This study investigates the allosteric coupling that exists between the intra- and extracellular parts of human β2-adrenergic receptor (β2-AR), in the presence of the intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies reported so far. Our recent 1 μs long MD run has revealed a transition to the so-called very inactive state of the receptor, in which ICL3 packed under the G protein’s binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. In the current study, we performed independent runs with a total duration of 4 μs to further investigate the very inactive state with packed ICL3 and the allosteric coupling event (three unrestrained runs and five runs with bond restraints at the ligand-binding site).
Results
In all three independent unrestrained runs (each 500 ns long), ICL3 preserved its initially packed/closed conformation within the studied time frame, suggesting an inhibition of the receptor’s activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained/closed binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free the ICL3 from its locked state due to the presence of persistent hydrogen bonds.
Conclusions
Overall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to the core of the receptor.
The interaction processes of trace amounts of N-methyl-2-pyrrolidinone (NMP), CS2/NMP (1:1 by volume) and pure NMP solvent with the hydrogen bond of OH?N in coal were constructed and simulated by density functional theory methods. The distances and bond orders between the main related atoms, and the hydrogen bond energy of OH?N were calculated. The calculated results show that pure NMP solvent does not weaken the hydrogen bond of OH?N in coal. However, trace amounts of NMP and CS2/NMP (1:1 by volume) have a strong capacity to weaken the hydrogen bond of OH?N in coal. The H2–N3 distances are elongated from 1.87 Å to 3.80 Å and 3.44 Å, the bond orders of H2–N3 all disappear, and the corresponding hydrogen bond energies of OH?N in coal decrease from 45.72 kJ mol?1 to 7.06 and 11.24 kJ mol?1, respectively. These results show that CS2 added to pure NMP solvent plays an important role in releasing the original capacity of NMP to weaken the hydrogen bond of OH?N in coal, in agreement with experimental observations. 相似文献
Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products.
Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study,
α-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions
of immobilization. The immobilized α-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum
temperature also shifted from 50°C to 57°C compared with soluble enzyme. Immobilized α-galactosidase was used in batch, repeated
batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the
fourth cycle. The performance of immobilized α-galactosidase was tested in a fluidized bed reactor at different flow rates
and 86% reduction of RO in soymilk was obtained at 25 ml h−1 flow rate. The study revealed that immobilized α-galactosidase in continuous mode is efficient in reduction of RO present
in soymilk. 相似文献
In the present investigation were evaluated the antifungal and antibacterial activities of Nano-silver (NS). Two separate experiments were done to evaluate the potential of silver nanoparticles in controlling the contamination of G × N15 micro-propagation. In the first experiment, a factorial experiment based on a completely randomized design with 15 treatments including five different NS concentrations (0, 50, 100, 150 and 200 ppm) and three soaking time of explants (3, 5 and 7 min) with five replications was conducted. In the other experiment, NS was added to Murashige and Skoog (MS) medium with concentrations of 0, 50, 100, 150 and 200 ppm in a completely randomized design. Results showed that the application of 100 and 150 ppm NS both as an immersion and as added directly to the culture medium significantly reduces internal and external contaminations compared with the control group. Using NS in culture medium was more efficient to reduce fungal and bacterial contamination than immersion. High concentrations of NS had an adverse effect on the viability of G × N15 single nodes and this effect was more serious in immersed explants in NS than directly added NS ones regarding the viability of buds and plantlet regeneration. In conclusion, due to high contamination of woody plants especially fruit trees and also adverse environmental effects of mercury chloride, the NS solution can be used as a low risk bactericide in micro-propagation of G × N15 and can be an appropriate alternative to mercury chloride in the future. 相似文献
StressEraser is a commercially marketed biofeedback device designed to enhance heart rate variability. StressEraser makes its internal calculations on beat-to-beat measures of finger pulse intervals. However, the accuracy and precision of StressEraser in quantifying interbeat intervals using finger pulse intervals has not been evaluated against standard laboratory equipment using R-R intervals. Accuracy was assessed by simultaneously recording interbeat intervals using StressEraser and a standard laboratory ECG system. The interbeat intervals were highly correlated between the systems. The average deviation in interbeat interval recordings between the systems was approximately 6 ms. Moreover, correlations approached unity between the systems on estimates of heart period, heart rate, and heart rate variability. Feedback from StressEraser is based on an interbeat time series that provides sufficient information to provide an excellent estimate of the dynamic changes in heart rate and heart rate variability. The slight variations between StressEraser and the laboratory equipment in quantifying heart rate and heart rate variability are due to features related to monitoring heart rate with finger pulse: (1) a lack in precision in the peak of the finger pulse relative to the clearly defined inflection point in the R-wave, and (2) contribution of variations in pulse transit time. 相似文献
The expression level of electrophoretically separated S- and F-allozymes of β-specific esterase (EC 3.1.1.2) in genotypes of wild-type Drosophila melanogaster (males and females) that are monozygous or heterozygous with respect to the locus β-Est is determined by means of computerized densitometry; α-naphthylacetate, β-naphthylacetate, and α-naphthylpropionate are used as the substrates. The intensity of the expression of the esterase is judged from the quantity of reaction product created as a result of simultaneous azo coupling between naphthol and diazonium in 4, 24, 44, and 64 min incubation times. Reliable differences in the expressions of the S- and F-allozymes as a function of the structure of the β-Est locus of genotypically distinct individuals are established. In all the variant experiments, a higher level of summary activity of the S- and F-allozymes of the β-esterase of the heterozygotes by comparison with the individual activity of the F-and S-allozymes of the corresponding homozygotes was demonstrated, independently of the sex of the Drosophila individual. A comparative estimate of the temporal dynamics of the expression of in vitro allozymes of the dominant homozygotes (β-EstS/β-EstS), heterozygotes (β-EstS/β-EstF), and recessive homozygotes (β-EstF/β-EstF) is performed. Possible mechanisms for the occurrence of heterosis according to the character of expression of S- and F-allozymes of β-esterase on the basis of the theory of biochemical enrichment of heterozygote genotypes are considered. 相似文献
The effects of antagonist of α2-adrenoceptors yohimbine and their agonist clonidine on Bax and Bcl-XL mRNA levels in neonatal rat brain were studied. Yohimbine decreased Bax mRNA level in the cerebellum, increased the ratio between Bcl-XL and Bax mRNA levels in the cerebellum, cortex, and hippocampus, and increased Bcl-XL mRNA level in the cortex and hippocampus of 6-day-old-rat pups 24 h after injection. Administration of clonidine 20 min after yohimbine administration abolished its effect on Bcl-XL mRNA level in the hippocampus. The data obtained indicate that the blockade of α2-adrenoceptors induces antiapoptotic changes in the developing rat brain, some of which can be abolished after coadministration with the agonist of these receptors. 相似文献
Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatGC). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatGN). The analogous variants of KatGC showed a much more profound loss of function as evaluated by their ability to return KatGN to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatGN reactivation, but with two substitutions, KatGC completely lost its capacity for the reactivation of KatGN. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality. 相似文献
Electronic, magnetic, and structural properties of pure and V-doped CrO2 were extensively investigated utilizing density functional theory. Usually, pure CrO2 is a half-metallic ferromagnet with conductive spin majority species and insulating spin minority species. This system remains in its half-metallic ferromagnetic phase even at 50% V-substitution for Cr within the crystal. The V-substituted compound Cr0.5V0.5O2 encounters metal–insulator transition upon the application of on-site Coulomb repulsion U?=?7 eV preserving its ferromagnetism in the insulating phase. It is revealed in this study that Cr3+-V5+ charge ordering accompanied by the transfer of the single V-3d electron to the Cr-3dt2g orbitals triggers metal–insulator transition in Cr0.5V0.5O2. The ferromagnetism of Cr0.5V0.5O2 in the insulating phase arises predominantly due to strong Hund’s coupling between the occupied electrons in the Cr-t2g states. Besides this, the ferromagnetic Curie temperature (Tc) decreases significantly due to V-substitution. Interestingly, a structural distortion is observed due to tilting of CrO6 or VO6 octahedra across the metal–insulator transition of Cr0.5V0.5O2.
Graphical abstract The V-doped compound Cr0.5V0.5O2 is found a half-metallic ferromagnet (HMF) in the absence of on-site Coulomb interaction (U). This HMF behavor maintains up to U?=?6 eV. Eventually, this system encounters metal-insulator transition (MIT) upon the application of U?=?7 eV with a band gap of Eg ~ 0.31 eV. Nevertheless, applications of higher U widen the band gaps. In this figure, calculated total (black), Cr-3d (red), V-3d (violet), and O-2p (blue) DOS of Cr0.5V0.5O2 for U?=?8 eV are illustrated. The system is insulating with a band gap of Eg ~ 0.7 eV.
Sodium salicylate (NaSA) increased induction of both intracellular and extracellular beta-1,3-glucanases in calluses of campion and duckweed. NaSA concentrations from 30 to 100 mM were optimal for induction of intracellular glucanase in the campion callus, and for induction of extracellular glucanase the optimal concentration varied from 5 to 100 mM. The glucanase activity in the duckweed callus was lower than in the campion callus, and co-cultivation of the campion callus with Trichoderma harzianum mycelium increased the production of intracellular and extracellular beta-1,3-glucanases and polygalacturonase in the callus. Biosynthesis by T. harzianum of glucanases, extracellular polygalacturonase and xylanase, and of intracellular galactosidase was increased. The co-cultivation was accompanied by increased activity of intracellular acidic isoform of glucanase Glu-3 secreted by the callus cells into the medium, whereas NaSA activated in the callus culture the extracellular acidic isoform Glu-1 and extracellular basic isoform Glu-5. These data indicate the induction of these isoforms and the specificity of protective response of plant cells to different factors. 相似文献
In the mountainous “Hohe Tauern” region of Salzburg (Austria), milk samples have been collected in a long-term montitoring
programme since 1988, at eight alpine sites used for extensive, seasonal stock farming. For this alpine environment with its
acidic soils developed on silicate bedrock, high soil-to-plant transfer factors and long-lasting 137Cs contamination levels in milk—the main product of seasonal agriculture at elevated altitudes—are characteristic features.
The decrease in 137Cs concentration in milk measured since 1988 turned out to be best described by one or two effective half-lives. For the period
from 1993 to 2007, which can be modelled with one effective half-life for all sites, effective half-lives between 3.7 and
15.0 years (ecological half-lives: 4.3–29.9 years) were obtained. The effective half-life increases with mean altitude of
the investigated graze pastures, probably due to reduced migration velocities of 137Cs and low 137Cs half-value depths of a few centimetres in the soil. 相似文献
Expression of surface antigen genes in Trypanosoma brucei occurs at expression sites located near telomeres. Since only one antigen is produced at a time, a mechanism must exist to prevent the simultaneous activity of multiple expression sites. Here we report that PstI and PvuII restriction sites in silent telomeric antigen genes are partially uncleavable , presumably as a consequence of DNA modification. The modification, which is absent in transcribed genes but returns after gene inactivation, may be specific for telomeric DNA because (1) it is not detected in non-telomeric genes; (2) modification is highest close to the telomere; (3) the level of modification in a telomeric gene is influenced by the size of the telomeric DNA segment downstream. Whether telomere modification is cause or consequence of antigen gene switch-off remains to be determined. 相似文献
The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses.
Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype
of C.albicans. Yeasts were isolated from 68.4% (91/133) individual’s dental prostheses users. Dental prostheses were found to be a significant
factor for the yeast colonization (P < 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals
(P < 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average
SAB for the entire collection of 47 C.␣albicans isolates were 0.779 ± 0.178. Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea
that the use of dental prostheses, independently of the host’s clinical condition, probably provides the necessary conditions
for these strains to gain a growth-specific advantage over others. 相似文献