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71.
A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly 总被引:1,自引:0,他引:1
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In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA. Binding causes a conformational change that is required for subsequent binding events. Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly. The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay. 相似文献
72.
Banning A Schnurr K Böl GF Kupper D Müller-Schmehl K Viita H Ylä-Herttuala S Brigelius-Flohé R 《Free radical biology & medicine》2004,36(2):135-144
Cytokines or hydroperoxides upregulate cell adhesion molecules (CAM) in early stages of atherosclerosis. VCAM-1 expression was therefore investigated in rabbit aortic smooth muscle cells (SMC) stably transfected either with phospholipid hydroperoxide glutathione peroxidase (PHGPx; SMCPHGPx) as a hydroperoxide-reducing enzyme or with 15-lipoxygenase (15-LOX; SMCLOX) as a hydroperoxide-producing enzyme. Transfected cells showed up to 3-fold enhanced PHGPx and a marked LOX activity, respectively, that was absent in controls. Intracellular hydroperoxides were 6-fold higher in SMCLOX than in SMC or SMCPHGPx. Intracellular protein thiols were decreased by 50 and 90% in SMCPHGPx and SMCLOX, respectively. Glutathione mixed disulfides were tentatively increased from SMC via SMCPHGPx to SMCLOX, accordingly. Thiol reduction with tris(2-carboxyethyl)phosphine completely restored protein thiols in SMCPHGPx, whereas in SMCLOX only 60% of control values were recovered. Basal VCAM-1 mRNA levels were decreased by 50% in SMCPHGPx and 75% in SMCLOX. VCAM-1-inducibility was abrogated in SMCLOX but not in SMCPHGPx. Accordingly, NFkappaB-driven reporter gene activation by IL-1 was unaffected in SMCPHGPx but abolished in SMCLOX. The data confirm that PHGPx overexpression dampens CAM expression either by lowering stimulatory hydroperoxides or by using hydroperoxides for protein modification. But hydroperoxides, when constitutively overproduced as in SMCLOX, inhibit CAM expression and render cells refractory to IL-1 stimulation likely due to oxidation of protein thiols of the signaling system. 相似文献
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74.
Canová N Kmonícková E Lincová D Vítek L Farghali H 《Alternatives to laboratory animals : ATLA》2004,32(1):25-35
A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes. In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2-3 days. Hepatocytes cultured in the FMB, specifically in a "sandwich" configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production. Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB. The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that minor changes could be observed at diverse time intervals and in a non-destructive way. The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity. In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications. 相似文献
75.
Kwiterovich PO Chen SC Virgil DG Schweitzer A Arnold DR Kratz LE 《Journal of lipid research》2003,44(6):1143-1155
Twelve obligate heterozygotes from two kindreds were ascertained through phytosterolemic probands homozygous for molecular defects in the ATP binding cassette (ABC) half transporter, ABCG8. The response of these heterozygotes to a Step 1 diet low in fat, saturated fat, and cholesterol, and to 2.2 g daily of plant sterols (as esters) was determined in Protocol I (16 weeks) and Protocol II (28 weeks) during three consecutive feeding periods: Step 1/placebo spread; Step 1/plant sterol spread; and Step 1/placebo spread (washout). At baseline, half the heterozygotes had moderate dyslipidemia and one-third had mildly elevated campesterol and sitosterol levels. On the Step 1/placebo spread, mean LDL cholesterol decreased significantly, 11.2% in Protocol I (n = 12), and 16.0% in Protocol II (n = 7). Substitution with plant sterol spread produced a significant treatment effect on LDL levels in Protocols I and II. Conversely, the mean levels of campesterol and sitosterol increased 119% and 54%, respectively, during the use of plant sterol spread for 6 weeks in Protocol I, an effect mirrored for 12 weeks in Protocol II. During the placebo spread washouts, LDL levels increased, while those of plant sterols decreased to baseline levels in both protocols. In conclusion, phytosterolemic heterozygotes respond well to a Step 1 diet, and their response to a plant sterol ester challenge appears similar to that observed in normals. 相似文献
76.
The mushroom bodies (MBs) within the brain of the honeybee, Apis mellifera, are prominent paired neuropil structures consisting of a lateral and a median subunit. The intrinsic MB neurons (Kenyon cells) of each of these subunits are generated in four distinct proliferation centers, each associated with a calyx. Previous BrdU studies revealed that neurogenesis of Kenyon cells starts at the first larval stage (L1) by symmetrical cell division of Kenyon precursor cells, and ceases abruptly at a midpupal stage (P5). In the present work, we confirmed these results using the antiphospho histone H3 mitosis marker to label mitotically active cells in a cell culture system, in histological sections, and in whole-mount brain preparations. To elucidate whether the steroid hormone ecdysone plays a role in the termination of Kenyon cell neurogenesis, we manipulated the hormone titer by injecting 20-hydroxyecdysone (20E) into animals of those pupal stages (P0/1, P3, P4) in which neurogenesis of Kenyon cells was still extensive. The effects of 20E were evaluated by determining the number of mitotically active cells in confocal microscopic images of squash preparations of the MB proliferation centers. In all pupal stages studied, 20E caused a reduction of mitotic activity, indicating its involvement in the cessation of Kenyon cell neurogenesis. 相似文献
77.
Klumpp S Bechmann G Mäurer A Selke D Krieglstein J 《Biochemical and biophysical research communications》2003,306(1):110-115
The first protein histidine phosphatase from vertebrates discovered recently was found in a variety of tissues, however, a physiological substrate protein was missing. Phosphorylation of liver extracts in the presence of EDTA, followed by SDS-PAGE and autoradiography showed labeling of three proteins. Acid- and alkaline-treatment revealed the existence of N-phosphates. Addition of histidine phosphatase exclusively resulted in dephosphorylation of a 110kDa protein (denaturing conditions). Gelfiltration revealed its native molecular mass of approximately 450kDa. That protein was purified and identified as ATP-citrate lyase. The results are in favor of histidine phosphatase playing an important yet unidentified role in metabolic processes. 相似文献
78.
79.
Arachidonic acid promotes phosphorylation of 5-lipoxygenase at Ser-271 by MAPK-activated protein kinase 2 (MK2) 总被引:3,自引:0,他引:3
Werz O Szellas D Steinhilber D Rådmark O 《The Journal of biological chemistry》2002,277(17):14793-14800
We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation. 相似文献
80.