the reactor coolant system under severe accident conditions. Revision 1

    SciTech Connect

    Heams, T J; Williams, D A; Johns, N A; Mason, A; Bixler, N E; Grimley, A J; Wheatley, C J; Dickson, L W; Osborn-Lee, I; Domagala, P; Zawadzki, S; Rest, J; Alexander, C A; Lee, R Y

    1992-12-01

    The VICTORIA model of radionuclide behavior in the reactor coolant system (RCS) of a light water reactor during a severe accident is described. It has been developed by the USNRC to define the radionuclide phenomena and processes that must be considered in systems-level models used for integrated analyses of severe accident source terms. The VICTORIA code, based upon this model, predicts fission product release from the fuel, chemical reactions involving fission products, vapor and aerosol behavior, and fission product decay heating. Also included is a detailed description of how the model is implemented in VICTORIA, the numerical algorithms used, and the correlations and thermochemical data necessary for determining a solution. A description of the code structure, input and output, and a sample problem are provided.

  13. Influence of the Pearl River estuary and vertical mixing in Victoria Harbor on water quality in relation to eutrophication impacts in Hong Kong waters.

    PubMed

    Yin, Kedong; Harrison, Paul J

    2007-06-01

    This study presents water quality parameters such as nutrients, phytoplankton biomass and dissolved oxygen based on 11 years of water quality data in Victoria Harbor and examined how the Pearl River estuary discharge in summer and year round sewage discharge influenced these parameters. Nutrients in Victoria Harbor were strongly influenced by both the Pearl River and sewage effluent, as indicated by the high NO(3) inputs from the Pearl River in summer and higher NH(4) and PO(4) in Victoria Harbor than both its sides. N:P ratios were low in the dry season, but increased to >16:1 in the wet season, suggesting that P is potentially the most limiting nutrient in this area during the critical period in the summer. Although there were generally high nutrients, the phytoplankton biomass was not as high as one would expect in Victoria Harbor. In fact, there were high concentrations of chl near the bottom well below the photic zone. Salinity near the bottom was lower in Victoria Harbor than at the two entrances to Victoria Harbor, suggesting strong vertical mixing within Victoria Harbor. Therefore, strong vertical mixing and horizontal advection appear to play an important role in significantly reducing eutrophication impacts in Victoria Harbor. Consequently, dissolved oxygen near the bottom was low in summer, but only occasionally dipped to 2 mgL(-1) despite the high organic loading from sewage effluent.

  14. Dual Fluorescence in GFP Chromophore Analogues: Chemical Modulation of Charge Transfer and Proton Transfer Bands.

    PubMed

    Chatterjee, Tanmay; Mandal, Mrinal; Das, Ananya; Bhattacharyya, Kalishankar; Datta, Ayan; Mandal, Prasun K

    2016-04-14

    Dual fluorescence of GFP chromophore analogues has been observed for the first time. OHIM (o-hydroxy imidazolidinone) shows only a charge transfer (CT) band, CHBDI (p-cyclicamino o-hydroxy benzimidazolidinone) shows a comparable intensity CT and PT (proton transfer) band, and MHBDI (p-methoxy o-hydroxy benzimidazolidinone) shows a higher intensity PT band. It could be shown that the differential optical behavior is not due to conformational variation in the solid or solution phase. Rather, control of the excited state electronic energy level and excited state acidity constant by functional group modification could be shown to be responsible for the differential optical behavior. Chemical modification-induced electronic control over the relative intensity of the charge transfer and proton transfer bands could thus be evidenced. Support from single-crystal X-ray structure, NMR, femtosecond to nanosecond fluorescence decay analysis, and TDDFT-based calculation provided important information and thus helped us understand the photophysics better.

  15. Membrane trafficking in higher plant cells: GFP and antibodies, partners for probing the secretory pathway.

    PubMed

    Satiat-Jeunemaitre, B; Boevink, P; Hawes, C

    1999-06-01

    Eukaryotic cells are characterised by the organised distribution of membrane bounded compartments in their cytoplasm. The endoplasmic reticulum (ER) and the Golgi apparatus (GA) are part of this endomembrane machinery. They are involved in protein flow, and are in charge of specific functions such as the assembly, sorting and transport of newly synthesised proteins, glycoproteins or polysaccharides to their final destination, where the macromolecules are recognised either for action, storage, deposition or degradation. The structural and functional relationship between the ER and GA in higher plants is still a matter of debate. Therefore, it was essential to develop probes that would specifically label proteins or glycoproteins of the endomembrane system in situ. Here we compare two complementary approaches to probe plant endomembranes; immunocytochemistry on fixed cells, and in vivo studies using the expression of GFP tagged chimeric proteins. The structural relationship between ER and GA as based on pharmacological approaches using the two systems is explored.

  16. From GFP to β-lactamase: advancing intact cell imaging for toxins and effectors

    PubMed Central

    Zuverink, Madison; Barbieri, Joseph T.

    2015-01-01

    Canonical reporters such as green fluorescent protein (GFP) and luciferase have assisted researchers in probing cellular pathways and processes. Prior research in pathogenesis depended on sensitivity of biochemical and biophysical techniques to identify effectors and elucidate entry mechanisms. Recently, the β-lactamase (βlac) reporter system has advanced toxin and effector reporting by permitting measurement of βlac delivery into the cytosol or host βlac expression in intact cells. βlac measurement in cells was facilitated by the development of the fluorogenic substrate, CCF2-AM, to identify novel effectors, target cells, and domains involved in bacterial pathogenesis. The assay is also adaptable for high-throughput screening of small molecule inhibitors against toxins, providing information on mechanism and potential therapeutic agents. The versatility and limitations of the βlac reporter system as applied to toxins and effectors are discussed in this review. PMID:26500183

  17. Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery

    NASA Astrophysics Data System (ADS)

    Hao, Yuzhi; Xu, Peng; He, Chuan; Yang, Xiaoyan; Huang, Min; Xing, James; Chen, Jie

    2011-07-01

    1-ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

  18. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching.

    PubMed

    Brakemann, Tanja; Stiel, Andre C; Weber, Gert; Andresen, Martin; Testa, Ilaria; Grotjohann, Tim; Leutenegger, Marcel; Plessmann, Uwe; Urlaub, Henning; Eggeling, Christian; Wahl, Markus C; Hell, Stefan W; Jakobs, Stefan

    2011-09-11

    Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

  19. Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot

    PubMed Central

    Armesto, Cecilia; Maia, Fernanda Gonçalves Martins; de Abreu, Mário Sobral; Figueira, Antonia dos Reis; da Silva, Bruno Marques; Monteiro, Fernando Pereira

    2012-01-01

    Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host × pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed. PMID:24031947

  20. Modular multiplication in GF(p) for public-key cryptography

    NASA Astrophysics Data System (ADS)

    Olszyna, Jakub

    Modular multiplication forms the basis of modular exponentiation which is the core operation of the RSA cryptosystem. It is also present in many other cryptographic algorithms including those based on ECC and HECC. Hence, an efficient implementation of PKC relies on efficient implementation of modular multiplication. The paper presents a survey of most common algorithms for modular multiplication along with hardware architectures especially suitable for cryptographic applications in energy constrained environments. The motivation for studying low-power and areaefficient modular multiplication algorithms comes from enabling public-key security for ultra-low power devices that can perform under constrained environments like wireless sensor networks. Serial architectures for GF(p) are analyzed and presented. Finally proposed architectures are verified and compared according to the amount of power dissipated throughout the operation.

  1. GFP-like fluorophores as DNA labels for studying DNA-protein interactions.

    PubMed

    Riedl, Jan; Ménová, Petra; Pohl, Radek; Orság, Petr; Fojta, Miroslav; Hocek, Michal

    2012-09-21

    GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.

  2. Quantitative GFP fluorescence as an indicator of arsenite developmental toxicity in mosaic heat shock protein 70 transgenic zebrafish

    SciTech Connect

    Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Kim, Dong-Jae; Na, Yi-Rang; Noh, Kyoung-Jin; Park, Sung-Hoon; Lee, Hyun-Kyoung; Lee, Byoung-Hee; Ryu, Doug-Young; Park, Jae-Hak

    2007-12-01

    In transgenic zebrafish (Danio rerio), green fluorescent protein (GFP) is a promising marker for environmental pollutants. In using GFP, one of the obstacles which we faced was how to compare toxicity among different toxicants or among a specific toxicant in different model species with the intensity of GFP expression. Using a fluorescence detection method, we first validated our method for estimating the amount of GFP fluorescence present in transgenic fish, which we used as an indicator of developmental toxicity caused by the well-known toxicant, arsenite. To this end, we developed mosaic transgenic zebrafish with the human heat shock response element (HSE) fused to the enhanced GFP (EGFP) reporter gene to indicate exposure to arsenite. We confirmed that EGFP expression sites correlate with gross morphological disruption caused by arsenite exposure. Arsenite (300.0 {mu}M) caused stronger EGFP fluorescence intensity and quantity than 50.0 {mu}M and 10.0 {mu}M arsenite in our transgenic zebrafish. Furthermore, arsenite-induced apoptosis was demonstrated by TUNEL assay. Apoptosis was inhibited by the antioxidant, N-acetyl-cystein (NAC) in this transgenic zebrafish. The distribution of TUNEL-positive cells in embryonic tissues was correlated with the sites of arsenite toxicity and EGFP expression. The EGFP values quantified using the standard curve equation from the known GFP quantity were consistent with the arsenite-induced EGFP expression pattern and arsenite concentration, indicating that this technique can be a reliable and applicable measurement. In conclusion, we propose that fluorescence-based EGFP quantification in transgenic fish containing the hsp70 promoter-EGFP reporter-gene construct is a useful indicator of development toxicity caused by arsenite.

  3. Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

    PubMed Central

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Uversky, Vladimir N.; Turoverov, Konstantin K.

    2016-01-01

    The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly mimicked by traditional physiological conditions, where low concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution containing high concentrations of various polymers that serve as model crowding agents. An expected outcome of the presence of such macromolecular crowding agents is their ability to increase conformational stability of a globular protein due to the excluded volume effects. In line with this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas the chemical nature of a macromolecular crowder should not play a role in its ability to modulate conformational properties. In this study, the effects of different crowding agents (polyethylene glycols (PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70) on the spectral properties and unfolding–refolding processes of the super-folder green fluorescent protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that, in addition to the expected excluded volume effects, there are some changes in the solvent properties. PMID:27801849

  4. An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins

    PubMed Central

    Ohashi, Tomoo; Galiacy, Stephane D.; Briscoe, Gina; Erickson, Harold P.

    2007-01-01

    We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of α-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66–68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor–acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied. PMID:17586775

  5. Coastal Louisiana Wetlands Restoration Monitoring with Global Fiducials Program (GFP) Imagery

    NASA Astrophysics Data System (ADS)

    Fisher, G.

    2012-12-01

    Coastal Louisiana has experienced dramatic landscape change over the past century due to human induced changes to the environment as well as an onslaught of major coastal storms. Coastal Louisiana loses on average 25-35 square miles of land per year. The USGS has partnered with the National Oceanographic and Atmospheric Administration (NOAA) - National Marine Fisheries Service to provide cyclical remote sensing data for selected restoration sites along the coast of Louisiana. Three of these sites are actively maintained in the GFP archive - Atchafalaya River Delta, East Timbalier Island, and Pecan Island. These three sites coincide with NOAA restoration sites that have been monitored since early 2000. The GFP has provided a consistent set of remote sensing data that has greatly benefited the long-term monitoring of these restoration sites. Long-term monitoring of these sites includes both pre- and post-hurricane season data collection used to identify landscape change along the coast. The long-term monitoring also has helped to identify areas of success in the restoration projects, as well as areas that have continued to decline in spite of restoration efforts. These three sites are significant to the program because they provide a variety of coastal landscape types: an open water barrier island environment at East Timbalier Island; coastal wetlands at Pecan Island, which have experienced subsidence of the marsh and convergence to an open water environment; and a deltaic marsh environment at Atchafalaya River Delta. Long-term monitoring of these sites has provided a wealth of knowledge about the changes occurring, as well as a valuable tool for reliable shoreline measurements. Continued monitoring is necessary to accurately assess the condition of these areas as environmental conditions continue to shape the landscape.

  6. Enhancers of Agrobacterium-mediated Transformation of Tibouchina semidecandra Selected on the Basis of GFP Expression.

    PubMed

    Yong, Wilson Thau Lym; Henry, Erle Stanley; Abdullah, Janna Ong

    2010-12-01

    Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 μM galactose and 100 μM tyrosine supplemented with 600 μM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 μM galactose and 50 μM tyrosine with 200 μM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers.

  7. Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice.

    PubMed

    Chen, Mei-Shu; Lin, Hua-Kuo; Chiu, Hsun; Lee, Don-Ching; Chung, Yu-Fen; Chiu, Ing-Ming

    2015-03-01

    FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B-GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (-540 to +31). Using the fresh brain sections of F1B-GFP transgenic mice, we found that the F1B-GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B-GFP(+) cells existed in the brains of F1B-GFP transgenic mice. We demonstrated that one population of F1B-GFP(+) cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B-GFP(+) cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B-GFP(+) cells and tyrosine hydroxylase indicated that a subpopulation of F1B-GFP(+) -neuronal cells was dopaminergic neurons. Importantly, these F1B-GFP(+) /TH(+) cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B-GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease.

  8. Flight muscle-specific expression of act88F: GFP in transgenic Culex quinquefasciatus Say (Diptera: Culicidae).

    PubMed

    Allen, Margaret L; Christensen, Bruce M

    2004-12-01

    A strategy to engineer a strain of Culex mosquitoes refractory to filarial transmission is described. A requirement for success of the strategy is identification of a flight muscle-specific promoter that functions in the mosquito. A GFP marker gene under the control of the promoter region of the Drosophila melanogaster act88F gene was inserted into the genome of Culex quinquefasciatus. Transformation was confirmed by Mendelian genetics. Hybridization of a genomic Southern blot to a radiolabeled probe verified that the entire donor plasmid integrated into the mosquito genome. GFP expression in the transgenic mosquitoes was restricted to the flight muscles.

  9. BDNF-GFP containing secretory granules are localized in the vicinity of synaptic junctions of cultured cortical neurons.

    PubMed

    Haubensak, W; Narz, F; Heumann, R; Lessmann, V

    1998-06-01

    The protein family of mammalian neurotrophins, comprising nerve-growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and -4/5 (NT-3, NT-4/5), supports the survival and the phenotype of neurons from the central as well as the peripheral nervous system (CNS, PNS). In addition, exogenous application of neurotrophins has recently been found to modulate synaptic transmission in the rodent CNS. However, to provide evidence for a role of neurotophins as endogenous fast acting modulators of synaptic transmission, the synaptic localization and secretion of neurotrophins needs to be shown. We have now constructed a fusion protein consisting of N-terminal BDNF (the most abundant neurotrophin in the rodent hippocampus and neocortex) and C-terminal green fluorescent protein (GFP) to elucidate the cellular localization of BDNF in cortical neurons. Transient expression of BDNF-GFP in COS-7 cells revealed that the cellular localization in the trans-Golgi network (TGN), the processing of precursor proteins and the secretion of mature BDNF-GFP is indistinguishable from the properties of untagged BDNF. Upon transient transfection of primary rat cortical neurons, BDNF-GFP was found in secretory granules of the regulated pathway of secretion, as indicated by colocalization with the secretory granule marker secretogranin II. BDNF-GFP vesicles were found in the neurites of transfected neurons with a pattern reminiscent of the localization of endogenous BDNF in untransfected cortical neurons. BDNF-GFP vesicles were found predominantly in the somatodendritic compartment of the neurons, whereas additional axonal localization was found less frequently. Immunocytochemical staining of synaptic terminals with synapsin I antibodies revealed that the density of BDNF-GFP vesicles is elevated in the vicinity of synaptic junctions, indicating that BDNF is localized appropriately to function as an acute modulator of synaptic transmission. These data suggest that BDNF-GFP will

  10. Problems encountered in bicistronic IRES-GFP expression vectors employed in functional analyses of GC-induced genes.

    PubMed

    Mansha, Muhammad; Wasim, Muhammad; Ploner, Christian; Hussain, Abrar; Latif, Asma Abdul; Tariq, Muhammad; Kofler, Anita

    2012-12-01

    Our laboratory has developed a series of Gateway(®) compatible lentiviral expression systems for constitutive and conditional gene knock-down and over-expression. For tetracycline-regulated transgenic expression, we constructed a lentiviral "DEST" plasmid (pHR-TetCMV-Dest-IRES-GFP5) containing a tetracycline-responsive minimal CMV promoter, followed by an attP site-flanked DEST cassette (for efficient cloning of cDNAs by "Gateway(®)" recombination cloning) and green fluorescent protein (GFP) driven by an internal ribosomal entry site (IRES).This lentiviral bicistronic plasmid allows immediate FACS identification and characterization of successfully transfected cell lines. Although this system worked well with several cDNAs, we experienced serious problems with SLA, Bam and BMF. Particularly, we cloned the cDNA for human SLA (Src-like adapter), a candidate gene in GC-induced apoptosis, into this plasmid. The resulting construct (pHR-TetCMV-SLA-IRES-GFP5) was transfected into HEK 293-T packaging cells to produce viral particles for transduction of CEM-C7H2-2C8 cells. Although the construct produced many green fluorescent colonies at the HEK 293-T and the CEM-C7H2-2C8 level, we could not detect any SLA protein with α-SLA antibody from corresponding cell lysates. In contrast, the antibody readily detected SLA in whole cell lysate of HEK 293-T cells transfected with a GST-flagged SLA construct lacking IRES-GFP. To directly address the potential role of the IRES-GFP sequence, we cloned the SLA coding region into pHR-TetCMV-Dest, a vector that differs from pHR-TetCMV-Dest-IRES-GFP5 just by the absence of the IRES-GFP cassette. The resulting pHR-TetCMV-SLA construct was used for transfection of HEK 293-T cells. Corresponding lysates were assayed with α-SLA antibody and found positive. These data, in concert with previous findings, suggest that the IRES-GFP cassette may interfere with translation of certain smaller size cDNAs (like SLA) or generate fusion proteins and

  11. Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS)

    PubMed Central

    Hendriks, William T.; Bros-Facer, Virginie; van Minnen, Jan; Martin, Joanne E.; Jackson, Graham S.; Greensmith, Linda; Schiavo, Giampietro; Fisher, Elizabeth M. C.

    2010-01-01

    Background Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts. Methodology/Principal Findings Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP. Conclusions/Significance Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag. PMID:20221404

  12. Tracking Monocyte Recruitment and Macrophage Accumulation in Atherosclerotic Plaque Progression Using a Novel hCD68GFP/ApoE−/− Reporter Mouse—Brief Report

    PubMed Central

    Iqbal, Asif J.; Jones, Daniel; Patel, Jyoti; Coutinho, Patricia; Taylor, Lewis; Greaves, David R.; Channon, Keith M.

    2017-01-01

    Objective— To create a model of atherosclerosis using green fluorescent protein (GFP)–targeted monocytes/macrophages, allowing analysis of both endogenous GFP+ and adoptively transferred GFP+ myeloid cells in arterial inflammation. Approach and Results— hCD68GFP reporter mice were crossed with ApoE−/− mice. Expression of GFP was localized to macrophages in atherosclerotic plaques and in angiotensin II–induced aortic aneurysms and correlated with galectin 3 and mCD68 expression. Flow cytometry confirmed GFP+ expression in CD11b+/CD64+, CD11c+/MHC-IIHI, and CD11b+/F4/80+ myeloid cells. Adoptive transfer of GFP+ monocytes demonstrated monocyte recruitment to both adventitia and atherosclerotic plaque, throughout the aortic root, within 72 hours. We demonstrated the biological utility of hCD68GFP monocytes by comparing the recruitment of wild-type and CCR2−/− monocytes to sites of inflammation. Conclusions— hCD68GFP/ApoE−/− mice provide a new approach to study macrophage accumulation in atherosclerotic plaque progression and to identify cells recruited from adoptively transferred monocytes. PMID:27908893

  13. The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

    PubMed

    Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

    2013-03-01

    Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

  14. Was Lates late? A null model for the Nile perch boom in Lake Victoria.

    PubMed

    Downing, Andrea S; Galic, Nika; Goudswaard, Kees P C; van Nes, Egbert H; Scheffer, Marten; Witte, Frans; Mooij, Wolf M

    2013-01-01

    Nile perch (Lates niloticus) suddenly invaded Lake Victoria between 1979 and 1987, 25 years after its introduction in the Ugandan side of the lake. Nile perch then replaced the native fish diversity and irreversibly altered the ecosystem and its role to lakeshore societies: it is now a prised export product that supports millions of livelihoods. The delay in the Nile perch boom led to a hunt for triggers of the sudden boom and generated several hypotheses regarding its growth at low abundances--all hypotheses having important implications for the management of Nile perch stocks. We use logistic growth as a parsimonious null model to predict when the Nile perch invasion should have been expected, given its growth rate, initial stock size and introduction year. We find the first exponential growth phase can explain the timing of the perch boom at the scale of Lake Victoria, suggesting that complex mechanisms are not necessary to explain the Nile perch invasion or its timing. However, the boom started in Kenya before Uganda, indicating perhaps that Allee effects act at smaller scales than that of the whole Lake. The Nile perch invasion of other lakes indicates that habitat differences may also have an effect on invasion success. Our results suggest there is probably no single management strategy applicable to the whole lake that would lead to both efficient and sustainable exploitation of its resources.

  15. Was Lates Late? A Null Model for the Nile Perch Boom in Lake Victoria

    PubMed Central

    Downing, Andrea S.; Galic, Nika; Goudswaard, Kees P. C.; van Nes, Egbert H.; Scheffer, Marten; Witte, Frans; Mooij, Wolf M.

    2013-01-01

    Nile perch (Lates niloticus) suddenly invaded Lake Victoria between 1979 and 1987, 25 years after its introduction in the Ugandan side of the lake. Nile perch then replaced the native fish diversity and irreversibly altered the ecosystem and its role to lakeshore societies: it is now a prised export product that supports millions of livelihoods. The delay in the Nile perch boom led to a hunt for triggers of the sudden boom and generated several hypotheses regarding its growth at low abundances – all hypotheses having important implications for the management of Nile perch stocks. We use logistic growth as a parsimonious null model to predict when the Nile perch invasion should have been expected, given its growth rate, initial stock size and introduction year. We find the first exponential growth phase can explain the timing of the perch boom at the scale of Lake Victoria, suggesting that complex mechanisms are not necessary to explain the Nile perch invasion or its timing. However, the boom started in Kenya before Uganda, indicating perhaps that Allee effects act at smaller scales than that of the whole Lake. The Nile perch invasion of other lakes indicates that habitat differences may also have an effect on invasion success. Our results suggest there is probably no single management strategy applicable to the whole lake that would lead to both efficient and sustainable exploitation of its resources. PMID:24204684

  16. Stretching out the Australasian microtektite strewn field in Victoria Land Transantarctic Mountains

    NASA Astrophysics Data System (ADS)

    Folco, Luigi; D'Orazio, Massimo; Gemelli, Maurizio; Rochette, Pierre

    2016-06-01

    Petrographic and geochemical studies of microtektites collected in newly explored summit plateaus of the Transantarctic Mountains (i.e., Schroeder Spur, Killer Nunatak, Miller Butte in the inland catchment of the Rennick Glacier, and Allan Hills, in the inland catchment of the Mackay-David Glaciers) document a regional distribution of Australasian microtektites in Victoria Land. A geochemical comparison with Australasian microtektites from deep sea sediments at lower latitudes identifies a possible projectile geochemical signature for the first time, and confirms that Transantarctic Mountains microtektites experienced higher thermal regimes. Ballistic calculations reveal that the extraordinary distance of the Transantarctic Mountains microtektites from the hypothetical impact location in Indochina (∼11,000 km) could be more efficiently attained at relatively low ejection angles (20°-40°). Finally, the occurrence of Australasian microtektites (∼0.8 Ma old) on specific glacial surfaces of the Antarctic bedrock constrains the glacial history of the East Antarctic Ice Sheet in Victoria Land. In particular, data from Allan Hills supports a glaciological scenario envisaging an extremely stable East Antarctic Ice Sheet over at least the last ∼1 Ma in the inland catchment of the Mackay/David glaciers. This is consistent with the large accumulation of meteorites in the adjacent blue ice fields.

  17. Getting that piece of paper: mental health nurses' experience of undertaking doctoral studies in Victoria, Australia.

    PubMed

    Welch, Tony; Happell, Brenda; Edward, Karen-Leigh

    2010-06-01

    The aim of this study was to explore the experience of mental health nurses undertaking doctoral studies. The study was conducted in Victoria, Australia. A descriptive-exploratory approach to inquiry was used for this study. Participants were mental health nurses who had successfully completed a doctoral qualification. Eligibility for inclusion required participants to be residing in Victoria (irrespective of where their doctoral studies were undertaken) and to have conducted their research within the domain of mental health and/or currently employed in the field of mental health nursing. Of the 20 potential participants invited, 16 accepted the invitation. Five emergent themes were explicated from narrative analyses. These themes were "being a trail blazer," "positioning for professional advancement," "achieving a balance between competing priorities," "maintaining a commitment to the development of the profession," and "a point of affirmation." An understanding of the experience of undertaking doctoral studies can be used to influence the development of strategies to encourage more mental health nurses to consider undertaking a doctoral degree.

  18. The diversity of male nuptial coloration leads to species diversity in Lake Victoria cichlids.

    PubMed

    Miyagi, Ryutaro; Terai, Yohey

    2013-01-01

    The amazing coloration shown by diverse cichlid fish not only fascinates aquarium keepers, but also receives great attention from biologists interested in speciation because of its recently-revealed role in their adaptive radiation in an African lake. We review the important role of coloration in the speciation and adaptive evolution of Lake Victoria cichlids, which have experienced adaptive radiation during a very short evolutionary period. Mature male cichlids display their colors during mate choice. The color of their skin reflects light, and the reflected light forms a color signal that is received by the visual system of females. The adaptive divergence of visual perceptions shapes and diverges colorations, to match the adapted visual perceptions. The divergence of visual perception and coloration indicates that the divergence of color signals causes reproductive isolation between species, and this process leads to speciation. Differences in color signals among coexisting species act to maintain reproductive isolation by preventing hybridization. Thus, the diversity of coloration has caused speciation and has maintained species diversity in Lake Victoria cichlids.

  19. Sunspots, El Niño, and the levels of Lake Victoria, East Africa

    NASA Astrophysics Data System (ADS)

    Stager, J. Curt; Ruzmaikin, Alexander; Conway, Declan; Verburg, Piet; Mason, Peter J.

    2007-08-01

    An association of high sunspot numbers with rises in the level of Lake Victoria, East Africa, has been the focus of many investigations and vigorous debate during the last century. In this paper, we show that peaks in the ~11-year sunspot cycle were accompanied by Victoria level maxima throughout the 20th century, due to the occurrence of positive rainfall anomalies ~1 year before solar maxima. Similar patterns also occurred in at least five other East African lakes, which indicates that these sunspot-rainfall relationships were broadly regional in scale. Although irradiance fluctuations associated with the sunspot cycle are weak, their effects on tropical rainfall could be amplified through interactions with sea surface temperatures and atmospheric circulation systems, including ENSO. If this Sun-rainfall relationship persists in the future, then sunspot cycles can be used for long-term prediction of precipitation anomalies and associated outbreaks of insect-borne disease in much of East Africa. In that case, unusually wet rainy seasons and Rift Valley Fever epidemics should occur a year or so before the next solar maximum, which is expected to occur in 2011-2012 AD.

  20. Risk factors for severe injury in cyclists involved in traffic crashes in Victoria, Australia.

    PubMed

    Boufous, Soufiane; de Rome, Liz; Senserrick, Teresa; Ivers, Rebecca

    2012-11-01

    This study examines the impact of cyclist, road and crash characteristics on the injury severity of cyclists involved in traffic crashes reported to the police in Victoria, Australia between 2004 and 2008. Logistic regression analysis was carried out to identify predictors of severe injury (serious injury and fatality) in cyclist crashes reported to the police. There were 6432 cyclist crashes reported to the police in Victoria between 2004 and 2008 with 2181 (33.9%) resulting in severe injury of the cyclist involved. The multivariate analysis found that factors that increase the risk of severe injury in cyclists involved in traffic crashes were age (50 years and older), not wearing a helmet, riding in the dark on unlit roads, riding on roads zoned 70 km/h or above, on curved sections of the road, in rural locations and being involved in head-on collisions as well as off path crashes, which include losing control of vehicle, and on path crashes which include striking the door of a parked vehicle. While this study did not test effectiveness of preventative measures, policy makers should consider implementation of programs that address these risk factors including helmet programs and environmental modifications such as speed reduction on roads that are frequented by cyclists.