Fatty acyl-Co A: sphingosine acyltransferase (ceramide synthase, EC 2.3.1.24) is mainly localized in the microsomal and mitochondrial membranes. Attempts to isolate the enzyme have failed, largely because there has been little or no detection of the enzyme activity in detergent extracts. In this study, we solubilized the membrane-bound enzyme from bovine brain mitochondria with a Tris-HCl buffer containing 2% Triton X-100 and, after removal of the detergent, reconstituted it with the membrane lipid liposomes. The specific activity of the reconstituted enzyme was approx. 8 times higher than that of the solubilized enzyme. We next examined the lipid dependence of the enzyme, using various phospholipid liposomes. The ability of phospholipids to enhance the activity of solubilized ceramide synthase was specific and structure-related. The most potent stimulator was phosphatidylserine liposomes, suggesting an important role of the net negative charges. This paper also describes a highly reproducible high-performance liquid chromatographic (HPLC) procedure for the determination of ceramide synthase activity. Combination of the HPLC method with the reconstituted enzyme system appears to be suitable for elucidating the characteristics of this enzyme.

What is known and Objective: It has been reported that ibuprofen interferes with the antiplatelet effect of low-dose aspirin. This interaction is ascribed to steric hindrance at the active site of cyclooxygenase-1 by ibuprofen, when aspirin is administered after ibuprofen. However, whether other nonsteroidal anti-inflammatory drugs (NSAIDs) interact with aspirin similarly is not well defined. The aim of this study was to assess the influence of nine NSAIDs on the antiplatelet effect of aspirin. Methods: We investigated the antiplatelet effect of NSAIDs using steady-state plasma concentration reported after usual doses. We studied the in vitro antiplatelet effect of NSAID alone, aspirin alone, aspirin before NSAID addition and aspirin after NSAID addition to platelet-rich plasma. The rates of platelet aggregation induced by collagen were determined. The final concentration of aspirin used was the 50% effective concentration (EC 50 ) previously estimated in vitro. Results and Discussion: Ibuprofen and mefenamic acid interfere with the antiplatelet effect of aspirin when added before the latter. The rate of platelet aggregation was reduced by 48AE1% and 22AE7%, respectively. The other NSAIDs tested did not significantly affect the aspirin antiplatelet effect when exposure was prior to aspirin. None of the nine NSAIDs altered the aspirin effect if administration followed that of aspirin. What is new and Conclusion: Naproxen and flurbiprofen have significant antiplatelet effects at plasma concentrations seen with usual doses. Our in vitro model suggests that the antiplatelet effect of aspirin is significantly diminished when taken after, but not before, ibuprofen or mefenamic acid. None of the other NSAIDs tested had any effect irrespective of the timing of dosing.

The present study underscores a regulatory role of intracellular ceramide in astrocytes for the release of an extracellular serine protease, tissue-type plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). Treatment of cultured human astrocytes with N-acetylsphingosine, a cell-permeable short-chain ceramide analogue or daunorubicin that could increase intracellular ceramide via activation of ceramide synthase or sphingomyelin hydrolysis increased the release of t-PA and conversely decreased the PAI-1 release. Interestingly, treatment of the astrocytes with tumor necrosis factor (TNF)-alpha also increased the intracellular ceramide levels but caused the elevation of PAI-1 release without altering the t-PA release. These data suggest that the generation of ceramide in astrocytes is linked at least with the regulation of PAI-1 release. We also demonstrate that the suppression of PAI-1 release with daunorubicin accelerates the cell death of neuronally differentiated PC12 cells and suggest an antiapoptotic role of PAI-1 in the nervous system.

The aim of this study was to establish a method to predict the antiplatelet effects of aspirin in vivo based on in vitro results. Aspirin in 5 different concentrations was added to the platelet-rich plasma samples, and the rates of platelet aggregation induced by collagen were determined in vitro. In addition, platelet aggregation and plasma drug concentration values were determined in vivo before and after the administration of aspirin (162 mg). The 50% effective concentration (EC50) values obtained from the in vivo and in vitro experiments were shown to have relevance, because the EC50 ratio for each subject was the same (0.23 ± 0.03). The actual and predicted values for the rate of inhibition of platelet aggregation were well correlated (P < .0001, r = .95) when the predicted rate was determined using the present method. Our results suggest that the antiplatelet effects of aspirin can be predicted using blood samples obtained before its administration.

A highly diastereoselective synthesis of 2-amino alcohol derivatives bearing a difluoromethylphosphonothioate group at the 3-position was achieved through LiAlH(O-t-Bu)(3)-mediated reduction of the corresponding alpha-amino ketones. The phosphonothioate moiety of the product was readily converted into the corresponding phosphonate by oxidation with m-CPBA, followed by aqueous workup. The developed methods should be useful for SAR studies of SMA-7, a potent inhibitor of SMases.