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1.
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Amine transporters are major target for development of various pharmacological agents to treat behavioral disorders. Serotonin
transporters (SERT) have been implicated in the etiology of depression and drugs acting on SERT can be effective in treating
depression. The aim of the present study was to study the in vivo effect of various antidepressants on [3H]paroxetine binding to SERT in regions of rat brain. Rats were treated with tricyclic antidepressant (TCAs) such as amitriptyline
(AMI), serotonin/norepinephrine reuptake inhibitor (SNRIs) such as clomipramine (CMI), and selective serotonin reuptake inhibitors
(SSRIs) such as fluoxetine (FLX) and citalopram (CIT) (10 mg/kg body wt.) for 30 days. Density of SERT was measured in cortex
and hippocampus using [3H]paroxetine (0.03–1.0 nM) in presence and absence of 10 μM fluoxetine as displacer. It was observed that the density of cortical
SERT was significantly decreased with CMI (68%, P < 0.0001), FLX (67%, P < 0.0001), CIT (54%, P < 0.0001), and AMI (52%, P < 0.0001) treatment, when compared to the density of 120.7 ± 4.0 fmol/mg protein in control rats, without altering the affinity
(Kd) of [3H]paroxetine to the transporters. The density of SERT in hippocampus was also significantly decreased with FLX (65%, P < 0.0001), CMI (54%, P < 0.0001), CIT (52%, P < 0.0001) and AMI (46%, P < 0.0001) treatment, when compared to the density of 74.0 ± 2.6 fmol/mg protein in control rats, without altering the affinity
of [3H]paroxetine to the transporters. Displacement study showed high affinity for CMI > CIT > FLX. The results suggest that chronic
antidepressant treatment significantly down-regulates both cortical and hippocampal SERT in rat brain and SSRIs have high
affinity for SERT than TCAs. 相似文献
3.
Based on the difference in the CD14 and CD16 expression, two subsets of monocytes were identified in human and other mammalian
blood. These subsets have different patterns of adhesion molecules and chemokine receptors that suggests the different mode
of their interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14+CD16+ and CD14++CD16− monocytes to adhere to endothelial cell monolayer in presence or absence of pro- and anti-inflammatory cytokines. We demonstrated
that CD14+CD16+ monocytes had a higher level of adhesion to intact monolayer of endothelial cells than CD14++CD16− monocytes. Adhesion of CD14++CD16− and CD14+CD16+ monocytes significantly increased in the presence of TNFα or its combination with other cytokines. IFNγ and IL-4 alone did
not affect the adhesion of monocytes. These results show that CD14++CD16− and CD14+CD16+ monocytes can be recruited to the inflamed endothelium, but CD14+CD16+ monocytes adhere to endothelial cells without inflammations twice as strongly as CD14++CD16− monocytes. 相似文献
4.
The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases. 相似文献
5.
Arginine side-chains are often key for enzyme catalysis, protein–ligand and protein–protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine 13Cζ-detected NMR experiment in which a double-quantum coherence involving the two 15Nη nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two 15Nη; this new approach is shown to eliminate the previously deleterious line broadenings of 15Nη resonances caused by the partially restricted rotation about the Cζ–Nε bond. Consequently, sharp and well-resolved 15Nη resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine 15Nη nuclei becomes possible using the double-quantum experiment. 相似文献
6.
E. Yu. Aleshina N. V. Pyndyk A. A. Moisa M. A. Sanzhakov O. N. Kharybin E. N. Nikolaev E. F. Kolesanova 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2008,2(3):288-292
The peptide RHDSGY, a fragment of the human β-amyloid Zn-binding site, and its isomers RH(D-Asp)SGY and RH(β-Asp)SGY have been obtained as amides by means of solid-phase synthesis and analyzed by HPLC and various mass spectrometric methods. The problem of low yield of the RHDSGY peptide and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of such side-products as RH(β-Asp)SGY (or RHDSGY during synthesis of RH(β-Asp)SGY) and RH(Asp-imide) SGY was solved via selection of individual reagents for removal of Fmoc groups from α-amino groups of the growing peptide chain. 相似文献
7.
In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of 15NO3 and 15NH4 were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately
1600 m2, were delimited by trenches in the Gleysols. K15NO3 was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition.
After the sampling and a one-year break, 15NH4Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood),
ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable
inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period,
the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO3− than NH4+ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in
the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of 15NO3− in runoff, with sharp 15N peaks corresponding to discharge peaks. NO3− leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these
ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline
of its C:N ratio. 相似文献
8.
We present a projected [1H,15N]-HMQC-[1H,1H]-NOESY experiment for observation of NOE interactions between amide protons with degenerate 15N chemical shifts in large molecular systems. The projection is achieved by simultaneous evolution of the multiple quantum
coherence of the nitrogen spin and the attached proton spin. In this way NOE signals can be separated from direct-correlation
peaks also in spectra with low resolution by fully exploiting both 1H and 15N frequency differences, such that sensitivity can be increased by using short maximum evolution times. The sensitivity of
the experiment is not dependent on the projection angle for projections up to 45° and no additional pulses or delays are required
as compared to the conventional 2D [1H,15N]-HMQC-NOESY. The experiment provides two distinct 2D spectra corresponding to the positive and negative angle projections,
respectively. With a linear combination of 1D cross-sections from the two projections the unavoidable sensitivity loss in
projection spectra can be compensated for each particular NOE interaction. We demonstrate the application of the novel projection
experiment for the observation of an NOE interaction between two sequential glycines with degenerate 15N chemical shifts in a 121.3 kDa complex of the linker H1 histone protein with a 152 bp linear DNA. 相似文献
9.
10.
The endomorphins (EM1 and EM2) are selective endogenous ligands for mu-opioid receptors (MOR1 and MOR2) with neurotransmitter
and neuromodulator roles in mammals. In the present study we investigated the potential actions of EMs on striatal GABA release
and the implication of different MORs in these processes. Rat striatal slices were preincubated with tritium-labelled GABA
([3H]GABA), pretreated with selective MOR1 and MOR2 antagonist beta-funaltrexamine and selective MOR1 antagonist naloxonazine
and then superfused with the selective MOR agonists, EM1 and EM2. EM1 significantly decreased the striatal [3H]GABA release induced by electrical stimulation. Beta-funaltrexamine antagonized the inhibitory action of EM1, but naloxonazine
did not affect it considerably. EM2 was ineffective, even in case of specific enzyme inhibitor diprotin A pretreatment. The
results demonstrate that EM1 decreases GABA release in the basal ganglia through MOR2, while EM2 does not influence it. 相似文献
11.
A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr2+ and Cl– and concentrations of stable isotope 18O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of 15N and 18O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in 15N and 18O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems. 相似文献
12.
The Na+/Mg2+ exchanger represents the main Mg2+ extrusion mechanism operating in mammalian cells including hepatocytes. We have previously reported that this exchanger, located in the basolateral domain of the hepatocyte, promotes the extrusion of intravesicular trapped Mg2+ for extravesicular Na+ with ratio 1. This electrogenic exchange is supported by the accumulation of tetraphenyl-phosphonium within the vesicles at the time when Mg2+ efflux occurs. In this present study, the role of extra- and intra-vesicular Cl? on the Na+/Mg2+ exchange ratio was investigated. The results reported here suggest that Cl? ions are not required for the Na+ to Mg2+ exchange to occur, but the stoichiometry ratio of the exchanger switches from electrogenic (1Na in + :1 Mg out 2+ ) in the presence of intravesicular Cl? to electroneutral (2Na in + :1 Mg out 2+ ) in their absence. In basolateral liver plasma membrane vesicles loaded with MgCl2 labeled with 36Cl?, a small but significant Cl? efflux (~30 nmol Cl?/mg protein/1 min) is observed following addition of NaCl or Na-isethionate to the extravesicular medium. Both Cl? and Mg2+ effluxes are inhibited by imipramine but not by amiloride, DIDS, niflumic acid, bumetanide, or furosemide. In vesicles loaded with Mg-gluconate and stimulated by Na-isethionate, an electroneutral Mg2+ extrusion is observed. Taken together, these results suggest that the Na+/Mg2+ exchanger can operate irrespective of the absence or the presence of Cl? in the extracellular or intracellular environment. Changes in trans-cellular Cl? content, however, can affect the modus operandi of the Na+/Mg2+ exchanger, and consequently impact "cellular" Na+ and Mg2+ homeostasis as well as the hepatocyte membrane potential. 相似文献
13.
We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides. 相似文献
14.
The limits of resolution that can be obtained in 1H–15N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide 1H resonances. Heteronuclear decoupling of 15N from the 1H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly 2H and 15N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the 15N nuclei. The combination of these techniques results in average 1H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and 15N decoupling are described for achieving the best possible performance in these experiments. 相似文献
15.
V. A. Belyaev D. A. Kozlov A. A. Terent’ev A. E. Trenin 《Plasma Physics Reports》2017,43(10):1039-1041
A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D? 2 and HD? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively. 相似文献
16.
The structures and stabilities of eleven N13
+ and N13
– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N13
+ isomers and six N13
– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N13
+ cation is structure C-2 with C2v symmetry, which contains a pentazole ring and two N4 open chains. It is different from those of the N7
+ and N9
+ clusters, but similar to the N11
+ cluster. Meanwhile, the most stable N13
– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N7
– and N9
– clusters, but also from the N11
– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species.
Figure Optimized geometrical parameters of N13
+ isomer C-2
相似文献
17.
Nishit Goradia Amelie Wißbrock Christoph Wiedemann Frank Bordusa Ramadurai Ramachandran Diana Imhof Oliver Ohlenschläger 《Biomolecular NMR assignments》2016,10(2):329-333
Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the 1H, 13C, and 15N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α. 相似文献
18.
Jorge A. Vila Pedro Serrano Kurt Wüthrich Harold A. Scheraga 《Journal of biomolecular NMR》2010,48(1):23-30
To evaluate sequential nearest-neighbor effects on quantum-chemical calculations of 13Cα chemical shifts, we selected the structure of the nucleic acid binding (NAB) protein from the SARS coronavirus determined
by NMR in solution (PDB id 2K87). NAB is a 116-residue α/β protein, which contains 9 prolines and has 50% of its residues
located in loops and turns. Overall, the results presented here show that sizeable nearest-neighbor effects are seen only
for residues preceding proline, where Pro introduces an overestimation, on average, of 1.73 ppm in the computed 13Cα chemical shifts. A new ensemble of 20 conformers representing the NMR structure of the NAB, which was calculated with an
input containing backbone torsion angle constraints derived from the theoretical 13Cα chemical shifts as supplementary data to the NOE distance constraints, exhibits very similar topology and comparable agreement
with the NOE constraints as the published NMR structure. However, the two structures differ in the patterns of differences
between observed and computed 13Cα chemical shifts, Δ
ca,i
, for the individual residues along the sequence. This indicates that the Δ
ca,i
-values for the NAB protein are primarily a consequence of the limited sampling by the bundles of 20 conformers used, as
in common practice, to represent the two NMR structures, rather than of local flaws in the structures. 相似文献
19.
20.
An extension to HN(CO-α/β-N,Cα-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants 1
J
N′(i)Cα(i), 2
J
HN(i)Cα(i), 2
J
Cα(i−1)N′(i), and 3
J
Cα(i−1)HN(i) in 15N,13C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the 2
J
CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase
and antiphase splittings possible with respect to both 2
J
CαN′ and 1
J
N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can
thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous
2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision.
Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point
Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are
exemplified using proteins of various size. 相似文献