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951.
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953.
Guo Z Benten WP Krücken J Wunderlich F 《The Journal of biological chemistry》2002,277(33):29600-29607
Steroid hormones exert genotropic actions through members of the nuclear receptor family. Here, we have demonstrated genotropic actions of testosterone that are independent of intracellular androgen receptors (iAR). Through plasma membrane androgen receptors (mAR), testosterone induces a rapid rise in the intracellular free Ca(2+) concentration of iAR-free murine RAW 264.7 macrophages. This nongenomic testosterone signaling, which is independent of both iAR and estrogen receptors, does not in itself activate either the mitogen-activated protein kinase (MAPK) families ERK1/2, p38, and JNK/SAPK, the stably and transiently transfected c-fos promoter, or NO production. In the context of lipopolysaccharide (LPS) signaling, however, testosterone attenuates LPS activation of the c-fos promoter and NO production, which is abolished by the intracellular Ca(2+) chelator BAPTA. Testosterone also attenuates the LPS activation of p38 but not that of ERK1/2 and JNK/SAPK, and this attenuation is abrogated by BAPTA. Moreover, the p38 inhibitor, SB 203580, largely reduces LPS activation of the c-fos promoter and NO production, and the remaining levels are no longer regulated by testosterone. This study is the first to provide information on genotropic actions of mAR-mediated nongenomic testosterone Ca(2+) signaling by cross-talk with the LPS signaling pathway through p38 MAPK with impact on cell function. 相似文献
954.
The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and time-resolved fluorescence approaches. We compared the environment and dynamics of tryptophan side chains of empty capsids and ribonucleoprotein particles of two icosahedral viruses from the comovirus group: cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV). We found a great difference between tryptophan fluorescence emission spectra of the ribonucleoprotein particles and the empty capsids of BPMV. For CPMV, time-resolved fluorescence revealed differences in the tryptophan environments of the capsid protein. The excited-state lifetimes of tryptophan residues were significantly modified by the presence of RNA in the capsid. More than half of the emission of the tryptophans in the ribonucleoprotein particles of CPMV originates from a single exponential decay that can be explained by a similar, nonpolar environment in the local structure of most of the tryptophans, even though they are physically located in different regions of the x-ray structure. CPMV particles without RNA lost this discrete component of emission. Anisotropy decay measurements demonstrated that tryptophans rotate faster in empty particles when compared with the ribonucleoprotein particles. The increased structural breathing facilitates the denaturation of the empty particles. Our studies bring new insights into the intricate interactions between protein and RNA where part of the missing structural information on the nucleic acid molecule is compensated for by the dynamics. 相似文献
955.
Ilari A Bonamore A Farina A Johnson KA Boffi A 《The Journal of biological chemistry》2002,277(26):23725-23732
The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 A and refined to an R-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species. 相似文献
956.
Nitric oxide induces degradation of the neutral ceramidase in rat renal mesangial cells and is counterregulated by protein kinase C 总被引:1,自引:0,他引:1
Franzen R Fabbro D Aschrafi A Pfeilschifter J Huwiler A 《The Journal of biological chemistry》2002,277(48):46184-46190
Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. In this study we show that the NO-triggered inhibition of neutral ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin II and ATP, resulting in a normalization of neutral ceramidase protein as well as activity. In vivo phosphorylation studies using (32)P(i)-labeled mesangial cells revealed that TPA, PDGF, angiotensin II, and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca(2+)-dependent isoforms, thus suggesting the involvement of a Ca(2+)-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-delta isoform and to a lesser extent the PKC-alpha isoform are efficient in directly phosphorylating neutral ceramidase. In summary, our data show that NO is able to induce degradation of neutral ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-delta isoenzyme, which can directly phosphorylate and thereby prevent neutral ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral ceramidase stability and subsequent ceramide accumulation. 相似文献
957.
Brashear KM Hunt CA Kucer BT Duggan ME Hartman GD Rodan GA Rodan SB Leu CT Prueksaritanont T Fernandez-Metzler C Barrish A Homnick CF Hutchinson JH Coleman PJ 《Bioorganic & medicinal chemistry letters》2002,12(23):3483-3486
A series of novel, highly potent alpha(v)beta(3) receptor antagonists with favorable pharmacokinetic profiles has been identified. In this series of antagonists, 2-aryl beta-amino acids function as potent aspartic acid replacements. 相似文献
958.
Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels
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Chirico G Cannone F Beretta S Diaspro A Campanini B Bettati S Ruotolo R Mozzarelli A 《Protein science : a publication of the Protein Society》2002,11(5):1152-1161
Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level. 相似文献
959.
Eight populations of Aster tripolium (Compositae) and six of Salicornia ramosissima (Chenopodiaceae) from inland, naturally salt-contaminated habitats and anthropogenic salt-polluted sites in central Germany (Thuringia, Anhalt-Saxony) were analysed using random amplified polymorphic DNA (RAPD) markers to investigate the patterns of genetic variation. In both species, the genetic diversity observed in the younger, anthropogenic sites caused by potash mines during the last century was found to be not significantly lower than in the older, naturally salt-contaminated habitats. Therefore, it is speculated that the loss of genetic diversity caused by founder effects on the anthropogenic habitats was balanced by successive colonization events, actual gene flow between populations, or the rapid growth of populations on the secondary habitats after colonization. Analyses of molecular variance (amova) of the RAPD markers, neighbour-joining clustering of populations based on Reynolds' co-ancestry distances, and Mantel tests indicate that: (i) anthropogenic habitats were colonized independently; (ii) genetic differentiation among populations of S. ramosissima is more pronounced than in A. tripolium, which is considered to be mainly due to biological differences between the two species; and (iii) the geographical pattern of genetic diversity was considerably modulated by historical events and/or population genetic effects. 相似文献
960.
Salvato P Battisti A Concato S Masutti L Patarnello T Zane L 《Molecular ecology》2002,11(11):2435-2444
The winter pine processionary moth has become an important pine pest in the last century, as a consequence of the spread of pine cultivation in the Mediterranean region. The pattern of genetic differentiation of this group, that includes two sibling species (Thaumetopoea pityocampa and Th. wilkinsoni), has been studied in nine populations using amplified fragment length polymorphism (AFLP) and single strand conformation polymorphism-sequence analysis (SSCP) of the mitochondrial cytochrome oxidase 1 (COI) and cytochrome oxydase 2 (COII). Results indicate the existence of strong genetic differentiation between the two species that became separated before the Quaternary ice ages. Moreover data indicate that Th. pityocampa has a strong geographical structure, particularly evident at the nuclear level, where all pairwise phiST resulted to be highly significant and individuals from the same population resulted to be strongly clustered when an individual tree was reconstructed. The estimates of the absolute number of migrants between populations (Nm), obtained from mitochondrial and nuclear DNA markers, suggest that gene flow is low and that a gender-related dispersal could occur in this species. The males appear to disperse more than females, contributing to the genetic diversity of populations on a relatively wide range, reducing the risks of inbreeding and the genetic loss associated with bottlenecks occurring in isolated populations. 相似文献