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11.
Eicosanoids and platelet-activating factor (PAF) are phospholipid-derived lipid mediators produced by various tissues and cells through a cascade pathway. For a comprehensive analysis of these lipid mediators, a simultaneous quantitation method with sensitivity and reliability is necessary. This article details a development of column-switching reversed-phase liquid chromatography-tandem mass spectrometry for multiplex quantitation of eicosanoids and PAF. The adsorptive nature of lipids caused significant loss of signal in a conventional column-switching configuration. The use of an online-dilution method allowed use of 100% methanol as a sample solvent, which prevented sample adsorption to contacting surfaces. Addition of 0.2% formic acid to the sample solvent was required for the successful introduction of LTC4 to the trapping column and minimizing its carryover. The optimized method provided rapid analysis of 14 lipid mediators with a throughput of 96 samples/24 h, lower limits of quantitation of 5 pg on column, and linear calibration ranges up to 2000-5000 pg. The system was highly compatible with solid-phase-extracted samples, as methanol-eluted fractions were directly injected without reconstitution. The analysis of lipid mediator production of macrophage-like RAW264.7 cells demonstrated that the cell-based assay can be performed in a 96-well format, suitable for metabolomics analyses and/or screening strategies. 相似文献
12.
Ryoichi Saiki Ai Nagata Naonori Uchida Tomohiro Kainou Hideyuki Matsuda Makoto Kawamukai 《European journal of biochemistry》2003,270(20):4113-4121
The analysis of the structure and function of long chain-producing polyprenyl diphosphate synthase, which synthesizes the side chain of ubiquinone, has largely focused on the prokaryotic enzymes, and little is known about the eukaryotic counterparts. Here we show that decaprenyl diphosphate synthase from Schizosaccharomyces pombe is comprised of a novel protein named Dlp1 acting in partnership with Dps1. Dps1 is highly homologous to other prenyl diphosphate synthases but Dlp1 shares only weak homology with Dps1. We showed that the two proteins must be present simultaneously in Escherichia coli transformants before ubiquinone-10, which is produced by S. pombe but not by E. coli, is generated. Furthermore, the two proteins were shown to form a heterotetrameric complex. This is unlike the prokaryotic counterparts, which are homodimers. The deletion mutant of dlp1 lacked the enzymatic activity of decaprenyl diphosphate synthase, did not produce ubiquinone-10 and had the typical ubiquinone-deficient S. pombe phenotypes, namely hypersensitivity to hydrogen peroxide, the need for antioxidants for growth on minimal medium and an elevated production of H2S. Both the dps1 (formerly dps) and dlp1 mutants could generate ubiquinone when they were transformed with a bacterial decaprenyl diphosphate synthase, which functions in its host as a homodimer. This indicates that both dps1 and dlp1 are required for the S. pombe enzymatic activity. Thus, decaprenyl diphosphate from a eukaryotic origin has a heterotetrameric structure that is not found in prokaryotes. 相似文献
13.
Hiro-oki Okamura Naonori Sugai Takahiro Kanno Tsuyoshi Shimizu Iwao Ohtani 《Histochemistry and cell biology》1996,106(2):257-260
Tissue specimens from guinea pigs were examined using an enzyme-histochemical reaction to explore the presence of carbonic
anhydrase (CA) activity in the trachea. CA activity was detected in a group of morphologically distinct epithelial cells,
in goblet cells, and in glands of the tracheal mucosa. The epithelial cells showing CA activity were distributed singly and
sparsely throughout the entire trachea. These cells showed a wide morphological variability and were clearly different from
those forming the pseudostratified ciliated epithelium. Their number was higher in sections closer to the tracheal bifurcation
than in those near the larynx. Although the nature of these cells is unknown, based on their morphological and histochemical
characteristics and their distribution, they may represent a specialized chemoreceptor. To our knowledge, this is the first
report of CA localized in tracheal epithelial cells.
Accepted: 6 March 1996 相似文献
14.
Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique 总被引:4,自引:0,他引:4
Hiro-oki Okamura Naonori Sugai Kazunori Suzuki Iwao Ohtani 《Histochemistry and cell biology》1996,106(4):425-430
We have made several improvements in the method of fixation of the inner ear and the enzyme-histochemical technique for carbonic
anhydrase (CA) detection. The results confirmed that CA is localized in the hair cells of the organ of Corti, Deiters' cells
or nerve endings, inner pillar cells, Boettcher's cells, stria vascularis, spiral ligament, spiral limbus, and spiral ganglion
cells. These results generally agree with previous histochemical observations but showed some differences. Our method preserved
tissue morphology and showed more detailed localization of CA activity in the inner ear. In particular, the marginal zone
of stria vascularis and the epithelial cells of spiral prominence, facing the endolymph, showed no CA activity, while the
suprastrial region of the spiral ligament and the supralimbal region of the spiral limbus, juxtaposed to the perilymph, showed
CA activity. In outer hair cells, the cuticular plate, which faces the endolymph showed CA activity, but the lateral membrane,
which faces the perilymph showed no CA activity. In contrast, the inner hair cell cytoplasm showed diffuse CA activity. These
results will be useful in considering ion exchange between endolymph and its adjacent cells, and between perilymph and its
adjacent structures. 相似文献
15.
Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is one of the key enzymes in lipid mediator generation. It preferentially hydrolyzes arachidonoyl-phospholipid in response to cellular stimuli, liberating arachidonic acid, the shared precursor of PGs and leukotrienes. Mice with disruption of the cPLA(2)alpha gene exhibit a more than 80% decrease in the generation of these lipid mediators, leading to dramatic phenotypes in various models of inflammatory and allergic disease. In this study, we use the cecal ligation and puncture model of sepsis along with multiplex quantitation systems to explore interactions between eicosanoids and protein mediators. cPLA(2)alpha-deficient mice exhibited significantly less weight loss accompanied by decreased generation of PGs, leukotriene B(4), IL-6, and CCL2. Despite these differences, genetic ablation of cPLA(2)alpha did not provide any survival advantage. Unexpectedly, abundant production of 12-hydroxy-eicosatetraenoic acid, another arachidonic acid-derived lipid mediator, was found to be unaffected by disruption of the cPLA(2)alpha gene. Eicosanoid production preceded the production of cytokines. Eicosanoid modulation of IL-6 and CCL2 expression was suggested by scattergram analyses. These results provide in vivo evidence for the rapid generation of eicosanoids, regulatory role(s) for cPLA(2)alpha-derived lipid mediators on protein mediator production, and the existence of a robust cPLA(2)alpha-independent pathway(s) of eicosanoid generation. 相似文献
16.
Asai K Hirabayashi T Houjou T Uozumi N Taguchi R Shimizu T 《The Journal of biological chemistry》2003,278(10):8809-8814
To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A(2) (PLA(2)), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca(2+)-independent group IVC PLA(2) isozyme (cPLA(2)gamma), a paralogue of cytosolic PLA(2)alpha (cPLA(2)alpha), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA(2)gamma increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H(2)O(2) and other hydroperoxides induced arachidonic acid release in cPLA(2)gamma-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA(2)gamma is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress. 相似文献
17.
Background
Genomic selection (GS) is a recent selective breeding method which uses predictive models based on whole-genome molecular markers. Until now, existing studies formulated GS as the problem of modeling an individual’s breeding value for a particular trait of interest, i.e., as a regression problem. To assess predictive accuracy of the model, the Pearson correlation between observed and predicted trait values was used.Contributions
In this paper, we propose to formulate GS as the problem of ranking individuals according to their breeding value. Our proposed framework allows us to employ machine learning methods for ranking which had previously not been considered in the GS literature. To assess ranking accuracy of a model, we introduce a new measure originating from the information retrieval literature called normalized discounted cumulative gain (NDCG). NDCG rewards more strongly models which assign a high rank to individuals with high breeding value. Therefore, NDCG reflects a prerequisite objective in selective breeding: accurate selection of individuals with high breeding value.Results
We conducted a comparison of 10 existing regression methods and 3 new ranking methods on 6 datasets, consisting of 4 plant species and 25 traits. Our experimental results suggest that tree-based ensemble methods including McRank, Random Forests and Gradient Boosting Regression Trees achieve excellent ranking accuracy. RKHS regression and RankSVM also achieve good accuracy when used with an RBF kernel. Traditional regression methods such as Bayesian lasso, wBSR and BayesC were found less suitable for ranking. Pearson correlation was found to correlate poorly with NDCG. Our study suggests two important messages. First, ranking methods are a promising research direction in GS. Second, NDCG can be a useful evaluation measure for GS. 相似文献18.
Aoki C Hidari KI Itonori S Yamada A Takahashi N Kasama T Hasebe F Islam MA Hatano K Matsuoka K Taki T Guo CT Takahashi T Sakano Y Suzuki T Miyamoto D Sugita M Terunuma D Morita K Suzuki Y 《Journal of biochemistry》2006,139(3):607-614
The interaction between cell surface receptors and the envelope glycoprotein (EGP) on the viral membrane surface is the initial step of Dengue virus infection. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate the molecular mechanisms of the interaction of EGP with receptor molecules. Here, using a TLC/virus-binding assay, we isolated and characterized a carbohydrate molecule on mammalian cell surfaces that is recognized by dengue virus type 2 (DEN2). Structural determination by immunochemical methods showed that the carbohydrate structure of the purified glycosphingolipid was neolactotetraosylceramide (nLc4Cer). This glycosphingolipid was expressed on the cell surface of susceptible cells, such as human erythroleukemia K562 and baby hamster kidney BHK-21. All serotypes of DEN viruses, DEN1 to DEN4, reacted with nLc4Cer, and the non-reducing terminal disaccharide residue Galbeta1-4GlcNAcbeta1- was found to be a critical determinant for the binding of DEN2. Chemically synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide, inhibited DEN2 infection of BHK-21 cells. These findings strongly suggested that multivalent nLc4 oligosaccharide could act as a competitive inhibitor against the binding of DEN2 to the host cells. 相似文献
19.
Tadashi Toyama Naonori Momotani Yuka Ogata Yuji Miyamori Daisuke Inoue Kazunari Sei Kazuhiro Mori Shintaro Kikuchi Michihiko Ike 《Applied and environmental microbiology》2010,76(20):6733-6740
We isolated three Sphingobium fuliginis strains from Phragmites australis rhizosphere sediment that were capable of utilizing 4-tert-butylphenol as a sole carbon and energy source. These strains are the first 4-tert-butylphenol-utilizing bacteria. The strain designated TIK-1 completely degraded 1.0 mM 4-tert-butylphenol in basal salts medium within 12 h, with concomitant cell growth. We identified 4-tert-butylcatechol and 3,3-dimethyl-2-butanone as internal metabolites by gas chromatography-mass spectrometry. When 3-fluorocatechol was used as an inactivator of meta-cleavage enzymes, strain TIK-1 could not degrade 4-tert-butylcatechol and 3,3-dimethyl-2-butanone was not detected. We concluded that metabolism of 4-tert-butylphenol by strain TIK-1 is initiated by hydroxylation to 4-tert-butylcatechol, followed by a meta-cleavage pathway. Growth experiments with 20 other alkylphenols showed that 4-isopropylphenol, 4-sec-butylphenol, and 4-tert-pentylphenol, which have alkyl side chains of three to five carbon atoms with α-quaternary or α-tertiary carbons, supported cell growth but that 4-n-alkylphenols, 4-tert-octylphenol, technical nonylphenol, 2-alkylphenols, and 3-alkylphenols did not. The rate of growth on 4-tert-butylphenol was much higher than that of growth on the other alkylphenols. Degradation experiments with various alkylphenols showed that strain TIK-1 cells grown on 4-tert-butylphenol could degrade 4-alkylphenols with variously sized and branched side chains (ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, tert-octyl, n-nonyl, and branched nonyl) via a meta-cleavage pathway but not 2- or 3-alkylphenols. Along with the degradation of these alkylphenols, we detected methyl alkyl ketones that retained the structure of the original alkyl side chains. Strain TIK-1 may be useful in the bioremediation of environments polluted by 4-tert-butylphenol and various other 4-alkylphenols.4-tert-Butylphenol is an alkylphenol with a tertiary branched side chain of four carbon atoms at the para position of phenol. It is an industrially important chemical and is abundantly and widely used for the production of phenolic, polycarbonate, and epoxy resins. Production of 4-tert-butylphenol in the European Union in 2001 was 25,251 tons (t) (9). In Japan, according to the National Institute of Technology and Evaluation (http://www.safe.nite.go.jp/english/sougou/view/ComprehensiveInfoDisplay_en.faces), production of 4-tert-butylphenol amounted to 27,761 t in 2007. 4-tert-Butylphenol is widely distributed in aquatic environments, including river waters (20), seawaters (17), river sediments (17), marine sediments (23), and effluent samples from sewage treatment plants and wastewater treatment plants (22). Furthermore, 4-tert-butylphenol interacts with estrogen receptors (29, 30, 34, 35, 39) and exhibits other toxic effects on aquatic organisms and humans (4, 15, 16, 25, 26, 42, 43). Therefore, it is necessary to study the biodegradation of 4-tert-butylphenol to understand its fate in the aquatic environment, to establish technologies to treat the waters polluted by it, and to remove it from contaminated environments.Studies of the biodegradation of alkylphenols have focused mainly on branched 4-nonylphenol. Several strains of sphingomonad bacteria, including Sphingomonas sp. strain TTNP3 (38), Sphingobium xenophagum Bayram (11), and Sphingomonas cloacae S-3T (10), have recently been isolated from activated sludge. These strains can degrade branched 4-nonylphenol and utilize it as a sole carbon source. In addition, several Pseudomonas strains that can degrade medium-chain 4-n-alkylphenols (e.g., 4-n-butylphenol) and utilize them as sole carbon sources have been isolated from activated sludge or contaminated soil; they include Pseudomonas veronii INA06 (1), Pseudomonas sp. strain KL28 (21), and Pseudomonas putida MT4 (36). Biodegradation of branched 4-nonylphenol and 4-n-butylphenol has been well studied, but little is known about the biodegradation of 4-tert-butylphenol, although this compound has a structure similar to those of branched 4-nonylphenol and 4-n-butylphenol. There is only one report on the biotransformation of 4-tert-butylphenol—by resting cells of S. xenophagum strain Bayram grown on technical nonylphenol—but this strain cannot grow on 4-tert-butylphenol (11, 14). To our knowledge, there are no reports of bacteria that utilize 4-tert-butylphenol as the sole carbon source, and the biochemical pathway of 4-tert-butylphenol utilization has not been described.Here we characterize three Sphingobium fuliginis strains—TIK-1, TIK-2, and TIK-3—isolated from rhizosphere sediment of the reed Phragmites australis. These strains could use 4-tert-butylphenol as a sole carbon source. On the basis of additional tests of strain TIK-1, we propose that it degrades 4-tert-butylphenol through 4-tert-butylcatechol along a meta-cleavage pathway. We also show that strain TIK-1 cells grown on 4-tert-butylphenol can degrade a wide range of 4-alkylphenols via a meta-cleavage pathway. 相似文献
20.
Yong-Zheng Wu Mohammad Abolhassani Mario Ollero Fariel Dif Naonori Uozumi Micheline Lagranderie Takao Shimizu Michel Chignard Lhousseine Touqui 《Respiratory research》2010,11(1):49