A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture

Caesar, Petula A.; Wilson, Susan J.; Normand, I. C. S.; Postle, Anthony D.

doi:10.1042/bj2530451

Cited by 41 publications

(24 citation statements)

References 36 publications

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“…showed no nuclear envelope at the corresponding higher magnification (d, scale bar 0.5 m). Western blots of nuclei (N) and whole cell (C) fractions probed with monoclonal anti-␤-tubulin (e) at equivalent cell loading displayed no immunoreactivity in the nuclear fraction, consistent with removal of endoplasmic reticulum and minimal nuclear envelope contamination (21,22).…”

Section: Resultsmentioning

confidence: 99%

“…Phospholipid Purification-Phospholipids from whole cells or isolated nuclei were extracted according to Bligh and Dyer (21), and the PtdCho component was separated in the chloroform:methanol (60:40; 1 ml) wash of 100-mg Bond Elut aminopropyl solid phase extraction cartridges (Varian) as previously described (22). Following a further methanol wash (1 ml) to elute PtdEtn, an acidic phospholipid fraction containing PtdOH, phosphatidylglycerol, phosphatidylserine, and PtdIns was eluted with methanol:water:phosphoric acid (96:4:1; 3 ϫ 1 ml) containing 40 mM choline chloride.…”

Section: Stockmentioning

confidence: 99%

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Highly Saturated Endonuclear Phosphatidylcholine Is Synthesizedin Situ and Colocated with CDP-choline Pathway Enzymes

Hunt

Clark

Attard

et al. 2001

Journal of Biological Chemistry

120

132

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Chromatin-associated phospholipids are well recognized. A report that catalytically active endonuclear CTP:choline-phosphate cytidylyltransferase ␣ 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.Considerable evidence supports the existence of an endonuclear pool of phospholipid, in association with the nuclear matrix and distinct from the nuclear envelope (1-4). This endonuclear phospholipid is remarkable for several reasons. Although all the major membrane phospholipids, predominantly phosphatidylcholine (PtdCho), 1 phosphatidylethanolamine (PtdEtn), phosphatidylserine, and phosphatidylinositol (PtdIns), may be present typically at 4 -10% of total cell content (2), transmission electron microscopy has failed to reveal endonuclear membranous systems (5). Hence the molecular organization of endonuclear phospholipids in eukaryotic cells is still unclear. Extensive histochemical and cytochemical studies suggest that their spatial distributions overlap that of decondensed chromatin domains (3, 4). A number of in vitro studies suggest a functional relationship between various endonuclear phospholipids and gene expression/transcription (6 -10). Moreover, cell studies have shown that the amounts of endonuclear phospholipids change during progression through the cell cycle (4). The potential physiological importance of intranuclear phospholipid has recently been highlighted by the recognition that the ␣ isoform (CCT␣) of CTP:choline-phosphate cytidylyltransferase, the principal regulatory enzyme of PtdCho biosynthesis (11), is confined to the nucleus throughout the ce...

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Section: Resultsmentioning

confidence: 99%

Section: Stockmentioning

confidence: 99%

Highly Saturated Endonuclear Phosphatidylcholine Is Synthesizedin Situ and Colocated with CDP-choline Pathway Enzymes

Hunt

Clark

Attard

et al. 2001

Journal of Biological Chemistry

120

132

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show abstract

“…Several SPE methods for lipids in plasma and tissue extracts have been published (6)(7)(8)(9)(10)(11)(12)(13)(14). However, none of these methods include BAs in their separation, nor have they been validated for the separation of lipids in intestinal fluids.…”

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confidence: 99%

Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods

Persson

Löfgren

Hansson

et al. 2007

Journal of Lipid Research

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The purpose of the study reported here was to develop a method for the determination of lipid classes in intestinal fluids, including bile acids (BAs). A solid-phase extraction (SPE) method using C18 and silica columns for the separation of BAs, phospholipids (PLs), and neutral lipids (NLs), including free fatty acids, has been developed and validated. Fed-state small intestinal fluid collected from humans was treated with orlistat to inhibit lipolysis and mixed with acetic acid and methanol before SPE to maximize lipid recoveries. BAs, PLs, and NLs were isolated using lipophilic and polar solvents to promote elution from the SPE columns. The different lipid classes were subsequently analyzed using three separately optimized HPLC methods with evaporative light-scattering detectors. High recoveries (.90%) of all lipids evaluated were observed, with low coefficients of variation (,5%). The HPLC methods developed were highly reproducible and allowed baseline separation of nearly all lipid classes investigated.

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“…Chloroform (2.0 mL) was then added and total plasma lipids were isolated [17]. PC and PE were purified by solid phase extraction using aminopropylsilica cartridges (100 mg) [18,19].…”

Section: Isolation Of Phospholipidsmentioning

confidence: 99%

Selective changes to phosphatidylcholine and phosphatidylethanolamine molecular species in the developing fetal guinea pig liver and plasma

Burdge

Postle

2004

Reprod. Nutr. Dev.

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-The molecular species composition of membrane phospholipids influences the activities of integral proteins and cell signalling pathways. We determined the effect of increasing gestational age on fetal guinea pig liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and plasma PC molecular species composition. The livers were collected from fetuses (n = 5/time point) at 5 day intervals between 40 and 65 days of gestation, and at term (68 days). Hepatic PC and PE molecular species composition was determined by electrospray ionisation mass spectrometry. An increasing gestational age was accompanied by selective changes in individual molecular species. The proportion of the sn-1 18:0 species increased relative to the sn-1 16:0 species in liver PC, but not PE, with an increasing gestational age. 1-O-alkyl-2-acyl PC species concentrations decreased significantly between 40 and 45 days of gestation (40%), and 65 and 68 days (54%). Total 1-Oalkenyl-2-acyl PE species concentration increased between days 60 and 65, due to a rise in 1-O-16:0 alkyl/20:4 content, and then decreased until term. Between day 40 and term, PC and PE sn-2 18:2n-6 species concentrations increased 3-fold. PC16:0/18:2 increased gradually throughout gestation, while PC18:0/18:2 content only increased after day 65. The overall increase in PE18:2n-6 content was due to PE18:0/18:2 alone. The composition of plasma PC essentially reflected hepatic PC. Overall, these data suggest differential regulation of hepatic PC and PE molecular species composition during development which is essentially independent of the maternal fatty acid supply.phospholipids / gestation / electrospray ionisation mass spectrometry / ether phospholipids

show abstract

A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture

Cited by 41 publications

References 36 publications

Highly Saturated Endonuclear Phosphatidylcholine Is Synthesizedin Situ and Colocated with CDP-choline Pathway Enzymes

Highly Saturated Endonuclear Phosphatidylcholine Is Synthesizedin Situ and Colocated with CDP-choline Pathway Enzymes

Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods

Selective changes to phosphatidylcholine and phosphatidylethanolamine molecular species in the developing fetal guinea pig liver and plasma

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