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61.
62.
A screen for dominant negative mutants of SEC18 reveals a role for the AAA protein consensus sequence in ATP hydrolysis
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An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro). Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. 相似文献
63.
Yonemitsu Y Kitson C Ferrari S Farley R Griesenbach U Judd D Steel R Scheid P Zhu J Jeffery PK Kato A Hasan MK Nagai Y Masaki I Fukumura M Hasegawa M Geddes DM Alton EW 《Nature biotechnology》2000,18(9):970-973
Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer. 相似文献
64.
Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly 总被引:8,自引:0,他引:8
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This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5' hexanethiol anchoring group with that of unfunctionalized oligonucleotides for lengths from 8 to 48 bases. Qualitatively, the results indicate that the thiol anchoring group strongly enhances oligonucleotide immobilization, but that the enhancement is reduced for longer strand lengths. Interestingly, examination of the probe coverage as a function of strand length suggests that adsorbed thiol-ssDNA oligonucleotides shorter than 24 bases tend to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. For strands longer than 24 bases, the surface coverage begins to decrease notably with probe length. The decrease is consistent with a less ordered arrangement of the DNA chains, presumably reflecting increasingly polymeric behavior. 相似文献
65.
Extended Survival and Persistence of Campylobacter spp. in Water and Aquatic Biofilms and Their Detection by Immunofluorescent-Antibody and -rRNA Staining 总被引:4,自引:0,他引:4
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Clive M. Buswell Yvonne M. Herlihy Lorna M. Lawrence James T. M. McGuiggan Philip D. Marsh C. William Keevil Steve A. Leach 《Applied microbiology》1998,64(2):733-741
In water microcosm experiments, the survival times of Campylobacter isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10°C (202 and 176 h, respectively) than at 22 and 37°C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora, Campylobacter isolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4°C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30°C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4°C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with a Campylobacter-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms. 相似文献
66.
Abstract. 1. This study evaluated the effect of dispersal on the density and feeding impact of a phytophagous insect in relation to the spatial distribution of its host plants.
2. The interaction between density, dispersal, and feeding impact of western flower thrips on flowering chrysanthemum was quantified at three spatial scales, with infested and uninfested plants either isolated in 0.25 m2 individual cages, or enclosed together in 2.25 m2 communal cages or 75 m2 greenhouses.
3. In individual cages, the rate of dispersal from chrysanthemum plants to blue sticky traps increased with the density of thrips for females but not males. Uninfested plants consistently had fewer thrips when they were individually caged rather than enclosed with plants infested with adults, indicating that dispersal mediates inter-plant distribution of thrips.
4. The feeding impact of thrips on inflorescences was evaluated using the absorbance of ethanol extracts at wavelengths characteristic of yellow carotenoid pigments associated with chrysanthemum inflorescences (415, 445, and 472 nm). Increasing absorbance of extracts with increasing density of thrips per inflorescence suggests that feeding by thrips results in ruptured cells leaching carotenoid pigments.
5. In communal cages, the distribution of thrips was uniform for infested and uninfested plants, whereas the density and feeding impact of thrips in greenhouses were higher for infested than uninfested plants. These results suggest that short-range dispersal by adults homogenises the density and feeding impact of thrips among host plants only on a small spatial scale. 相似文献
2. The interaction between density, dispersal, and feeding impact of western flower thrips on flowering chrysanthemum was quantified at three spatial scales, with infested and uninfested plants either isolated in 0.25 m
3. In individual cages, the rate of dispersal from chrysanthemum plants to blue sticky traps increased with the density of thrips for females but not males. Uninfested plants consistently had fewer thrips when they were individually caged rather than enclosed with plants infested with adults, indicating that dispersal mediates inter-plant distribution of thrips.
4. The feeding impact of thrips on inflorescences was evaluated using the absorbance of ethanol extracts at wavelengths characteristic of yellow carotenoid pigments associated with chrysanthemum inflorescences (415, 445, and 472 nm). Increasing absorbance of extracts with increasing density of thrips per inflorescence suggests that feeding by thrips results in ruptured cells leaching carotenoid pigments.
5. In communal cages, the distribution of thrips was uniform for infested and uninfested plants, whereas the density and feeding impact of thrips in greenhouses were higher for infested than uninfested plants. These results suggest that short-range dispersal by adults homogenises the density and feeding impact of thrips among host plants only on a small spatial scale. 相似文献
67.
Gloria Omosa-Manyonyi Juliet Mpendo Eugene Ruzagira William Kilembe Elwyn Chomba Fran?ois Roman Patricia Bourguignon Marguerite Koutsoukos Alix Collard Gerald Voss Dagna Laufer Gwynn Stevens Peter Hayes Lorna Clark Emmanuel Cormier Len Dally Burc Barin Jim Ackland Kristen Syvertsen Devika Zachariah Kamaal Anas Eddy Sayeed Angela Lombardo Jill Gilmour Josephine Cox Patricia Fast Frances Priddy 《PloS one》2015,10(5)
Background
Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses.Methods
In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01E or F4/AS01B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01B; or three co-administrations of Ad35-GRIN and F4/AS01B. T cell and antibody responses were measured.Results
The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.Conclusion
Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.Trial Registration
ClinicalTrials.gov NCT01264445 相似文献68.
Previous studies have suggested that negative feedback is more effective in driving learning than positive feedback. We investigated the effect on learning of providing varying amounts of negative and positive feedback while listeners attempted to discriminate between three identical tones; an impossible task that nevertheless produces robust learning. Four feedback conditions were compared during training: 90% positive feedback or 10% negative feedback informed the participants that they were doing equally well, while 10% positive or 90% negative feedback informed them they were doing equally badly. In all conditions the feedback was random in relation to the listeners’ responses (because the task was to discriminate three identical tones), yet both the valence (negative vs. positive) and the probability of feedback (10% vs. 90%) affected learning. Feedback that informed listeners they were doing badly resulted in better post-training performance than feedback that informed them they were doing well, independent of valence. In addition, positive feedback during training resulted in better post-training performance than negative feedback, but only positive feedback indicating listeners were doing badly on the task resulted in learning. As we have previously speculated, feedback that better reflected the difficulty of the task was more effective in driving learning than feedback that suggested performance was better than it should have been given perceived task difficulty. But contrary to expectations, positive feedback was more effective than negative feedback in driving learning. Feedback thus had two separable effects on learning: feedback valence affected motivation on a subjectively difficult task, and learning occurred only when feedback probability reflected the subjective difficulty. To optimize learning, training programs need to take into consideration both feedback valence and probability. 相似文献
69.
Mette Nyegaard Nanna D. Rendtorff Morten S. Nielsen Thomas J. Corydon Ditte Demontis Anna Starnawska Anne Hedemand Annalisa Buniello Francesco Niola Michael T. Overgaard Suzanne M. Leal Wasim Ahmad Friedrik P. Wikman Kirsten B. Petersen Dorthe G. Crüger Jaap Oostrik Hannie Kremer Niels Tommerup Morten Fr?din Karen P. Steel Lisbeth Tranebj?rg Anders D. B?rglum 《PLoS genetics》2015,11(7)
70.
Yulia Anopa Alex D. McMahon David I. Conway Graham E. Ball Emma McIntosh Lorna M. D. Macpherson 《PloS one》2015,10(8)
Dental caries is one of the most common diseases of childhood. The aim of this study was to compare the cost of providing the Scotland-wide nursery toothbrushing programme with associated National Health Service (NHS) cost savings from improvements in the dental health of five-year-old children: through avoided dental extractions, fillings and potential treatments for decay.