Hélène Juguelin

A quantitative developmental study of the peripheral nerve lipid composition during myelinogenesis in normal and trembler mice

The quantitative evolution of 10 polar lipids was examined in the sciatic nerves of normal and trembler mice between the ages of 3 days and 60 days. In normal nerves, the polar lipids accumulated slowly until the age of 9 days. A period of rapid accumulation then took place until 18 days of age, after which the phospholipids plateaued, while the glycolipid content continued to increase at a slower rate. The results obtained for the sciatic nerves of trembler mice show that the accumulation of all the polar lipids studied, except phosphatidylcholine and hydroxysulfatides, is abnormal from the earliest stages of postnatal development, and strongly support the view that the primary disorder in the trembler peripheral nervous system is one of dysmyelination. With the exception of cardiolipin, all the lipids in the trembler nerves stopped accumulating at the age of 18 days. The cerebrosides were the lipids the most affected severely at all ages.

A quantitative developmental study of neutral lipids during myelinogenesis in the peripheral nervous system of normal and trembler mice

The quantitative accumulation of neutral lipids during the period of myelination in the peripheral nervous system was studied in normal and trembler mouse sciatic nerves, between the ages of 5 and 27 days. Neutral lipids were resolved by high-performance thin-layer chromatography, using the solvent mixture hexane/diethyl ether/acetic acid (90:15:2, v/v/v). The lipids were quantitated, after copper acetate/phosphoric acid charring, by densitometric scanning, using an external standard technique. Cholesterol and triacylglycerols accumulated in normal nerves throughout the period studied, while cholesteryl esters were not observed at any age. In trembler nerves, the accumulation of cholesterol took place at a much lower rate than in normal nerves and this lipid was deficient from the earliest stages of development. Triacylglycerols were not significantly deficient in trembler nerves during the first 2-3 weeks, but, after the age of 18 days, their quantity diminished significantly. Cholesteryl esters were first detected in the mutant nerves at the age of 18 days. These results, in agreement with those of a previous developmental study of the polar lipids, are strongly in favour of the view that the trembler mutation directly induces a process of dysmyelination and that demyelination is a secondary event.

Correlation between the morphology and the lipid and protein compositions in the peripheral nervous system of individual 8-day-old normal and trembler mice

The hereditary, hypertrophic interstitial neuropathy which afflicts the trembler mouse manifests itself about two weeks after birth. Consequently, the identification of these mutant mice was not possible before this age, except when double mutants were available. We show that the trembler mice can be easily distinguished from their normal littermates before the clinical symptoms appear by using an HPTLC/densitometry technique that allows the simple and rapid analysis of the polar lipids extracted from one sciatic nerve. The results presented in this paper demonstrate important differences between the polar lipid compositions of sciatic nerves from 8-day-old normal and trembler littermates, whose phenotypes were confirmed by the morphological analysis of the contralateral sciatic nerves. The small amount of material that is needed for this identification makes it possible to use the remaining nerve material for other studies. Furthermore, important differences between the sciatic nerve protein compositions of normal and trembler mice, identified according to their polar lipid composition, were also observed and these differences can, therefore, also be employed for the identification of the mutants before the manifestation of the clinical symptoms of the trembler neuropathy.

Plasma membrane biogenesis in higher plants: In vivo transfer of lipids to the plasma membrane☆

Abstract The intermembrane transfer of lipids and particularly of very long chain fatty acids (VLCFA) from a light membrane fraction (1.08–1.09 g/cm 3 ) to By means of biochemical markers (glucan synthetase II, IDPase, CDP-choline diglyceride transferase and NADPH cyt c reductase), it was shown that th The biosynthesis of VLCFA was assayed in vitro in the purified plasma membrane and the other membranes after phase partition of the microsomal pell

Intermembrane transfer of long chain fatty acid synthesized by etiolated leek seedlings

Abstract Etiolated leek seedlings provided with [1-14C]acetate in vivo, synthesized very long chain fatty acids (VLCFA) that were incorporated into the phospholipids (chiefly PC and PE) and the neutral lipids (including free fatty acids). After a labeling period and various chase times, the seedlings were homogenized and a 150 000 g pellet was prepared. The latter was then subfractionated on a linear sucrose gradient (0.35–1.7 M). Four membrane fractions, banding at 0.65±0.05 M (A), 0.85±0.05 M (B), 1.05±0.05 M (C)and 1.30±0.10 M (D), were obtained. The lipids of each membrane fraction were extracted and their radioactivity determined. Nearly 60% of the total labeled lipids of the lightest membrane fraction (A) were transferred to heavier membranes during the chase. The fatty acid analysis of each membrane fraction showed that the labeled VLCFA of the membrane fraction A were integrally transferred to heavier membranes during the chase period.

Molecular basis for low temperature compartment formation by transitional endoplasmic reticulum of rat liver

The molecular basis for temperature compartment formation was investigated using a cell‐free system from rat liver. The donor was from liver slices prelabeled with [3H]acetate. Unlabeled Golgi apparatus membranes were immobilized on nitrocellulose as the acceptor. When transfer was determined as a function of temperature, a transition in transfer activity was observed at low temperatures (≤ 20°C) similar to that seen in vivo. The decrease in transfer efficiency correlated with a decrease in phosphatidylethanolamine and phosphatidylserine content of the transition vesicles formed. By adding lipid mixtures enriched in these lipids to the vesicles, their ability to fuse with the cis Golgi apparatus was reconstituted. These findings provide evidence for a role for lipids in low temperature compartment formation.

Monensin-induced accumulation of neosynthesized lipids and fatty acids in a Golgi fraction prepared from etiolated leek seedlings

Abstract The effects of monensin on the intracellular distribution of neosynthesized lipids and fatty acids between different membrane fractions prepared from etiolated leek seedlings, were investigated. The penetration of the drug into the seedlings was measured after resolution of monensin by thin-layer chromatography with a solvent system consisting of hexane/diethyl ether/acetic acid (20:80:2, v/v). The concentration of monensin in the microsomal membranes as a function of the external monensin concentration was quantified. The effects of various amounts of the drug on lipid metabolism were studied to determine monensin concentrations having only slight effects on the overall lipid synthesis. Using such conditions, an accumulation of neosynthesized lipids was found in a subcellular membrane fraction enriched in Golgi membranes.

Acylation of endogenous acyl acceptors by mouse sciatic nerve microsomes.

Phospholipid (chiefly phosphatidylcholine) labeling from radioactive acyl-CoAs by mouse sciatic nerve microsomes is observed in the absence of added acyl acceptors. The maximal acylation (ca 10% of administered) for 10 micrograms microsomal proteins is observed at relatively low amounts of oleoyl-CoA (0.2-0.3 nmol) and decreases as the acyl-CoA amount increases. Labeled lysophosphatidylcholine (almost exclusively esterified at position 2) is also observed, particularly when the [1-14C]oleoyl-CoA concentration is higher than 0.2-0.3 nmol/50 microliters. The labeled acyl group is mainly inserted in position 2] of the glycerophosphorylcholine. With 0.15 nmol labeled oleoyl-CoA, phosphatidylcholine acylation increases as a function of the protein amount and reaches 25% of the added label at 40 microgram proteins. It is evaluated that, in the presence of 10 microgram proteins, 2% of the microsomal phosphatidylcholine molecules are acylated from 0.1 nmol acyl-CoA. The acylation mechanism seems to involve an acyl exchange between acyl-CoA and phosphatidylcholine.

Monensin: A Tool to Study Intracellular Transport of Lipids in Higher Plants

The biogenesis of the plasma membrane has been extensively studied in both animal and plant cells by following the biosynthesis, maturation and intracellular transport of glycoproteins. However, little is yet available concerning the intracellular transfer of lipids to the plasma membrane, and particularly in the plant cells.