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1.
Summary We have analyzed a combined use of the two-dimensional nuclear Overhauser effect in the laboratory frame (NOESY) and in the rotating frame (ROESY) to determine interproton distances and correlation time in medium-sized rigid molecules (Davis, 1987). This method can be applied in the intermediate motional regime, 0.2 < oc, < 5, (c, correlation time, (o resonance frequency). Error limits depend on the motional regime and are smallest near oc=1.14.The method was tested on six geminal proton pairs in the bicyclic octapeptide (S-deoxo--[R]-OH-Ile3 amaninamide, Mw =870) for which at 297 K in DMSO, a correlation time of 1.0 ns, with a standard deviation of 0.12 ns, and an interproton distance of 1.87 Å, with standard deviation of 0.04 Å, are obtained.  相似文献   

2.
Summary A method for measuring three-bond 13C-1H scalar coupling constants across glycosidic bonds in a cyclic (12)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2) protons permits the selective observation of the inter-residue heteronuclear couplings 3JC1H2 and 3JC2H1 in a natural-abundance 13C-1-half-filtered {1H,1H} ROESY experiment.Abbreviations COSY scalar correlated spectroscopy - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - ROESY rotating-frame NOE spectroscopy  相似文献   

3.
A stretch-activated (SA) Cl channel in the plasma membrane of the human mast cell line HMC-1 was identified in outside-out patch-clamp experiments. SA currents, induced by pressure applied to the pipette, exhibited voltage dependence with strong outward rectification (55.1 pS at +100 mV and an about tenfold lower conductance at −100 mV). The probability of the SA channel being open (P o) also showed steep outward rectification and pressure dependence. The open-time distribution was fitted with three components with time constants of τ1o = 755.1 ms, τ2o = 166.4 ms, and τ3o = 16.5 ms at +60 mV. The closed-time distribution also required three components with time constants of τ1c = 661.6 ms, τ2c = 253.2 ms, and τ3c = 5.6 ms at +60 mV. Lowering extracellular Cl concentration reduced the conductance, shifted the reversal potential toward chloride reversal potential, and decreased the P o at positive potentials. The SA Cl currents were reversibly blocked by the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) but not by (Z)-1-(p-dimethylaminoethoxyphenyl)-1,2-diphenyl-1-butene (tamoxifen). Furthermore, in HMC-1 cells swelling due to osmotic stress, DIDS could inhibit the increase in intracellular [Ca2+] and degranulation. We conclude that in the HMC-1 cell line, the SA outward currents are mediated by Cl influx. The SA Cl channel might contribute to mast cell degranulation caused by mechanical stimuli or accelerate membrane fusion during the degranulation process.  相似文献   

4.
The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with 13C and 15N were studied by 13C spin relaxation experiments. R1, R and the 13C-{1H} steady-state NOE of C6 and C1′ in pyrimidine and C8 and C1′ in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time τc of 2.31±0.13 ns and an axial ratio of 1.35±0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported 13C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539–10551; Fiala et al., 2000 J. Biomol. NMR 16, 291–302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165–190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S2 < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S2 = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of 13C relaxation rates were found to be in agreement with 15N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702–709). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.  相似文献   

5.
A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring 15N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone 15N R 1, R , 15N–{1H} NOE, and 15N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsATM, exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, τc = 38 ± 2.5 ns, at 50 °C and a diffusion anisotropy, , corresponding to an aspect ratio a/b ≥ 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S 2 values in the 0.2–0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps–ns time scale for the 5-residue selectivity filter, which selects K+ ions to enter the channel. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . An erratum to this article can be found at  相似文献   

6.
Voltage clamp experiments, which determine the kinetic parameters of calcium conductance of cardiac muscle, (d ,f , τ d and τ f ) are analyzed with a generally accepted expression for slow inward currentI s=g sdf (E-E R). Activation (d) and inactivation (f) reach the final valuesd andf with time constants τ d and τ f respectively. The analysis indicates that the measuredf agrees with the theoreticalf , but the measuredd differs from the theoreticald by a factor which depends on τ d . The peak tension can be made to correlate closely with the theoreticald after a correction factor is applied to the raw measurements of activation. It can be shown that experiments designed to measure τ f can also be used to determine τ d with greater accuracy.  相似文献   

7.
A Gram-negative, non-motile, non-spore-forming, small, orange, rod-shaped bacterium was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequence examination revealed that strain PB323T belongs to the family Sphingomonadaceae. The highest degree of sequence similarity was found with Sphingomonas kaistensis PB56T (98.9%), followed by Sphingomonas astaxanthinifaciens TDMA-17T (98.3%). Chemotaxonomic characteristics (the G+C content of the genomic DNA 69.0 mol%, Q-10 quinone system, C18:1 ω7c/ω9t/ω12t, C16:1 ω7c/C15:0 iso 2OH, C17:1 ω6c, and C16:0 as the major fatty acids) corroborated assignment of strain PB323T to the genus Sphingomonas. Results of physiological and biochemical tests clearly demonstrate that strain PB323T represents a distinct species and support its affiliation with the genus Sphingomonas. Based on these data, PB323T (=KCTC 12341T =JCM 16603T =KEMB 9004-003T) should be classified as a type strain of a novel species, for which the name Sphingomonas humi sp. nov. is proposed.  相似文献   

8.
Tyrammonium 4-nitrophthalate has been synthesized and its structural and spectroscopic properties elucidated by single-crystal X-ray diffraction, solid-state polarized IR-spectroscopy of oriented colloids in a nematic host, HPLC with tandem mass spectrometry (HPLC ESI-MSMS), and TGV and DSC methods. The compound crystallizes in the monoclinic P21/c space group and its structure consists of a 3D network of molecules joined by intermolecular interactions with the participation of cations, anions and two solvent molecules. The tyrammonium cation adopts a T trans configuration with corresponding angles of ϕ 1 = 76.0(4)°, ϕ = 54.8(1)° and ϕ 3 = 63.4(1)°, respectively. In the 4-nitrophthalate anion, the COO and COOH groups are turned off the plane of the benzene ring at angles of τ 1 = 88.1(5)° and τ 2= 22.1(7)°, respectively.  相似文献   

9.
Strain BS12T, a Gram-negative motile bacterium, was isolated from soil in South Korea and characterized to determine its taxonomic position. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain belonged to the family Alcaligenaceae in the class Betaproteobacteria. The highest degree of sequence similarities of strain BS12T were found with Pigmentiphaga litoralis JSM 061001T (98.3%), Pigmentiphaga daeguensis K110T (98.2%), and Pigmentiphaga kullae K24T (98.1%). Chemotaxonomic data revealed that strain BS12T possessed ubiquinone-8, which is common in the family Alcaligenaceae, and the predominant fatty acids were C16:0, C17:0 cyclo, summed feature 3 (C16:1 ω6c/ω7c), and summed feature 8 (C18:1 ω6c/ω7c). The major polar lipids of strain BS12T were phosphatidylethanolamine and phosphatidylglycerol. Based on these data, BS12T (=KCTC 23577T =JCM 17666T =KEMB 9004-082T) should be classified as a type strain of a novel species, for which the name Pigmentiphaga soli sp. nov. is proposed.  相似文献   

10.
Two-dimensional 1H/31P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving 31P have not been previously pursued to a great extent in lipid bilayers due to the long 1H-31P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different 1H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these 1H/31P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional 1H/13C INEPT HETCOR experiments with NOE mixing support the 1H/31P dipolar HETCOR results and confirm the presence of a H2O environment that has nonvanishing dipolar interactions with the SM backbone.  相似文献   

11.
Twenty pregnane glycosides, tuberoside A1–L5, were isolated from the diethyl ether-soluble fraction of the MeOH extract from the aerial parts of Asclepias tuberosa (Asclepiadaceae). The pregnane glycosides were composed of 8,12;8,20-diepoxy-8,14-secopregnane as aglycon, and d-cymarose, d-oleandrose, d-digitoxose and/or d-glucose as the component sugars. Their structures were established using NMR spectroscopic analysis and chemical methodologies.  相似文献   

12.
A Gram-negative, aerobic, motile and slightly curved rod-shaped bacterium (BFLP-8T) was isolated from cutaneous mucus of wild long-snouted seahorses (Hippocampus guttulatus) captured in northwest Spain (Toralla, Galicia). Strain BFLP-8T grew at 10–35 °C and pH 5–9 (optimally at 25 °C and pH 7.0) and with 1–6 % (w/v) NaCl (optimally with 2 % NaCl). The predominant respiratory quinone (90 %) was ubiquinone with ten isoprene units (Q-10) and the major fatty acids identified were C18:1 ω7c (54.8 % of the total), C19:0 cyclo ω8c (11.6 %), C16:0 (9.5 %), C18:1 2-OH (7.1 %) and C16:1 ω11c (6.7 %). The G+C content of the DNA was 57.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-8T formed a distinct clade within the family Sneathiellaceae but is not specifically associated with any species in the family. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-8T represents a novel species within a new genus, for which the name Oceanibacterium hippocampi gen. nov., sp. nov. is proposed. The type strain is BFLP-8T (=CECT 7691T = DSM 23444T).  相似文献   

13.
Model-free analysis is a technique commonly used within the field of NMR spectroscopy to extract atomic resolution, interpretable dynamic information on multiple timescales from the R 1, R 2, and steady state NOE. Model-free approaches employ two disparate areas of data analysis, the discipline of mathematical optimisation, specifically the minimisation of a χ2 function, and the statistical field of model selection. By searching through a large number of model-free minimisations, which were setup using synthetic relaxation data whereby the true underlying dynamics is known, certain model-free models have been identified to, at times, fail. This has been characterised as either the internal correlation times, τ e , τ f , or τ s , or the global correlation time parameter, local τ m , heading towards infinity, the result being that the final parameter values are far from the true values. In a number of cases the minimised χ2 value of the failed model is significantly lower than that of all other models and, hence, will be the model which is chosen by model selection techniques. If these models are not removed prior to model selection the final model-free results could be far from the truth. By implementing a series of empirical rules involving inequalities these models can be specifically isolated and removed. Model-free analysis should therefore consist of three distinct steps: model-free minimisation, model-free model elimination, and finally model-free model selection. Failure has also been identified to affect the individual Monte Carlo simulations used within error analysis. Each simulation involves an independent randomised relaxation data set and model-free minimisation, thus simulations suffer from exactly the same types of failure as model-free models. Therefore, to prevent these outliers from causing a significant overestimation of the errors the failed Monte Carlo simulations need to be culled prior to calculating the parameter standard deviations.  相似文献   

14.
Summary We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with 15N. The requirement of 13C-/15N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., 0c~1). The technique was applied to an unstructured 15N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.  相似文献   

15.
An important criterion used to detect adaptive evolution in DNA sequence data is ωi > 1, where ωi is the ratio of nonsynonymous to synonymous substitution rates in lineage i. However, the evaluation of multiple ωi within a phylogenetic tree can easily inflate the statistical type I error rate. We developed two rigorous methods of analysis that avoid this and other potential pitfalls. We applied these methods to four published examples of adaptive evolution. One case was strongly supported by our reanalysis (abalone sperm lysin), and one was weakly supported (baboon α-globin), but two examples (primate lysozyme and Antarctic fish β-globin) did not show significant evidence of adaptive evolution. Our first method is a “bottom-up” hierarchical maximum likelihood approach, which (1) tests for significant heterogeneity in ω across the phylogeny, (2) locates its source using a sequence of planned comparisons, and (3) tests homogeneous groups of ω for ω > 1, using a modified level of significance that incorporates the pretesting. The second method is a “top-down” log-linear analysis based on estimates of nonsynonymous and synonymous substitutions in pairs of lineages. The log-linear test is applied to pairs of lineages joined at progressively deeper nodes. For each pair, the analysis simultaneously tests for adaptive evolution (ω > 1), a shift in natural selection (ω1 ≠ω2), and unequal evolution rate (the relative rate test). In both tests, we emphasized that the criterion ω1 ≠ ω2 is an important additional indicator of a phylogenetic shift in the balance between natural selection and genetic drift between two related lineages. [Reviewing Editor: Dr. John Huelsenbeck]  相似文献   

16.
Eukaryotic algae were studied to determine their ability to biotransform HgII under aerated and pH controlled conditions. All algae converted HgII into β-HgS and Hg0 to varying degrees. When HgII was administered as HgCl2 to the algae, biotransformation by species of Chlorophyceae (Selenastrum minutum and Chlorella fusca var. fusca) was initiated with β-HgS synthesis (K 1/2 of hours) and concomitant Hg° evolution occurred in the first hour. Hg° synthesis was impeded by the formation of β-HgS and this inhibition was released in C. fusca var. fusca when cellular thiols were oxidized by the addition of dimethylfumarate (DMF). The diatom, Navicula pelliculosa (Bacillariophyceae), converted a substantially greater proportion of the applied HgII into Hg0, whereas the thermophilic alga, Galdieria sulphuraria (Cyanidiophyceae), rapidly biotransformed as much as 90% of applied HgII into β-HgS (K 1/2 ≈ 20 min). This thermophile was also able to generate Hg0 even after all exogenously applied HgCl2 had been biotransformed. The results suggest that β-HgS may be the major dietary mercurial for grazers of contaminated eukaryotic algae.  相似文献   

17.
A novel Gram-negative and rod-shaped bacterium, designated N8T, was isolated from tidal flat sediment. Phylogenetic analysis based on 16S rRNA gene sequences showed that N8T strain is associated with the family Phyllobacteriaceae: two uncultured clones (98.4 and 99.8% 16S rRNA gene sequence similarity) and the genus Mesorhizobium (≤97.0%). The novel strain formed a separate clade with uncultured clones in the phylogenetic tree based on 16S rRNA gene sequences. Cellular fatty acid profiles predominately comprised C18:1 ω7c and C19:0 cyclo ω8c. The major isoprenoid quinone is ubiquinone-10 and genomic DNA G+C content is 53.4 mol%. The polyphasic taxonomic study indicates that the novel strain N8T represents a novel species of the new genus in the family Phyllobacteriaceae, named Aliihoeflea aestuarii. The type strain is N8T (= KCTC 22052T= JCM 15118T= DSM 19536T).  相似文献   

18.
A novel Gram-negative, catalase- and oxidase-positive, strictly aerobic, non spore-forming, rod-shaped bacterium, designated strain JSM 083058T, was isolated from non-saline forest soil in Hunan Province, China. Growth occurred with 0–8% (w/v) NaCl (optimum, 0.5–3%) at pH 6.0–10.0 (optimum, pH 7.0) and at 5–35°C (optimum, 25–30°C). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 083058T fell within the cluster comprising species of the genus Sphingomonas, clustering with Sphingomonas aestuarii K4T, with which it shared highest 16S rRNA gene sequence similarity (99.2%). The chemotaxonomic properties of strain JSM 083058T were consistent with those of the genus Sphingomonas. The predominant respiratory quinone was ubiquinone Q-10, and the major cellular fatty acids were summed feature 8 (C18:1ω7c/C18:1ω6c), C16:0, summed feature 3 (C16:1ω7c/C16:1ω6c) and C17:1ω6c. The polar lipids consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. The genomic DNA G+C content of strain JSM 083058T was 65.5 mol%. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 083058T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas hunanensis sp. nov. is proposed. The type strain is JSM 083058T (=CCTCC AA 209011T = DSM 22213T).  相似文献   

19.
 Dynamical models of two coupled biological oscillators interpret the detuning term as an arithmetic difference between the uncoupled frequencies, Δω= (ω1−ω2). This Δω interpretation of detuning was addressed in four experiments in which human subjects oscillated pendulums in their right and left hands in 1 : 1 frequency locking in antiphase (Experiments 1–3) or inphase (Experiment 4). Differences between the uncoupled frequencies were manipulated through differences in the equivalent simple pendulum lengths, and the effects of this manipulation on the detuning of relative phase from π or 0 and the standard deviation of relative phase SDφ were measured. In Experiment 1, the same values of ω i were satisfied by several different physical configurations. The experiment confirmed that the detuning term is related strictly to the uncoupled frequencies rather than to other physical characteristics of the oscillators. Experiments 2, 3 and 4 showed, however, that the particular dependency of fixed point drift and SDφ on Δω depends on the particulars of ω1 and ω2. With variations in Δω brought about by different ω1 and ω2 that always formed a constant ratio, fixed point drift related inversely to Δω, and SDφ varied with Δω in ways that depended on the magnitude of the constant ratio. These outcomes do not conform to expectations from models of coordination dynamics that interpret detuning as (ω1−ω2). Received: 18 October 1993/Accepted in revised form: 2 December 1994  相似文献   

20.
The kinetics of the torque-velocity (T-ω) relationship after aerobic exercise was studied to assess the effect of fatigue on the contractile properties of muscle. A group of 13 subjects exercised until fatigued on a cycle ergometer, at an intensity which corresponded to 60% of their maximal aerobic power for 50 min (MAP60%); ten subjects exercised until fatigued at 80% of their maximal aerobic power for 15 min (MAP80%). Of the subjects 7 exercised at both intensities with at least a 1-week interval between sessions. Pedalling rate was set at 60 rpm. The T-ω relationship was determined from the velocity data collected during all-out sprints against a 19 N · m braking torque on the same ergometer, according to a method proposed previously. Maximal theoretical velocity (ω0) and maximal theoretical torque (T 0) were estimated by extrapolation of the linear T-ω relationship. Maximal power (P max) was calculated from the values of T 0 and ω0 (P max = 0.25 ω0T 0). The T-ω relationships were determined before, immediately after and 5 and 10 min after the aerobic exercise. The kinetics of ω0, T 0 and P max was assumed to express the effects of fatigue on the muscle contractile properties (maximal shortening velocity, maximal muscle strength and maximal power). Immediately after exercise at MAP60% a 7.8% decrease in T 0 and 8.8% decrease in P max was seen while the decrease in ω0 was nonsignificant, which suggested that P max decreased in the main because of a loss in maximal muscle strength. In contrast, MAP80% induced a 8.1% decrease in ω0 and 12.8% decrease in P max while the decrease in T 0 was nonsignificant, which suggested that the main cause of the decrease in P max was probably a slowing of maximal shortening velocity. The short recovery time of the T-ω relationship suggests that the causes of the decrease of torque and velocity are processes which recover rapidly. Accepted: 25 November 1996  相似文献   

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