PubMed · 10563321
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B Liu, Y A Hannun. 2000. Sphingomyelinase assay using radiolabeled substrate.. https://doi.org/10.1016/s0076-6879(00)11077-8
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As a prelude to detailed flux control analysis of lipid synthesis in plants, we have examined the latter in tissue cultures from two important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). Temperature was used to manipulate the overall rate of lipid formation in order to characterize and validate the system to be used for analysis. With [1-14C]acetate as a precursor, an increase in temperature from 20 to 30 degrees C produced nearly a doubling of total lipid labelling. This increase in total lipids did not change the radioactivity in the intermediate acyl-(acyl carrier protein) or acyl-CoA pools, indicating that metabolism of these pools did not exert any significant constraint for overall synthesis. In contrast, there were some differences in the proportional labelling of fatty acids and of lipid classes at the two temperatures. The higher temperature caused a decrease in polyunsaturated fatty acid labelling and an increase in the proportion of triacylglycerol labelling in both calli. The intermediate diacylglycerol was increased in olive, but not in oil palm. Overall the data indicate the suitability of olive and oil-palm cultures for the study of lipid synthesis and indicate that de novo fatty acid synthesis may exert more flux control than complex lipid assembly. In olive, diacylglycerol acyltransferase may exert significant flux control when lipid synthesis is rapid.
The effects of soil depth and moisture on pesticide photolysis were studied. Moist soil at depths of 3, 2.5, 2, 1.5, 1, and 0.5 mm were each dosed at 2.5 microg/g with (14)C-niclosamide and photolyzed under a xenon lamp at constant temperature. Samples were removed after 20, 40, 110, and 153 h of continuous irradiation. The decrease in percent of niclosamide and the appearance of degradates were followed by analyzing the soil extracts by HPLC. A corresponding set of experiments used air-dried soil. An experiment was also performed using initially moist soil which was permitted to dry during photolysis but returned to moist conditions at each sampling. Qualitative and quantitative differences were found in the rate and route of degradation of niclosamide under these conditions. These differences have resulted from a combination of reduced photochemical activity and microbial population in dry soil. The half-lives of niclosamide in the dry soils were 2 to 5 times longer than those in the moisture-maintained soil. There was also a noticeable difference in the half-lives in soil of different depths. Moisture-maintained soil showed a uniform linear increase in half-life from 95 to 195 h as soil depth increased from 0.5 mm to 3.0 mm. With air-dried soil the half-lives were greatly dependent on soil depth, showing a much broader range of 199 h at 0.5-mm to 1064 h in 3.0-mm soil. An experimental design is described which maintains soil temperature and moisture to preset conditions.
OBJECTIVE: To develop a non-high-performance liquid chromatography method for the determination of thiopurine- S-methyltransferase (TPMT) phenotype using thin-layer chromatography and quantitative scanning. METHODS: TPMT reaction was performed using a radiochemical assay. The reaction product [(14)C]-6-methylmercaptopurine was separated using thin-layer chromatography and quantified by means of radioactive scanning. Day-to-day variance was determined to validate results. RESULTS: Determination of TPMT phenotype using thin-layer chromatography and quantitative scanning is reliable (day-to-day variance 8.5+/-1.7%, mean+/-SEM). Mean TPMT activity in 314 randomly selected patient samples was 11.8+/-3.3 units/ml red blood cells (mean+/-SD, range 3.5-25 units/ml). CONCLUSION: We developed a new assay variant for the determination of TPMT phenotype that is easy to perform, reliable and reduces production of radioactive waste. This may lead to more frequent pretreatment determination of TPMT phenotype and increase drug safety and efficacy by individualising thiopurine doses.