Using membrane stress to our advantage.

The nature of the bilayer motif coupled with the ability of lipids and proteins to diffuse freely through this structure is crucial to the viability of cells and their ability to compartmentalize domains contained therein. It seems surprising to find then that biological as well as model membranes exist in a dynamic state of mechanical stress. The stresses within such membranes are surprisingly large, typically reaching up to 50 atm (1 atm=101.325 kPa) at the core of the membrane and vary as a function of depth. The uneven distribution of lateral pressures within monolayer leaflets causes them to bend away from or towards the water interface. This can result in the formation of complex, self-assembled mesophases, many of which occur in vivo. Our knowledge of the principles underlying membrane mechanics has reached the point where we are now able to manipulate them and create nano-structures with reasonable predictability. In addition, they can be used both to explain and control the partitioning of amphipathic proteins on to membranes. The dependence of the dynamics of membrane-bound proteins and the chemical reactivity of amphipathic drug molecules on membrane stresses suggests that Nature itself takes advantage of this. Understanding and manipulating these internal forces will be a key element in creating self-assembled, biocompatible, nanoscale cell-like systems.

Regulation of cellular processes by PPARgamma ligands in neuroblastoma cells is modulated by the level of retinoblastoma protein expression.

Neuroblastoma is a childhood cancer, which spontaneously regresses. This has led to a search for agents that mimic this process. We show that both natural and synthetic ligands of PPARgamma (peroxisome-proliferator-activated receptor gamma) inhibit the growth of neuroblastoma cells in vitro. The degree of PPAR activation was attenuated however in the presence of the retinoblastoma protein. Addition of trichostatin A, a histone deacetylase inhibitor, abolished retinoblastoma protein repression of PPAR activity. Moreover, enhanced growth inhibition was observed when neuroblastoma cells were treated with a PPARgamma ligand and a histone deacetylase inhibitor, suggesting a combination therapy to treat neuroblastoma might prove more effective than using either agent alone.

Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells.

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d(6)-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241(-) and m/z 247(-) respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ.

Dynamic lipidomics using ESI-MS (tandem electrospray ionization mass spectrometry) of 9-deuterated choline (choline-d(9)) incorporation into mammalian cell PtdCho (phosphatidylcholine) permits assessment of the molecular specificity of synthesis. Bulk cell PtdCho synthesis occurs in spatially distinct locations, using separate CPTs (1,2 diacylglycerol CDP:choline cholinephosphotransferases). We assessed whether in vitro molecular selectivity of DAG (diacylglycerol) incorporation between CPTs is manifest in situ, by monitoring choline-d(9) incorporation into PtdCho and lyso-PtdCho molecular species over 3 h in control cells and in CHO-K1 cells overexpressing hCEPT1. Compared with controls, the basal molecular species composition of hCEPT1 overexpressors was significantly enriched in arachidonate. This was not due to net accretion of cellular PtdCho arguing against effects of inadequate unsaturated PtdCho degradation or remodelling. Rather, time-course analyses of PtdCho and lyso-PtdCho pools showed that both arachidonate-containing DAG incorporation and turnover of PtdCho is increased in hCEPT1 overexpressors. Increased choline-d(9) incorporation into arachidonyl lyso-PtdCho shows that both phospholipase A(1)- and A(2)-mediated turnover is involved. Spatially distinct molecular specificity of DAG incorporation into cellular PtdCho at the level of hCEPT1 exists in situ.

Use of mass spectrometry-based lipidomics to probe PITPalpha (phosphatidylinositol transfer protein alpha) function inside the nuclei of PITPalpha+/+ and PITPalpha-/- cells.

The mammalian phospholipid exchange protein PITPalpha (phosphatidylinositol transfer protein alpha), found in both extranuclear and endonuclear compartments, is thought in part to facilitate nuclear import of the PtdIns (phosphatidylinositol) consumed in the generation of proliferation-associated endonuclear diacylglycerol accumulations. Unlike phosphatidylcholine, endonuclear PtdIns is not synthesized in situ. However, despite progressive postnatal lethality of PITPalpha ablation in mice, PITPalpha(-/-) MEF (mouse embryonic fibroblasts) lack an obviously impaired proliferative capacity. We used ESI-MS (tandem electrospray ionization-MS) to monitor incorporation of the deuterated phospholipid precursors, choline-d(9) and inositol-d(6), into molecular species of whole cell and endonuclear phosphatidylcholine and PtdIns over 24 h to assess the contribution of PITPalpha to the nuclear import of PtdIns into MEF cells. In cells labelled for 1, 3, 6, 12 and 24 h fractional inositol-d(6) incorporation into whole-cell PtdIns species was consistently higher in PITPalpha(-/-) MEF implying greater flux through its biosynthetic pathway. Moreover, endonuclear accumulation of PtdIns-d(6) was apparent in the PITPalpha(-/-) cells and mirrored that in PITPalpha(+/+) cells. Together, these results suggest that the essential endonuclear PtdIns import via PITPalpha can be accommodated by other mechanisms.

Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes.

Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase alpha is necessary for cell survival questions whether endonuclear, CDP-choline pathway phosphatidylcholine synthesis may occur in situ. We report that chromatin from human IMR-32 neuroblastoma cells possesses such a biosynthetic pathway. First, membrane-free nuclei retain all three CDP-choline pathway enzymes in proportions comparable with the content of chromatin-associated phosphatidylcholine. Second, following supplementation of cells with deuterated choline and using electrospray ionization mass spectrometry, both the time course and molecular species labeling pattern of newly synthesized endonuclear and whole cell phosphatidylcholine revealed the operation of spatially separate, compositionally distinct biosynthetic routes. Specifically, endogenous and newly synthesized endonuclear phosphatidylcholine species are both characterized by a high degree of diacyl/alkylacyl chain saturation. This unusual species content and synthetic pattern (evident within 10 min of supplementation) are maintained through cell growth arrest by serum depletion and when proliferation is restored, suggesting that endonuclear disaturated phosphatidylcholine enrichment is essential and closely regulated. We propose that endonuclear phosphatidylcholine synthesis may regulate periodic nuclear accumulations of phosphatidylcholine-derived lipid second messengers. Furthermore, our estimates of saturated phosphatidylcholine nuclear volume occupancy of around 10% may imply a significant additional role in regulating chromatin structure.

Modulation of CTP:phosphocholine cytidylyltransferase by membrane curvature elastic stress.

The activity of CTP:phosphocholine cytidylyltransferase, a rate-limiting enzyme in phosphatidylcholine biosynthesis, is modulated by its interaction with lipid bilayers [Kent, C. (1997) Biochim. Biophys. Acta 1348, 79-90]. Its regulation is of central importance in the maintenance of membrane lipid homeostasis. Here we show evidence that the stored curvature elastic stress in the lipid membrane's monolayer modulates the activity of CTP:phosphocholine cytidylyltransferase. Our results show how a purely physical feedback signal could play a key role in the control of membrane lipid synthesis.

Phospholipid composition of neonatal guinea pig liver and plasma: effect of postnatal food restriction.

Preterm guinea pigs were delivered on day 65 of gestation (term = 68 d) and were allowed either free or restricted access to food for the subsequent 48 h. Plasma phosphatidylcholine (PC) concentration increased postnatally from 190 (range 144-307) to 751 (426-1039) and 883 (758-977) microM for fed and starved pups, respectively. Plasma PC composition in both groups of pups was characterized by selective and equivalent relative increases to individual molecular species containing 18:0 at the sn-1 position. Hepatic PC concentration increased from 6.75 (5.41-8.20) to 8.65 (6.54-10.63) and 9.23 (8.18-10.17) mumol/g for fed and starved pups, respectively, and, under all conditions, hepatic PC molecular composition closely mirrored that of plasma PC. These results support the hypothesis that the molecular species composition of plasma PC for the guinea pig in the immediate postnatal period is determined largely by the composition of the hepatic PC pool destined for lipoprotein secretion. Hepatic PC composition and concentration of the starved neonatal guinea pig were maintained independently of any dietary nutrient intake, at the expense of mobilization of extra hepatic lipid reserves. While this adaptive mechanism has inherent limited survival potential in neonatal starvation, it has implications for studies measuring plasma phospholipid fatty acid compositions as biochemical markers of dietary fat intake in preterm infants.

Mechanisms of hepatic phosphatidylcholine synthesis in adult rat: effects of pregnancy.

Late pregnancy in the rat (gestational ages 16-21 days) was accompanied by a specific increase in hepatic phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species containing C16:0 at the sn-1 position and polyunsaturated essential fatty acids (PUFA), in particular C22:6(n-3), at the sn-2 position. Incorporation of either CDP:[Me-14C]choline or CDP:[1,2-14C]-ethanolamine into hepatic microsomal sn-1 C16:0 PC or PE molecular species in vitro was greater at term than in non-pregnant animals, suggesting modifications to the composition of specific diacylglycerol (DAG) pools destined for synthesis of either PC or PE. Also, incorporation of [Me-14C]choline or [Me-14C]methionine into hepatic PC in vivo over 6 h in term pregnant rats was consistent with decreased phospholipase A1-dependent acyl remodelling of sn-1 C16:0 to sn-1 C18:0 molecular species. There was, however, no evidence to support any change to the specificity of acyl remodelling. The rate of PC synthesis by the de novo pathway in vivo was increased in term liver compared with non-pregnant animals, accompanied by increased choline-phosphotransferase activity in vitro in d21 liver microsomes. The rate of PC synthesis by PE N-methylation did not appear to change during pregnancy. Changes in composition of plasma PC species at term reflected those of newly synthesized hepatic PC. Our data suggest supply of PUFA to the developing fetal rat is the result of specific adaptations to maternal hepatic phospholipid biosynthesis rather than passive transfer from the maternal diet.

Developmental variation in whole human lung phosphatidylcholine molecular species: a comparison with guinea pig and rat.

Detailed analysis of the pattern of human and rodent lung phosphatidylcholine (PC) species during fetal development revealed a progressive increase in two disaturated species. The rise in the fractional content of dipalmitoyl PC (PC16:0/16:0) and myristoylpalmitoyl PC (PC14:0/16:0) was accompanied at each time point by a fall of similar magnitude in palmitoyloleoyl PC (PC16:0/18:1). Up to 20% of term lung PC was PC14:0/16:0. The temporal increase in rodent lung PC saturation began later in gestation than the human, and in the rat a significant increase in PC saturation only occurred postnatally. In this respect the guinea pig more closely resembled the human. For each mammal, a ratio of whole lung PC16:0/16:0 to PC16:0/18:1 (the P/O ratio) provided a sensitive marker of fetal lung maturity. The PC composition of whole adult lung and its saturation enrichment in bronchoalveolar lavage samples were similar in human, guinea pig and rat. We propose that the guinea pig provides a useful model for human lung prematurity studies.

Biochemical maturation of the guinea pig lung and survival following premature delivery.

1. Viable preterm guinea pigs were delivered by Caesarean section from 62 day gestation (term = 68 days). 2. Survival rates (24 hr) were greater than 90%, greater than 55% and greater than 35% respectively at 65, 63 and 62 days gestation. Guinea pig pups experienced increasing respiratory difficulty with progressive prematurity. 3. Lung phosphatidylcholine concentration increased steadily from 0.52 +/- 0.09 mumol/mg DNA at day 50 to 3.9 +/- 0.5 mumol/mg DNA at term. The relative contribution of the disaturated dipalmitoyl species increased over this time from 24.5 to 42.9%. 4. Pulmonary antioxidant capacity increased markedly over the final eight days of gestation, individual increases being manganese superoxide dismutase 68%, copper/zinc superoxide dismutase 48%, glutathione peroxide 37% and catalase 198%.

CTP:cholinephosphate cytidylyltransferase in human and rat lung: association in vitro with cytoskeletal actin.

CTP:cholinephosphate cytidylyltransferase activities were compared in saline homogenates of immature fetal (15-16 weeks gestation) and adult human lung. There were no differences in subcellular enzyme distribution, in Vmax activity, or in the phosphatidylglycerol-mediated stimulation of soluble enzyme activity. These results provide no support for a developmental translocation of cytidylyltransferase from a cytosolic to a microsomal location in human lung, such as that proposed to accompany the maturation of pulmonary surfactant phosphatidylcholine biosynthesis in rat. Soluble cytidylyltransferase activity from human but not rat lung was increased after manipulation in vitro. Resolution of human H form (greater than 10(3) kDa) and L form (200 kDa) enzyme by gel filtration led to an activity increase of 200%. Incubation at 37 degrees C for 2 h increased soluble enzyme recovery, although prior centrifugal removal of generated actin-rich aggregates was necessary in adult lung fractions. In contrast, 85% of soluble rat lung cytidylyltransferase was actin aggregate-associated after incubation. The apparent heteroassociation of rat and human lung enzyme with actin in the presence of poly(ethylene glycol) at 4 degrees C strongly suggested close in vitro and potential in vivo linkage. A partial co-purification of adult human lung cytidylyltransferase with actin was also consistent with this idea. We propose that some reported cytidylyltransferase translocation phenomena may be mediated by cytoskeletal interactions in vitro.

The proteins of human lung surfactant.

Human pulmonary surfactant was purified from bronchoalveolar lavage of patients. The proteins present in surfactant were analyzed by SDS-polyacrylamide gel electrophoresis into serum and non-serum components. One non-serum surfactant protein (Mr = 43 000) was then identified in the 100 000 X g supernatant of a lung homogenate on the basis of phospholipid binding. This lung protein was purified and partially characterized. The presence of 3-methyl histidine and reaction in Western blot analysis with antibody against chicken muscle actin both strongly suggested that the 43 000 Da protein of human surfactant is indeed cytoplasmic actin. It is proposed that this surfactant protein is involved in the secretion and not necessarily in the function of surfactant.