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A new method was developed for the separation and determination of inosine monophosphate (IMP) by ion chromatography (IC) with suppressed conductivity detection. Separation was achieved on an anion-exchange column Ionpac AS11-HC of high capacity within a short time. 30 mmol/L KOH produced by an EGC-KOH eluent generator was used for isocratic elution. No interferences existed between the seven common inorganic anions and IMP. Under the optimum conditions, the linear range of the calibration curve for IMP was 1. 0-200 mg/L, with correlation coefficient ( R2 ) of 0. 999. The relative standard deviations (RSDs) for retention time, peak height and peak area of IMP were 0. 16%, 0. 94% and 0. 86%, respectively, indicating good reproducibility of the method. The method was successfully applied to the determination of IMP in meat products, with spiked recovery ranging from 86. 0% to 110. 0%. This simple, accurate and reliable method could be served as a rapid and effective analytical tool for meat flavoring research.
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Objective To study the effect of inosine monophosphate dehydrogenase inhibitor (IMPDHI) on maturation, migration, endocytosis and allostimulatory properties of human peripheral myeloid dendritic cell (MDC). Methods PBMC from healthy donors were isolated. MDC were cocultured with PBMC and exposed to mycophenolic acid (MPA) for 48 h. The expression of co-stimulatory and adhesion molecules as well as chemokine receptors on MDC was analyzed by flow cytometry. In separate experiments,MDC were cultured with or without MPA, and their endocytosis function was estimated by means of FITC dextran uptake. MDC migration experiments were performed in Transwell chambers. Inflammatory chemo kines were added to the lower chambers and MDC numbers were analyzed by flow cytometry. MPA treated (48 h) BDCA-1 + DC served as stimulator cells in MLR. Allogenic healthy CD4 T responder cells were labeled with fluorescent dye CFSE and measured by flow cytometry. Results Maturation: compared to the control group, the expression of CD40, CD62L, HLA-DR, CD54, CD80, CD83 and CD86 on MDC in study group were significantly down-regulated ( P ＜ 0.05 ). Chemokine receptor and migration: compared to control group, the expression of CCR1 on MDC in study group was up-regulated significantly (17.02 ±3.23 vs 30.63 ± 9.13, P ＜ 0.05 ), the expression of CCR3 ( 10.26 ± 2.25 vs 5.81 ± 0.97, P ＜ 0.05 ) and CCR7(9.56 ± 1.84 vs 5.18 ±0.60, P ＜0. 05) on MDC were down-regulated significantly in the study group.MDC in study group showed enchanced migratory response to inflammatory chemokine CCL2, CCL3, CCL4,CCL7, CXCL12 (P＜0.05). Endocytotic capacity: the capacity of endocytosis in study group was signifi cantly higher than that in control group( P ＜ 0.05 ). Llostimulatory capacity: MPA-treated MDC exhibited a markedly reduced ability to stimulate allogenic CD4+ T cell proliferation. Conclusion Treatment of MDC with MPA exhibited an immature phenotype, a propensity to migrate in response to inflammatory chemokines, increased endocytotic capacity and impaired allogenic ability of MDC.
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Objective To study the effect of inosine monophosphate dehydrogenase inhibitor (IMPDHI) on chemotaxis, migration and endocytosis of human peripheral myeloid dendritic cells (MDCs). Methods Freshly isolated peripheral blood mononuclear cells(PBMC)collected from healthy volunteers (N=15) and the study group were treated with IMPDHI. CC chemokine receptors on MDCs were analyzed by flow cytometry. The study group, control group and different chemokines were added via trans-well approach for different chemokines, stained by Lin-1/CD11c/HLA-DR and counted by flow cytometry. The migration index was calculated as a percentage of MDC migrated in response to the tested chemokine. After isolation of blood dendritic cell antigen-1+ (BDCA-1+ ), mannose receptor-mediated endocytosis was measured as the cellular uptake of FITC-dextran by the flow cytometry. Results (1) Compared to the control group, the expression of CCR1 in the study group was up-regulated significantly(17.02±3.23～30.63±9.13, P＜0.05) and the expressions of CCR3(10.26±2.25～5.81±0.97 P＜0.05) and CCR7 (9.56± 1.84～5.18±0.60 P＜0.05)were downregulated significantly. MDCs in the study group showed enhanced migratory response to inflammatory chemokine CCL2, CCL3, CCL4, CCL7 and CXCL12 (P＜0.05). (2)The endocytosis capacity in the study group was significantly higher than that in control group (P ＜0.05). Conclusion IMPDHI enhances the endocytotic capacity of MDCs and impairs the migratory response of peripheral MDCs to lymphocytic tissue by up-regulating the expression of chemokine receptor in MDCs and enhancing migratory response to inflammatory chemokines.
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PURPOSE: Mesangial cell extracellular matrix (ECM) synthesis plays an important role in various renal diseases. Mycophenolic acid (MPA), which is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), inhibits mesangial cell proliferation and ECM synthesis. However, the exact mechanism of MPA has not been clearly elucidated in mesangial cells. To examine the relative importance of IMPDH on the inhibitory action of MPA, we compared the effects of MPA or IMPDH2 siRNA on platelet-derived growth factor (PDGF)-induced fibronectin secretion and cellular reactive oxygen species (ROS) in mouse mesangial cells (MMC). METHODS: MMC were stimulated with PDGF 10 ng/ml with or without MPA 0.1~10micrometer, IMPDH2 siRNA 10~50 nM, or N-acetylcystein (NAC). IMPDH2 siRNA was transiently transfected by lipofectamine for 24 hours. MPA 0.1~10micrometer, ribavirin 10~100micrometer, and NAC 5 mM were administered 1 hour before the stimulation. Cell viability was measured by methylthiazoletetrazolium (MTT) assay, fibronectin secretion by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by flow cytometry. RESULTS: PDGF 10 ng/ml effectively increased fibronectin secretion and cellular ROS in MMC. MPA and NAC at concentration without affecting basal level of fibronectin and cellular ROS ameliorated PDGF-induced fibronectin secretion and cellular ROS. However, IMPDH2 siRNA only partially reduced PDGF- induced fibronectin secretion and cellular ROS in MMC. CONCLUSION: These results suggest that MPA may inhibit PDGF-induced fibronectin secretion partly through IMPDH2 or cellular ROS in MMC, and there may be other mechanisms on the inhibitory action of MPA in mesenchymal cells.