Fas-induced apoptosis of T cells occurs independently of ceramide generation

The Fas receptor is one of a number of important physiological inducers of programmed cell death (apoptosis). Current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases. Induced changes in cellular levels of such sphingosine-based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate by treatment of cellular lipid extracts with bacterial diacylglycerol kinase (DAGK). To allow direct study of cellular sphingosine- and sphinganine-based ceramide levels, we developed a mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations. Contrary to current models, we detected no changes in cellular ceramide levels up to 2 hr poststimulation of Jurkat T cells with an anti-Fas IgM, although this treatment did induce apoptosis. We also determined in the same system that, when utilizing the DAGK assay, increased phosphorylation of substrates that comigrated with ceramide standards was apparent but that this effect was due to an enhancement of DAGK activity rather than increases in levels of cellular ceramides as substrates per se. Thus, the first direct measurement of ceramides present in cells undergoing apoptosis indicates that, insofar as it can be measured, the induction of apoptosis does not involve the generation of sphingosine-based ceramides, contrary to many published accounts.

Fluorescence-intensity distribution analysis and its application in biomolecular detection technology

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5′-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.