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41.
Analysis of mannose selection used for transformation of sugar beet 总被引:39,自引:0,他引:39
Joersbo Morten Donaldson Iain Kreiberg Jette Petersen Steen Guldager Brunstedt Janne Okkels Finn T. 《Molecular breeding : new strategies in plant improvement》1998,4(2):111-117
Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed. 相似文献
42.
Paul W. D'Alvise Jette Melchiorsen Cisse H. Porsby Kristian F. Nielsen Lone Gram 《Applied and environmental microbiology》2010,76(7):2366-2370
The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (107 CFU/cm2) resulted in significant reduction or complete killing of the pathogen inoculated at 102 to 104 CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces. 相似文献
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44.
Juan López Gejo Narayanapillai Manoj Sarojiniamma Sumalekshmy Hartmut Glieman Thomas Schimmel Michael W?rner André M Braun 《Photochemical & photobiological sciences》2006,5(10):948-954
Vacuum-ultraviolet (VUV) irradiation (lambda(exc): 172 +/- 12 nm) of polystyrene films in the presence of oxygen produced not only oxidatively functionalized surfaces, but generated also morphological changes. Whereas OH- and C[double bond, length as m-dash]O-functionalized surfaces might be used for e.g. secondary functionalization, enhanced aggregation or printing, processes leading to morphological changes open new possibilities of microstructurization. Series of experiments made under different experimental conditions brought evidence of two different reaction pathways: introduction of OH- and C[double bond, length as m-dash]O-groups at the polystyrene pathways is mainly due to the reaction of reactive oxygen species (hydroxyl radicals, atomic oxygen, ozone) produced in the gas phase between the VUV-radiation source and the substrate. However, oxidative fragmentation leading to morphological changes, oxidation products of low molecular weight and eventually to mineralization of the organic substrate is initiated by electronic excitation of the polymer leading to C-C-bond homolysis and to a complex oxidation manifold after trapping of the C-centred radicals by molecular oxygen. The pathways of oxidative functionalization or fragmentation could be differentiated by FTIR-ATR analysis of irradiated polystyrene surfaces before and after washing with acetonitrile and microscopic fluorescence analysis of the surfaces secondarily functionalized with the N,N,N-tridodecyl-triaza-triangulenium (TATA) cation. Ozonization of the polystyrene leads to oxidative functionalization of the polymer surface but cannot initiate the fragmentation of the polymer backbone. Oxidative fragmentation is initiated by electronic excitation of the polymer (contact-mode AFM analysis), and evidence of the generation of intermediate C-centred radicals is given e.g. by experiments in the absence of oxygen leading to cross-linking (solubility effects, optical microscopy, friction-mode AFM) and disproportionation (fluorescence). 相似文献
45.
Tyrosine O-sulfation is a posttranslational modification of secretory and membrane proteins transported through the Golgi apparatus, which is widespread among higher eukaryotes. O-Sulfated tyrosines are not immediately identified during sequencing of peptides and proteins, because the sulfate ester is acid labile and rapidly hydrolyses to tyrosine in strong acidic solutions. Little is known about the hydrolysis at mildly acidic solutions, which are used during several protein purification and analysis procedures. We have examined the stability of tyrosine sulfate using sulfated gastrin-17, caerulein, and drosulfokinin as models for tyrosine O-sulfated peptides. The peptides were incubated in acidic solutions in a pH range of 1 to 3 at different temperatures and time spans. Only marginal hydrolysis of gastrin-17 was observed in triflouroacetic acid at room temperature or below. Comparison of the acid hydrolysis of the three peptides showed that hydrolysis rate depends mainly on the primary amino acid composition of the peptide. The activation energy (E(a)) for the hydrolysis of sulfated gastrin-17 was found to be E(a)=98.7+/-5 kJ mol(-1). This study serves as a general reference for handling tyrosine sulfated peptides in aqueous acidic solutions. We conclude that tyrosine sulfate is more stable under normal protein purification conditions than previously assumed. 相似文献
46.
Hald H Naur P Pickering DS Sprogøe D Madsen U Timmermann DB Ahring PK Liljefors T Schousboe A Egebjerg J Gajhede M Kastrup JS 《The Journal of biological chemistry》2007,282(35):25726-25736
More than 50 structures have been reported on the ligand-binding core of the ionotropic glutamate receptor iGluR2 that belongs to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid-type of receptors. In contrast, the ligand-binding core of the kainic acid-type receptor iGluR5 has only been crystallized with three different ligands. Hence, additional structures of iGluR5 are needed to broaden the understanding of the ligand-binding properties of iGluR5, and the conformational changes leading to channel opening and closing. Here, we present two structures of the ligand-binding core of iGluR5; one as a complex with the partial agonist (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)-5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid (domoic acid) and one as a complex with the antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid ((S)-ATPO). In agreement with the partial agonist activity of domoic acid, the ligand-binding core of the iGluR5 complex is stabilized by domoic acid in a conformation that is 11 degrees more open than the conformation observed in the full agonist (S)-glutamic acid complex. This is primarily caused by the 5-carboxy-1-methyl-hexa-1,3-dienyl moiety of domoic acid and residues Val685-Thr690 of iGluR5. An even larger domain opening of 28 degrees is introduced upon binding of the antagonist (S)-ATPO. It appears that the span of domain opening is much larger in the ligand-binding core of iGluR5 (30 degrees) compared with what has been observed in iGluR2 (19 degrees ). Similarly, much larger variation in the distances between transmembrane linker residues in the two protomers comprising the dimer is observed in iGluR5 as compared with iGluR2. 相似文献
47.
Functional and crystal structure analysis of active site adaptations of a potent anti-angiogenic human tRNA synthetase 总被引:1,自引:0,他引:1
Yang XL Guo M Kapoor M Ewalt KL Otero FJ Skene RJ McRee DE Schimmel P 《Structure (London, England : 1993)》2007,15(7):793-805
Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion. 相似文献
48.
49.
Nexø BA Christensen T Frederiksen J Møller-Larsen A Oturai AB Villesen P Hansen B Nissen KK Laska MJ Petersen TS Bonnesen S Hedemand A Wu T Wang X Zhang X Brudek T Maric R Søndergaard HB Sellebjerg F Brusgaard K Kjeldbjerg AL Rasmussen HB Nielsen AL Nyegaard M Petersen T Børglum AD Pedersen FS 《PloS one》2011,6(2):e16652
We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis. 相似文献
50.
Nielsen BB Pickering DS Greenwood JR Brehm L Gajhede M Schousboe A Kastrup JS 《The FEBS journal》2005,272(7):1639-1648
The X-ray structure of the ionotropic GluR2 ligand-binding core (GluR2-S1S2J) in complex with the bicyclical AMPA analogue (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid [(S)-4-AHCP] has been determined, as well as the binding pharmacology of this construct and of the full-length GluR2 receptor. (S)-4-AHCP binds with a glutamate-like binding mode and the ligand adopts two different conformations. The K(i) of (S)-4-AHCP at GluR2-S1S2J was determined to be 185 +/- 29 nM and at full-length GluR2(R)o it was 175 +/- 8 nM. (S)-4-AHCP appears to elicit partial agonism at GluR2 by inducing an intermediate degree of domain closure (17 degrees). Also, functionally (S)-4-AHCP has an efficacy of 0.38 at GluR2(Q)i, relative to (S)-glutamate. The proximity of bound (S)-4-AHCP to domain D2 prevents full D1-D2 domain closure, which is limited by steric repulsion, especially between Leu704 and the ligand. 相似文献