Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.

Green tea contains various antioxidative flavan-3ols (tea catechins), such as (-)-epigallocatechin gallate (EGCg, the major catechin), which exert potent inhibitory effects on LDL oxidation in vitro and ex vivo in humans. In this study, the antiatherogenic effects of tea catechins were examined in atherosclerosis-susceptible C57BL/6J, apoprotein (apo)E-deficient mice. Male apoE-deficient mice (10 wk old) were fed an atherogenic diet for 14 wk; during that time, one group (tea) was supplied drinking water supplemented with green tea extract (0.8 g/L), and another group (control) was offered the vehicle only. The tea extract consisted of the following (g/100 g): EGCg, 58.4; (-)-epigallocatechin (EGC), 11.7; (-)-epicatechin (EC), 6.6; (-)-gallocatechingallate (GCg), 1.6; (-)-epicatechin gallate (ECg), 0.5; and caffeine, 0.4. The estimated actual intake of tea catechin was 1.7 mg/(d. mouse). Tea ingestion did not influence plasma cholesterol or triglyceride concentrations. Plasma lipid peroxides were reduced in the tea group at wk 8, suggesting that the in vivo oxidative state is improved by tea ingestion. Atheromatous areas in the aorta from the arch to the femoral bifurcation and aortic weights were both significantly attenuated by 23% in the tea group compared with the control group. Aortic cholesterol and triglyceride contents were 27 and 50% lower, respectively, in the tea group than in the control group. These results suggest that chronic ingestion of tea extract prevents the development of atherosclerosis without changing the plasma lipid level in apoE-deficient mice, probably through the potent antioxidative activity of the tea.

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Na-Ca exchange current was measured at various concentrations of internal Na ([Na]i) and Ca ([Ca]i) using intracellular perfusion technique and whole-cell voltage clamp in single cardiac ventricular cells of guinea pig. Internal Ca has an activating effect on Na~-Cao exchange beginning at ~10 nM and saturating at ~50 nM with a half maximum 14 mM was obtained. When comparing internal and external Km values, the external value is markedly larger than the internal one and thus we conclude that binding sites of the Na-Ca exchange molecule are at least apparently asymmetrical between the inside and outside of the membrane.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.