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排序方式: 共有190条查询结果,搜索用时 15 毫秒
171.
Ho M Goh CH Brothers MC Wang S Young RL Ou Y Lui JN Kalafatis M Lan X Wolf AE Rienstra CM Wilson BA 《Protein science : a publication of the Protein Society》2012,21(3):318-326
The light chain of botulinum neurotoxin A (BoNT/A‐LC) is a Zn‐dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C‐terminus of SNAP25 (residues 141–206) retains full activity for BoNT/A‐LC‐catalyzed cleavage at P1‐P1' (Gln197‐Arg198). Using the structure of a complex between the C‐terminus of SNAP25 and BoNT/A‐LC as a model to design SNAP25‐derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala‐Asn‐Gln‐Arg (residues 195–198) at the substrate cleavage site, with the intent to identify possible side‐chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1‐P1' residues to explore the spatial tolerance within the active‐site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A‐LC. Among the SNAPIs tested, several known cleavage‐resistant, single‐point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly‐insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A‐LC. Two of the Gly‐insertion mutants exhibited 10‐fold lower IC50 values than the wildtype 66‐mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed. 相似文献
172.
Kloepper KD Zhou DH Li Y Winter KA George JM Rienstra CM 《Journal of biomolecular NMR》2007,39(3):197-211
The protein α-synuclein (AS) is the primary fibrillar component of Lewy bodies, the pathological hallmark of Parkinson’s disease.
Wild-type human AS and the three mutant forms linked to Parkinson’s disease (A53T, A30P, and E46K) all form fibrils through
a nucleation-dependent pathway; however, the biophysical details of these fibrillation events are not yet well understood.
Atomic-level structural insight is required in order to elucidate the potential role of AS fibrils in Parkinson’s disease.
Here we show that low temperature acquisition of magic-angle spinning NMR spectra of wild type AS fibrils-greatly enhances
spectral sensitivity, enabling the detection of a substantially larger number of spin systems. At 0 ± 3°C sample temperature,
cross polarization (CP) experiments yield weak signals. Lower temperature spectra (−40 ± 3°C) demonstrated several times greater
signal intensity, an effect further amplified in 3D 15N–13C–13C experiments, which are required to perform backbone assignments on this sample. Thus 3D experiments enabled assignments
of most amino acids in the rigid part of the fibril (approximately residues 64 to 94), as well as tentative site-specific
assignments for T22, V26, A27, Y39, G41, S42, H50, V52, A53, T54, V55, V63, A107, I112, and S129. Most of these signals were
not observed in 2D or 3D spectra at 0 ± 3°C. Spectra acquired at low temperatures therefore permitted more complete chemical
shift assignments. Observation of the majority of residues in AS fibrils represents an important step towards solving the
3D structure. 相似文献
173.
Background
Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.Results
Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.Conclusion
A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1017) contains supplementary material, which is available to authorized users. 相似文献174.
Background
To obtain predictions that are not biased by selection, the conditional mean of the breeding values must be computed given the data that were used for selection. When single nucleotide polymorphism (SNP) effects have a normal distribution, it can be argued that single-step best linear unbiased prediction (SS-BLUP) yields a conditional mean of the breeding values. Obtaining SS-BLUP, however, requires computing the inverse of the dense matrix G of genomic relationships, which will become infeasible as the number of genotyped animals increases. Also, computing G requires the frequencies of SNP alleles in the founders, which are not available in most situations. Furthermore, SS-BLUP is expected to perform poorly relative to variable selection models such as BayesB and BayesC as marker densities increase.Methods
A strategy is presented for Bayesian regression models (SSBR) that combines all available data from genotyped and non-genotyped animals, as in SS-BLUP, but accommodates a wider class of models. Our strategy uses imputed marker covariates for animals that are not genotyped, together with an appropriate residual genetic effect to accommodate deviations between true and imputed genotypes. Under normality, one formulation of SSBR yields results identical to SS-BLUP, but does not require computing G or its inverse and provides richer inferences. At present, Bayesian regression analyses are used with a few thousand genotyped individuals. However, when SSBR is applied to all animals in a breeding program, there will be a 100 to 200-fold increase in the number of animals and an associated 100 to 200-fold increase in computing time. Parallel computing strategies can be used to reduce computing time. In one such strategy, a 58-fold speedup was achieved using 120 cores.Discussion
In SSBR and SS-BLUP, phenotype, genotype and pedigree information are combined in a single-step. Unlike SS-BLUP, SSBR is not limited to normally distributed marker effects; it can be used when marker effects have a t distribution, as in BayesA, or mixture distributions, as in BayesB or BayesC π. Furthermore, it has the advantage that matrix inversion is not required. We have investigated parallel computing to speedup SSBR analyses so they can be used for routine applications.Electronic supplementary material
The online version of this article (doi:10.1186/1297-9686-46-50) contains supplementary material, which is available to authorized users. 相似文献175.
A Geert Heidema Jolanda MA Boer Nico Nagelkerke Edwin CM Mariman Daphne L van der A Edith JM Feskens 《BMC genetics》2006,7(1):1-15
Background
Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates.Results
Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales (<100 km). SSCP coupled with targeted DNA sequencing was used to obtain genotypic, genic and genealogical information from six loci (three per species). Phylogeographic analysis identified introns as being most informative.Conclusion
The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation. 相似文献176.
Esther C.H.E.T. Lucassen Alfons J.P. Smolders Ger Boedeltje Piet J J. van den Munckhof Jan CM. Roelofs 《植被学杂志》2006,17(4):425-434
Question: How does groundwater input affect plant distribution in Alnus glutinosa (black alder) carrs? Location: Alder carrs along the river Meuse, SE Netherlands. Methods: Three types of site, characterized by groundwater flow, were sampled in 17 A. glutinosa carrs. Vegetation and abiotic data (soil and water chemistry) were collected and analysed using a Canonical Correspondence Analysis. Based on the results, a laboratory experiment tested the effect of groundwater input (Ca2+) on pore water chemistry (NH4+ availability). Results: Environmental factors indicating groundwater input (Ca2+ and Fe2+), correlating with the NH+4 concentration in the pore water, best explained the variation in plant distribution. NH4+ availability was determined by Ca2+ input via the groundwater and subsequent competition for exchange sites in the sediment. As a result, nutrient‐poor seepage locations fully fed by groundwater were dominated by small iron resistant plants such as Caltha palustris and Equisetum fluviatile. More nutrient‐rich locations, fed by a combination of groundwater and surface water, allowed the growth of taller iron resistant plant species such as Carex paniculata. Nutrient‐rich locations with stagnating surface water were hardly fed by groundwater, allowing the occurrence of fast growing and less iron tolerant wetland grasses such as Glyceria fluitans and G. maxima. Conclusion: Groundwater input affects plant composition in A. glutinosa carrs along the river Meuse by determining nutrient availability (ammonium) and concentrations of toxic iron. 相似文献
177.
The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster 总被引:3,自引:1,他引:3
Schug MD; Hutter CM; Wetterstrand KA; Gaudette MS; Mackay TF; Aquadro CF 《Molecular biology and evolution》1998,15(12):1751-1760
In a recent study, we reported that the combined average mutation rate of
10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster
was 6.3 x 10(-6) mutations per locus per generation, a rate substantially
below that of microsatellite repeat units in mammals studied to date (range
= 10(-2)-10(-5) per locus per generation). To obtain a more precise
estimate of mutation rate for dinucleotide repeat motifs alone, we assayed
39 new dinucleotide repeat microsatellite loci in the mutation accumulation
lines from our earlier study. Our estimate of mutation rate for a total of
49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only
slightly higher than the estimate from our earlier study. We also estimated
the relative difference in microsatellite mutation rate among di-, tri-,
and tetranucleotide repeats in the genome of D. melanogaster using a method
based on population variation, and we found that tri- and tetranucleotide
repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide
repeats, respectively. The slower mutation rates of tri- and
tetranucleotide repeats appear to be associated with a relatively short
repeat unit length of these repeat motifs in the genome of D. melanogaster.
A positive correlation between repeat unit length and allelic variation
suggests that mutation rate increases as the repeat unit lengths of
microsatellites increase.
相似文献
178.
John M. Boettcher Mary C. Clay Benjamin J. LaHood James H. Morrissey Chad M. Rienstra 《Biomolecular NMR assignments》2010,4(2):183-185
Backbone 1H, 13C and 15N resonance assignments are presented for the extracellular domain of tissue factor. Tissue factor is the integral membrane protein that initiates blood coagulation through the formation an enzymatic complex with the plasma serine protease, factor VIIa. 相似文献
179.
Several studies have indicated that olfactory responses are impeded by
amiloride. Therefore, it was of interest to see whether, and if so which,
olfactory epithelial cellular compartments have amiloride- sensitive
structures. Using ultrastructural methods that involved rapid freezing,
freeze-substitution and low temperature embedding of olfactory epithelia,
this study shows that, in the rat, this tissue is immunoreactive to
antibodies against amiloride sensitive Na(+)- channels. However, microvilli
of olfactory supporting cells, as opposed to receptor cilia, contained most
of the immunoreactive sites. Apices from which the microvilli sprout and
receptor cell dendritic knobs had much less if any of the
amiloride-antibody binding sites. Using a direct ligand-binding
cytochemical method, this study also confirms earlier ones that showed that
olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that
supporting cell microvilli and the receptor cilia themselves have
mechanisms, different but likely complementary, that participate in
regulating the salt concentration around the receptor cell cilia. In this
way, both structures help to provide the ambient mucous environment for
receptor cells to function properly. This regulation of the salt
concentration of an ambient fluid environment is a function that the
olfactory epithelium shares with cells of transporting epithelia, such as
those of kidney.
相似文献
180.