1,720,982 research outputs found
Analysis of lung surfactant phosphatidylcholine metabolism in transgenic mice using stable isotopes
Full text linkStable isotope labelling of lipid precursors coupled with mass spectrometry-based lipidomic analyses and determination of isotope enrichment in substrate, intermediate and product pools provide the parameters needed to determine absolute flux rates through lipid pathways in vivo. Here, as an illustration of the power of such analyses we investigated lung phosphatidylcholine (PC) synthesis in Surfactant Protein-D (SP-D) null mice. These animals develop emphysema, foamy alveolar macrophages and an alveolar lipoproteinosis with increasing age. We used the incorporation of methyl-9-[2H] choline chloride coupled with ESI-MS/MS to quantify absolute rates of lung surfactant PC synthesis and secretion in an SP-D-/? mouse model, together with an analysis of the molecular specificity of lung PC synthesis. PC synthetic rates were comparable in control (0.52 ?moles/lung/h) and SP-D-/? (0.69 ?moles/lung/h) mice, as were rates of surfactant PC secretion (29.8 and 30.6 nmoles/lung/h respectively). Increased lung PC in the SP-D-/? mouse was due to impaired catabolism, with a rate of accumulation of 0.057 ?moles/lung/h. The relatively low rates of surfactant PC secretion compared with total lung PC synthesis were compatible with a suggested ABCA1-mediated basolateral lipid efflux from alveolar type II epithelial cells. Finally, PC molecular species analysis suggested that a proportion of newly-synthesised PC is secreted rapidly into the lung air spaces in both control and SP-D-/? mice before significant PC acyl remodelling occurs<br/
Phospholipid composition of neonatal guinea pig liver and plasma: Effect of postnatal food restriction
No full textPreterm 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) μM 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 thesn-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) μmol/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
The proteins of human lung surfactant
No full textHuman 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
CTP:cholinephosphate cytidylyltransferase in human and rat lung: Association in vitro with cytoskeletal actin
No full textCTP: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
Mechanisms of hepatic phosphatidylcholine synthesis in adult rat: Effects of pregnancy
Full text linkLate 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
No full textDetailed 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
Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes
No full textChromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase α 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
An in vivo ratio control mechanism for phospholipid homeostasis: evidence from lipidomic studies
No full textWhile it is widely accepted that the lipid composition of eukaryotic membranes is under homeostatic control, the mechanisms through which cells sense lipid composition are still the subject of debate. It has been postulated that membrane curvature elastic energy is the membrane property that is regulated by cells, and that lipid composition is maintained by a ratio control function derived from the concentrations of type II and type 0 lipids, weighted appropriately. We assess this proposal by seeking a signature of ratio control in quantified lipid composition data obtained by electrospray ionization mass spectrometry from over 40 independent asynchronous cell populations. Our approach revealed the existence of a universal 'pivot' lipid, which marks the boundary between type 0 lipids and type II lipids, and which is invariant between different cell types or cells grown under different conditions. The presence of such a pivot species is a distinctive signature of the operation in vivo, in human cell lines, of a control function that is consistent with the hypothesis that membrane elastic energy is homeostatically controlled
Use of mass spectrometry-based lipidomics to probe PITP? (phosphatidylinositol transfer protein ?) function inside the nuclei of PITP?+/+ and PITP?-/- cells
No full textThe mammalian phospholipid exchange protein PITPa (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 PITPa ablation in mice, PITPa-/- 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-d9 and inositol-d6, into molecular species of whole cell and endonuclear phosphatidylcholine and PtdIns over 24 h to assess the contribution of PITPa to the nuclear import of PtdIns into MEF cells. In cells labelled for 1, 3, 6, 12 and 24 h fractional inositol-d6 incorporation into whole-cell PtdIns species was consistently higher in PITPa-/- MEF implying greater flux through its biosynthetic pathway. Moreover, endonuclear accumulation of PtdIns-d6 was apparent in the PITPa-/- cells and mirrored that in PITPa+/+ cells. Together, these results suggest that the essential endonuclear PtdIns import via PITPa can be accommodated by other mechanisms
Acute respiratory distress syndrome and acute lung injury
No full textAcute respiratory distress syndrome (ARDS) is a life threatening respiratory failure due to lung injury from a variety of precipitants. Pathologically ARDS is characterised by diffuse alveolar damage, alveolar capillary leakage, and protein rich pulmonary oedema leading to the clinical manifestation of poor lung compliance, severe hypoxaemia, and bilateral infiltrates on chest radiograph. Several aetiological factors associated with the development of ARDS are identified with sepsis, pneumonia, and trauma with multiple transfusions accounting for most cases. Despite the absence of a robust diagnostic definition, extensive epidemiological investigations suggest ARDS remains a significant health burden with substantial morbidity and mortality. Improvements in outcome following ARDS over the past decade are in part due to improved strategies of mechanical ventilation and advanced support of other failing organs. Optimal treatment involves judicious fluid management, protective lung ventilation with low tidal volumes and moderate positive end expiratory pressure, multi-organ support, and treatment where possible of the underlying cause. Moreover, advances in general supportive measures such as appropriate antimicrobial therapy, early enteral nutrition, prophylaxis against venous thromboembolism and gastrointestinal ulceration are likely contributory reasons for the improved outcomes. Although therapies such as corticosteroids, nitric oxide, prostacyclins, exogenous surfactants, ketoconazole and antioxidants have shown promising clinical effects in animal models, these have failed to translate positively in human studies. Most recently, clinical trials with ?2 agonists aiding alveolar fluid clearance and immunonutrition with omega-3 fatty acids have also provided disappointing results. Despite these negative studies, mortality seems to be in decline due to advances in overall patient care. Future directions of research are likely to concentrate on identifying potential biomarkers or genetic markers to facilitate diagnosis, with phenotyping of patients to predict outcome and treatment response. Pharmacotherapies remain experimental and recent advances in the modulation of inflammation and novel cellular based therapies, such as mesenchymal stem cells, may reduce lung injury and facilitate repair
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