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Guanghua Zhu
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Fluorescence spectroscopic determination of dipyridamole binding on pancreas-1 tumor cell membrane.
BACKGROUND: The traditional method to determine the binding sites of inhibitors bound to nucleoside transporters on cell membrane is the radioactive assay. This method suffers from radiolabel instability and the need to dispose of reactive materials. Fluorescence spectroscopy has been increasingly applied to biochemical analysis due to its high sensitivity and selectivity. We describe fluorescence spectroscopy for the determination of the binding sites of dipyridamole bound to nucleoside transporters on pancreas-1 tumor cell membrane. METHODS: Pancreas-1 tumor cell was cultured in Dulbecco's modified Eagle medium under appropriate conditions. The cell plasma membrane was separated from the ultrasonically dissolved cell mixture by gradient centrifugation. After dipyridamole was bound to the cell membrane, the test sample was obtained by ultrasonically dissolving the labeled membrane in 10 mmol/l tris-HCl membrane lysis solution. Under selected experimental conditions, the fluorescence intensity of dipyridamole was determined. RESULTS: At lambda em of 295 nm and lambda em of 485 nm, dipyridamole was proportional to its concentration ranging from 1.0 x 10(-12) to 5.0 x 10(-11) mol/l with a detection limit of 2.8 x 10(-13) mol/l (0.14 pg/ml) at 3sigma. The value of affinity constant of the cell membrane to dipyridamole was 4.7(+/-0.5) x 10(10) l/mol and the average total number of binding sites of a pancreas-1 tumor cell was 1.9(+/-0.2) x 10(6). CONCLUSIONS: The proposed method can be used in the study of binding characters of dipyridamole on the cell membrane.
Binding Sites↗
[Effect of fluoroacetamide on cardiomyocytes of rat and the antidotal effect of acetamide].
OBJECTIVE: To observe the effect of fluoroacetamide on cardiomyocytes of rat and the antidotal effect of acetamide. METHODS: 4 groups of SD rats were treated with various dosages of fluoroacetamid(p.o.) and 2 groups of them were treated with acetamide(i.p.). The changes of cardiomyocytes and serum AST, LDH, CK, CK-MB and HBDH were measured at different intervals after poisoning. RESULTS: In the group treated with fluoroacetamid 8 mg/kg. bw, serum AST[(589.58 +/- 821.72) U/L], CK[(916.78 +/- 343.55) U/L], HBDH[(504.47 +/- 148.88) U/L] raised obviously compared with control[(187.70 +/- 46.87), (755.65 +/- 498.90), (347.25 +/- 228.40) U/L respectively] (P < 0.01), and the pathological findings such as degeneration, liquefactive necrosis and filtration of inflammatory cells in cardiac muscles were observed 24 hours later, while all the male dead within 3 days. In the group treated with fluoroacetamid 4 mg/kg. bw, serum LDH and HBDH rose significantly compared with control(P < 0.01) 5 day later. On the day of 10, myocardial enzymes restored in all experiment groups with some interstitial fibroblastic proliferation. The pathological changes were reduced in the group treated with acetamide synchronously (100 mg/kg. bw). CONCLUSION: Acute intoxication of fluoroacetamide could damage cardiomyocytes while acetamide could reduce the injury of them, but the injury was reversible. The levels of serum myocardial enzymes could be a usable index for early diagnosis.
Acetamides↗