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981.
Kamila Pszon-Bartosz Jesper S. Hansen Karin B. Stibius Jesper S. Groth Jenny Emnéus Oliver Geschke Claus Hélix-Nielsen 《Biochemical and biophysical research communications》2011,406(1):96
Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 105 FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm2 within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications. 相似文献
982.
Athenstaedt K 《Biochimica et biophysica acta》2011,1811(10):587-596
The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylglycerol accumulation. Most interestingly, when oleic acid serves as the carbon source the ratio of triacylglycerol accumulation in mutants to wild-type is significantly increased in strains defective in DGA1 but not in lro1Δ. In vitro experiments revealed that under these conditions an additional acyl-CoA dependent triacylglycerol synthase contributes to triacylglycerol synthesis in the respective mutants. Taken together, evidence is provided that Yarrowia lipolytica contains at least four triacylglycerol synthases, namely Lro1p, Dga1p and two additional triacylglycerol synthases whereof one is acyl-CoA dependent and specifically induced upon growth on oleic acid. 相似文献
983.
The interaction of Pseudomonas aeruginosa with surfaces has been described as a two-stage process requiring distinct signaling events and the reciprocal modulation of small RNAs (sRNAs). However, little is known regarding the relationship between sRNA-modulating pathways active under planktonic or surface-associated growth conditions. Here, we demonstrate that SagS (PA2824), the cognate sensor of HptB, links sRNA-modulating activities via the Gac/HptB/Rsm system postattachment to the signal transduction network BfiSR, previously demonstrated to be required for the development of P. aeruginosa. Consistent with the role of SagS in the GacA-dependent HtpB signaling pathway, inactivation of sagS resulted in hyperattachment, an HptB-dependent increase in rsmYZ, increased Psl polysaccharide production, and increased virulence. Moreover, sagS inactivation rescued attachment but abrogated biofilm formation by the ΔgacA and ΔhptB mutant strains. The ΔsagS strain was impaired in biofilm formation at a stage similar to that of the previously described two-component system BfiSR. Expression of bfiR but not bfiS restored ΔsagS biofilm formation independently of rsmYZ. We demonstrate that SagS interacts directly with BfiS and only indirectly with BfiR, with the direct and specific interaction between these two membrane-bound sensors resulting in the modulation of the phosphorylation state of BfiS in a growth-mode-dependent manner. SagS plays an important role in P. aeruginosa virulence in a manner opposite to that of BfiS. Our findings indicate that SagS acts as a switch by linking the GacA-dependent sensory system under planktonic conditions to the suppression of sRNAs postattachment and to BfiSR, required for the development of P. aeruginosa biofilms, in a sequential and stage-specific manner. 相似文献
984.
985.
Xu S Schlüter PM Scopece G Breitkopf H Gross K Cozzolino S Schiestl FP 《Evolution; international journal of organic evolution》2011,65(9):2606-2620
Floral isolation is an important component of pollinator-driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators' mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F(1) hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination. 相似文献
986.
Bakker HC Switt AI Cummings CA Hoelzer K Degoricija L Rodriguez-Rivera LD Wright EM Fang R Davis M Root T Schoonmaker-Bopp D Musser KA Villamil E Waechter H Kornstein L Furtado MR Wiedmann M 《Applied and environmental microbiology》2011,77(24):8648-8655
In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods. 相似文献
987.
Short guanine-rich sequences have a tendency to form quadruplexes that are stabilized by G-quartets with specific cation coordination. Quadruplexes are part of telomeres at the ends of chromosomes and play an important role in the regulation of gene expression. In addition, there is a strong interest in the therapeutic and biotechnological potential of quadruplex oligonucleotides. The HIV-integrase aptamer, d(GGGT)(4), demonstrated unusually favorable van't Hoff thermodynamics, and based on NMR studies the aptamer was proposed to fold into an antiparallel structure. Here we probed an apparent discrepancy between the NMR structure and the quadruplex topology suggested by circular dichroism (CD). Systematic thermodynamic analyses of d(GGGT)(4) and variants containing sequence modifications or missing specific nucleotides are consistent with a parallel quadruplex fold. CD studies carried out over a wide concentration range did not support a possible structural transition upon increasing strand concentration. Taken together, both optical and thermodynamic studies performed here strongly support a parallel fold for the d(GGGT)(4) aptamer. 相似文献
988.
Beich-Frandsen M Vecerek B Konarev PV Sjöblom B Kloiber K Hämmerle H Rajkowitsch L Miles AJ Kontaxis G Wallace BA Svergun DI Konrat R Bläsi U Djinovic-Carugo K 《Nucleic acids research》2011,39(11):4900-4915
The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec). 相似文献
989.
Dohnt K Sauer M Müller M Atallah K Weidemann M Gronemeyer P Rasch D Tielen P Krull R 《Journal of microbiological methods》2011,87(3):302-308
Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min-1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation. 相似文献
990.
Hayashi AA Webb J Choi J Baker C Lino M Trigatti B Trajcevski KE Hawke TJ Adeli K 《American journal of physiology. Gastrointestinal and liver physiology》2011,301(2):G326-G337
Intestinal lipid dysregulation is a common feature of insulin-resistant states. The present study investigated alterations in gene expression of key proteins involved in the active absorption of dietary fat and cholesterol in response to development of insulin resistance. Studies were conducted in two diet-induced animal models of insulin resistance: fructose-fed hamster and high-fat-fed mouse. Changes in the mRNA abundance of lipid transporters, adenosine triphosphate cassette (ABC) G5, ABCG8, FA-CoA ligase fatty acid translocase P4, Niemann-Pick C1-Like1 (NPC1L1), fatty acid transport protein 4 (FATP4), and Scavenger Receptor Class B Type I (SR-BI), were assessed in intestinal fragments (duodenum, jejunum, and ileum) using quantitative real-time PCR. Of all the transporters evaluated, SR-B1 showed the most significant changes in both animal models examined. A marked stimulation of SR-B1 expression was observed in all intestinal segments examined in both insulin-resistant animal models. The link between SR-BI expression and intestinal lipoprotein production was then examined in the Caco-2 cell model. SR-B1 overexpression in Caco-2 cells increased apolipoprotein B (apoB) 100 and apoB48 secretion, whereas RNAi knock down of SR-B1 decreased secretion of both apoB100 and apoB48. We also observed changes in subcellular distribution of SR-B1 in response to exogenous lipid and insulin. Confocal microscopy revealed marked changes in SR-BI subcellular distribution in response to both exogenous lipids (oleate) and insulin. In summary, marked stimulation of intestinal SR-BI occurs in vivo in animal models of diet-induced insulin resistance, and modulation of SR-BI in vitro regulates production of apoB-containing lipoprotein particles. We postulate that apical and/or basolateral SR-BI may play an important role in intestinal chylomicron production and may contribute to chylomicron overproduction normally observed in insulin-resistant states. 相似文献