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Nuclear magnetic resonance-visible mobile lipids (ML) have been reported to accumulate during cell apoptosis in vitro and in vivo. The biogenesis, biochemical nature and structure of these lipids are still under debate. In this study, a human lymphoblastoid cell line, HuT 78, was induced to apoptosis by exposure to anti-Fas monoclonal antibodies (alpha-Fas mAb) followed by incubation for different time intervals (1-24 h, hypodiploid cell fraction, H, varying from 1% to over 60%) either in the presence or in the absence of 5.0 microM Triacsin C (TRC), specific inhibitor of long-chain acyl-CoA synthetase (ACS). The increase of ML in apoptotic cells correlated linearly with H and was associated with: (a) accumulation of intracellular lipid bodies, detected by confocal laser scanning microscopy in lipophilic dye-stained cells; (b) increases, detected by thin-layer chromatography in total lipid extracts, in the relative abundance of triacylglycerides (TAG) and cholesteryl esters (CE), with corresponding decreases of phospholipids (PL). TRC completely abolished both ML and lipid body formation in anti-Fas-treated apoptotic cells, with concomitant reversion of TAG, CE and PL to control levels, but did not alter cell viability nor did it inhibit apoptosis. ML signals detected during anti-Fas-induced apoptosis therefore appear to originate from neutral lipids assembled in intracellular lipid bodies, synthesised from cellular acyl-CoA pools.

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.

Elevated contents of phosphoethanolamine (Etn-P) and/or phosphocholine (Cho-P), a common feature of most tumours with respect to normal counterparts, may also occur in non-cancerous proliferating tissues. The significance of these alterations in relation to cell proliferation, differentiation and maturation is scarcely understood. In this work, the Cho-P and Etn-P pools were measured by (31)P-NMR in extracts of chick embryo pectoral muscle at different days of development. The average concentration of these metabolites exhibited the highest values (respectively, 1.5 and 3.0 micromol/mg DNA) on days 9-11 and decreased at later stages of myogenesis. While, however, Cho-P maintained substantial levels (above 1.0 micromol/mg DNA) also during myotube formation (days 11-18) and stepwise decreased (to about 0.5 micromol/mg DNA) upon fibres' maturation, Etn-P gradually decreased between day 11 and hatching time (down to about 0.2 micromol/mg DNA). These results demonstrate that significant changes may occur in the steady-state pools of these metabolites during normal in vivo cellular development and differentiation, and are consistent with: (a) high rates of phospholipid biosynthesis reported in the literature for proliferating myoblasts; (b) sustained phosphatidylcholine synthesis maintained also during myoblast fusion; and (c) decreased requirement of phospholipid synthesis in the last phase of in ovo myofibre maturation.

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3-H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and h4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3-H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1-0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.

High resolution proton spin-lattice relaxation times (T1), spin-spin relaxation times (T2) and resonance linewidths were measured above the gel-to-liquid crystal transition temperature (Tm), in phosphatidylcholine bilayers possessing various degrees of intramolecular motional anisotrophy at the level of various alkyl chain proton groups. The experiments were designed to test the hypothesis that coupled trans-gauche isomerizations along the chains can be responsible for the anisotropic motion of phosphatidylcholine proton groups in bilayer membranes (Horwitz, A.F., Horsley, W.J. and Klein, M.O. (1972) Proc. Natl. Acad. Sci. U.S. 69,500). Systematic series of structural perturbations of the bilayer were achieved in mixed phosphatidylcholine/fatty acid and in mixed phosphatidylcholine bilayers where the degree of motional anisotrophy of the chains' proton groups was gradually reduced by progressively increasing the chain length disparity of the two components. The systematic T1 and T2 variations observed were interterpreted on the basis of the Woessner's treatment for computing the relaxation times of a spin pair reorienting randomly about an axis which, in turn, tumbles randomly (Woessner, D.E. (1962) J. Chem. Phys. 36, 1). The results confirmed in a qualitative sense the original hypothesis made by Horwitz et al. The time-averaged structural interpretations suggested by the mangetic relaxation studies are in agreement with low-angle X-ray diffraction results obtained below Tm. In addition, the T1 values evaluated at various temperatures in dipalmitoyl phosphatidylcholine vesicles incorporated with either 2H-labeled or unlabeled palmitic acid chains indicated that the average intermolecular contribution to the spin-lattice relaxation rate of the proton groups of the phosphatidylcholine chains appears comparable to the intramolecular term at temperatures moderately higher than Tm, but becomes less and less important as the temperature is further increased above the thermal transition.