目的:探讨三氧化二砷(arsen ic trioxide,ATO)对白血病耐药细胞株K562/A02细胞表达的基质金属蛋白酶2,9(MMP-2、9)活性的影响。方法:用MTT法检测不同浓度ATO对K562/A02细胞的增殖抑制率;用明胶酶谱法检测ATO对K562/A02细胞MMP-2、9活性...目的:探讨三氧化二砷(arsen ic trioxide,ATO)对白血病耐药细胞株K562/A02细胞表达的基质金属蛋白酶2,9(MMP-2、9)活性的影响。方法:用MTT法检测不同浓度ATO对K562/A02细胞的增殖抑制率;用明胶酶谱法检测ATO对K562/A02细胞MMP-2、9活性的影响。结果:K562/A02细胞的MMP-2、9活性条带在对照组以MMP-2(72KD)最宽,MMP-9次之,MMP-2(62KD)最窄。MMP-2、9经0.05μMATO处理后在24、48小时与对照组比较无明显差异(P>0.05),72小时差异才具有显著性(P<0.05)。MMP-2、9的活性经0.4μM和3.2μM的ATO处理后随作用时间延长而抑制作用也逐渐增强,与对照组比较差异均有显著性(P<0.05)。结论:ATO可以有效抑制K562/A02细胞的MMP-2、9的活性,但其作用的强度与剂量及作用时间有关。ATO对MMP-2、9的活性的抑制可能是其抗白血病细胞血管新生的机制之一。展开更多
Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are over-expressed in joints of rheumatoid arthritis (RA) patients. However, the contribution of specific MMPs for the development of arthritic joints...Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are over-expressed in joints of rheumatoid arthritis (RA) patients. However, the contribution of specific MMPs for the development of arthritic joints is unknown. This study is aimed at studying the role of matrix metalloproteinase-9 (MMP-9) in mice, using the K/BxN serum-transfer model of RA. Arthritis was induced in Balb/c mice by injecting K/BxN serum. Development of arthritis was followed in these mice by measuring ankle thickness and clinical index score. MMP-9 expression in the joints of mice killed at various time points during the disease progression was determined by gelatin zymography using ankle lysates. We found that MMP-9 expression increased with the severity of arthritis. Importantly MMP-9 deficient mice injected with K/BxN serum showed a milder form of arthritis in comparison to the control C57BL/6 mice injected with K/BxN serum. We therefore conclude that MMP-9 promotes arthritis in mice.展开更多
AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quen...AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.展开更多
文摘目的:探讨三氧化二砷(arsen ic trioxide,ATO)对白血病耐药细胞株K562/A02细胞表达的基质金属蛋白酶2,9(MMP-2、9)活性的影响。方法:用MTT法检测不同浓度ATO对K562/A02细胞的增殖抑制率;用明胶酶谱法检测ATO对K562/A02细胞MMP-2、9活性的影响。结果:K562/A02细胞的MMP-2、9活性条带在对照组以MMP-2(72KD)最宽,MMP-9次之,MMP-2(62KD)最窄。MMP-2、9经0.05μMATO处理后在24、48小时与对照组比较无明显差异(P>0.05),72小时差异才具有显著性(P<0.05)。MMP-2、9的活性经0.4μM和3.2μM的ATO处理后随作用时间延长而抑制作用也逐渐增强,与对照组比较差异均有显著性(P<0.05)。结论:ATO可以有效抑制K562/A02细胞的MMP-2、9的活性,但其作用的强度与剂量及作用时间有关。ATO对MMP-2、9的活性的抑制可能是其抗白血病细胞血管新生的机制之一。
文摘Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are over-expressed in joints of rheumatoid arthritis (RA) patients. However, the contribution of specific MMPs for the development of arthritic joints is unknown. This study is aimed at studying the role of matrix metalloproteinase-9 (MMP-9) in mice, using the K/BxN serum-transfer model of RA. Arthritis was induced in Balb/c mice by injecting K/BxN serum. Development of arthritis was followed in these mice by measuring ankle thickness and clinical index score. MMP-9 expression in the joints of mice killed at various time points during the disease progression was determined by gelatin zymography using ankle lysates. We found that MMP-9 expression increased with the severity of arthritis. Importantly MMP-9 deficient mice injected with K/BxN serum showed a milder form of arthritis in comparison to the control C57BL/6 mice injected with K/BxN serum. We therefore conclude that MMP-9 promotes arthritis in mice.
基金Supported by A postdoctoral fellowship (Van den Steen PE) and a research assistantship (Geurts N) of the Fund for Scientific Research,Belgium (FWO-Vlaanderen)
文摘AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.
