Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, is released by either acid sphingomyelinase or neutral sphingomyelinase (nSMase) and has emerged as a potential lipid signaling molecule. nSMase is regarded as a key enzyme in the regulated activation of the “sphingomyelin cycle” and cell signaling. We report here the cloning, identification, and functional characterization of murine and human nSMase, a ubiquitously expressed integral membrane protein, which displays all established properties of the Mg 2+ -dependent nSMase of the plasma membrane. Stably nSMase-overexpressing U937 and human embryonic kidney cell lines have been generated for the study of the role of nSMase in signal transduction pathways. Their stimulation by tumor necrosis factor α leads only to a moderately elevated ceramide concentration. Activation of Jun kinase and NFκB and poly(ADP-ribose) polymerase cleavage are identical in mock- and nSMase-transfected cells. Tumor necrosis factor α triggers the ERK1 pathway in none of the cell lines. The cloned nSMase will facilitate further controlled experiments aiming at the definition of a possible role of ceramide as signal transduction molecule.

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Acid sphingomyelinase-deficient (asmase 7/7 ) mice generated by gene targeting abundantly store sphingomyelin in the reticuloendothelial system of liver, spleen, bone marrow, and in brain. Liver cells of asmase 7/7 mice accumulate sphingomyelin and glycosphingolipids in purified lipid bilayers of microsomes, Golgi, and the plasma membrane, but cholesterol is depleted in the plasma membrane. Detergent-insoluble glycolipid-enriched membrane microdomains (GEM) can be isolated from hepatocytes, embryonic fibroblasts, and splenocytes of wild-type, but not of asmase 7/7 mice, by sucrose gradient density centrifugation. Lck and other Src-family kinases are reduced in isopycnic fractions of asmase 7/7 splenocytes compared to GEMcontaining fractions of wild-type cells. The proliferation of asmase 7/7 T lymphocytes is reduced, whereas their susceptibility to Fas-induced apoptosis is increased after T cell receptor (TCR) stimulation. TNF receptor I signaling remains unimpaired. The perturbation of GEM impairs tyrosine phosphorylation and, consequently, mitogenic signaling of the TCR. Reduced MAPK activity-dependent FLICE-like inhibitory protein (FLIP) expression in asmase 7/7 T lymphocytes increases their sensitivity towards Fasmediated apoptosis. Cell Death and Differentiation (2000) 7, 413 ± 424.

Human embryonic stem (hES) cells provide an important tool for the study of human development, disease, and tissue regeneration. Technologies for efficient genetic modification are required to exploit hES cells fully for these applications. Here we present a customized protocol for the transfection of hES cells with the Nucleofector technology and compare its efficiency with conventional electroporation and lipofection. Cell survival and transfection efficiency were quantified using an enhanced green fluorescent protein (EGFP) reporter construct. Our optimized nucleofection parameters yielded survival rates >70%. Under these conditions, 66% of the surviving cells showed transgene expression 24 h after nucleofection. Transfected cells maintained expression of the pluripotency- associated markers Tra-1-60, Tra-1-81, and Oct4 and could be expanded to stably transgene-expressing clones. The low quantities of hES cells and DNA required for nucleofection could make this method an attractive tool for miniaturized high throughput screening (HTS) applications.

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