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1.
Norepinephrine is N-methylated to epinephrine by the catalytic effect of the terminal enzyme in catecholamine biosynthesis, phenylethanolamine N-methyltransferase (PNMT). PNMT has been covalently immobilized onto a silica-based liquid chromatographic support, glutaraldehyde-P (Glut-P). The resulting PNMT-Glut-P stationary phase (PNMT-SP) was enzymatically active, stable, and reusable. Standard Michaelis-Menten kinetic studies were performed with both free and immobilized PNMT and known substrates and inhibitors were examined. The results demonstrate that the PNMT-SP can be utilized for the rapid screening of potential PNMT substrates as well as the screening of compounds for PNMT inhibitory activity. 相似文献
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Markoglou N Hsuesh R Wainer IW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,804(2):295-302
Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC(50) values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties. 相似文献
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Dexamethasone (DEX) at physiologically elevated (stress) concentration (1 µM) decreased K+-evoked glutamate release from rat hippocampal slices under superfusion in the presence of Ca2+. On the contrary 10 µM DEX increased this K+-evoked glutamate release while 0.1 µM DEX had no effect. The glucocorticoid antagonist for the classic receptor, RU 486, completely reversed the effect of 1 µM DEX. Actinomycin D had no effect. Dexamethasone at 1 µM had no effect on the Ca2+-independent (10 µM Mg2+ replacing 1 mM Ca2+) K+-evoked glutamate release. Dexamethasone at 1 µM or 10 µM had no effect on the phosphate-activated glutaminase—the key enzyme for the biosynthesis of neurotransmitter glutamate. These results suggest that the effect of DEX on K+-evoked glutamate release: (i) depends on its concentration; (ii) is exerted on the Ca2+-dependent (neurotransmitter release), at least at physiological stress concentrations; and (iii) is exerted via the classical receptor but is nongenomic. 相似文献
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RY Zhao NA Mifsud K Xiao KF Chan S Oveissi HM Jackson N Dimopoulos P Guillaume AJ Knights T Lowen NC Robson SE Russell E Scotet ID Davis E Maraskovsky J Cebon IF Luescher W Chen 《PloS one》2012,7(9):e44707
NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8+ T cell epitope, NY-ESO-188–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-188–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1157–165. On the other hand, NY-ESO-1157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A26–35; whereas NY-ESO-188–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-188–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-188–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed. 相似文献
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Binding Rather Than Metabolism May Explain the Interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative ά-Zearalenol
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Hani El-Nezami Nektaria Polychronaki Seppo Salminen Hannu Mykknen 《Applied microbiology》2002,68(7):3545-3549
The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative ¯α-zearalenol (¯α-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 μg ml−1) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and ¯α-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or ¯α-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with ¯α-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds. 相似文献
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El-khalil Bendriss Nektaria Markoglou Irving W. Wainer 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2000,746(2)
An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase. 相似文献
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Jackson H Dimopoulos N Mifsud NA Tai TY Chen Q Svobodova S Browning J Luescher I Stockert L Old LJ Davis ID Cebon J Chen W 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(10):5908-5917
Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject. 相似文献
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Markoglou N Wainer IW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,766(1):145-151
Immobilized enzyme reactors (IMERs) based upon dopamine beta-hydroxylase (DBH) have been developed. Immobilized artificial membrane (IAM) and glutaraldehyde-P (Glut-P) stationary phases have been used to immobilize DBH. When DBH is immobilized on the Glut-P interphase the enzyme is outside the stationary phase whereas with the IAM interphase the enzyme is embedded within the interphase surroundings. The activity of each IMER and their ability for on-line hydroxylation has been investigated. The resulting IMERs are enzymatically active and reproducible. The IMERs can be utilized through the use of coupled chromatography to characterize the cytosolic (DBH-Glut-P-IMER) and membrane-bound (DBH-IAM-IMER) forms of the enzyme. The substrate is injected onto the individual IMERs and the reactants and products are eluted onto a phenylboronic acid column for on-line extraction. The substrates and products are then transported via a switching valve to coupled analytical columns. The results demonstrate that enzyme-substrate and enzyme-inhibitor interactions can be investigated with the on-line system. These IMERs can be utilized for the discovery and characterization of new drug candidates specific for the soluble form and membrane-bound form of DBH. The effects of flow-rate, contact time, pH and temperature have also been investigated. 相似文献
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Nicholaou T Chen W Davis ID Jackson HM Dimopoulos N Barrow C Browning J Macgregor D Williams D Hopkins W Maraskovsky E Venhaus R Pan L Hoffman EW Old LJ Cebon J 《Cancer immunology, immunotherapy : CII》2011,60(11):1625-1637