Claude Cassagne

Myelin synthesis in the peripheral nervous system

By imposing saltatory conduction on the nervous impulse, the principal role of the myelin sheath is to allow the faster propagation of action potentials along the axons which it surrounds. Peripheral nervous system (PNS) myelin is formed by the differentiation of the plasma membrane of Schwann cells. One of the biochemical characteristics that distinguishes myelin from other biological membranes is its high lipid-to-protein ratio. All the major lipid classes are represented in the myelin membrane, while several myelin-specific proteins have been identified. During development, the presence of axons is required for the initiation of myelination, but the nature of the axonal signal is still unknown. The only certainties are that this signal is synthesized by axons whose diameter is greater than 0.7 microm, and that the signal(s) include(s) a diffusible molecule. Morphological studies have provided us with information concerning the timing of myelination, the mechanism by which immature Schwann cells differentiate into a myelinating phenotype and lay down the myelin sheath around the axon, and the accumulation and the structure of the myelin membrane. The last 20 years have seen the identification and the cDNA and gene cloning of the major PNS myelin proteins, which signalled the beginning of the knock-out decade: transgenic null-mutant mice have been created for almost every protein gene. The study of these animals shows that the formation of myelin is considerably less sensitive to molecular alterations than the maintenance of myelin. During the same period, important data has been gathered concerning the synthesis and function of lipids in PNS myelin, although this field has received relatively little attention compared with that of their protein counterparts.

The C16:3\C18:3 fatty acid balance in photosynthetic tissues from 468 plant species

Abstract Two kinds of plants may be distinguished according to their (n–3) trienoic fatty acid composition in photosynthetic tissues. The cis -7,10,13-hexadecatrienoic acid\ cis -9,12,15-octadecatrienoic acid balance directly reflects the biosynthesis pathways (a plastidial one and an extra-plastidial one) of chloroplastic lipids. We analysed the correlation between the existence of these pathways and the evolutionary classification of Cormophytes (particularly Angiosperms). By using cis -7,10,13-hexadecatrienoic acid as a marker for the existence of the plastidial pathway, we studied the overall fatty acid composition of 468 plant species (280 already described in the literature and 188 new ones) distributed among 141 botanical families. The data strongly suggest that the plastidial pathway was lost during evolution and that, in the case of dicotyledonous plants, this loss probably occurred independently and at different rates. The data are also discussed from an environmental and chemotaxonomic point of view.

Regulation of Oleoyl‐CoA Synthesis in the Peripheral Nervous System: Demonstration of a Link with Myelin Synthesis

Abstract: We studied the regulation of oleic acid synthesis in the PNS. During mouse postnatal development, the proportion of 18:1 rises in the sciatic nerve from 17% at 5 days of age to 33% at 25 days. However, this rise does not occur in the dysmyelinating mutant mouse trembler. In normal mouse development, the total stearoyl‐CoA desaturase (SCD) activity measured in sciatic nerve homogenates is high during the first 3 weeks. Yet in trembler nerves, this SCD activity represents only 15% of normal values. Using the RT‐PCR technique, we demonstrate that the SCD2 isoform is predominantly expressed in the PNS. Northern blot analysis showed that the mRNA levels for SCD2 parallel those of other specific myelin proteins in both normal mouse and trembler mutant development. Similar experiments in a rat demyelination‐remyelination model confirmed that SCD2 mRNA levels are regulated in the PNS in a similar manner to myelin‐specific proteins.

Taxonomy of gymnospermae: multivariate analyses of leaf fatty acid composition

The fatty acid composition of photosynthetic tissues from 137 species of gymnosperms belonging to 14 families was determined by gas chromatography. Statistical analysis clearly discriminated four groups. Ginkgoaceae, Cycadaceae, Stangeriaceae, Zamiaceae, Sciadopityaceae, Podocarpaceae, Cephalotaxaceae, Taxaceae, Ephedraceae and Welwitschiaceae are in the first group, while Cupressaceae and Araucariaceae are mainly in the second one. The third and the fourth groups composed of Pinaceae species are characterized by the genera Larix, and Abies and Cedrus, respectively. Principal component and discriminant analyses and divisive hierarchical clustering analysis of the 43 Pinaceae species were also performed. A clear-cut separation of the genera Abies, Larix, and Cedrus from the other Pinaceae was evidenced. In addition, a mass analysis of the two main chloroplastic lipids from 14 gymnosperms was performed. The results point to a great originality in gymnosperms since in several species and contrary to the angiosperms, the amount of digalactosyldiacylglycerol exceeds that of monogalactosyldiacylglycerol.

Ceramide excluded from cell-free vesicular lipid transfer from endoplasmic reticulum to Golgi apparatus. Evidence for lipid sorting

The distribution and cell-free transfer of ceramide and other lipids were compared using highly purified fractions of endoplasmic reticulum, transitional endoplasmic reticulum, transition vesicles and Golgi apparatus from rat liver. Ceramides were present in both endoplasmic reticulum and Golgi apparatus where they represented between 0.3 and 1% of the total lipids. Ceramides, however, were much reduced or absent (< 0.05%) from transition vesicles. Transition vesicles were induced to form from transitional endoplasmic reticulum by incubation with ATP and a cytosol fraction. When transfer of [14C]choline-labeled phosphatidylcholine from transitional endoplasmic reticulum to Golgi apparatus was followed, transition vesicles were more efficient in transfer than the transitional endoplasmic reticulum from which they were derived. This transfer was temperature- and ATP-dependent and inhibited by N-ethylmaleimide. When transfer of [3H]ceramide was followed, there was little or no transfer via transition vesicles and that transfer which occurred was temperature-, ATP- and N-ethylmaleimide independent. Transfer of ceramide in the cell-free system did occur from endoplasmic reticulum to Golgi apparatus but via a non-vesicular mechanism that was temperature-dependent but not dependent on ATP or cytosol, alone, or in combination, nor was it inhibited by N-ethylmaleimide. A component of phosphatidylcholine transfer exhibited similar characteristics. The results provide evidence for two distinct mechanisms for cell-free transfer of lipids from endoplasmic reticulum to Golgi apparatus. The first is via 50 to 70 nm transition vesicles which is temperature- and ATP-dependent, inhibited by N-ethylmaleimide and from which ceramides are excluded. The second is non-vesicular, temperature-dependent, and neither ATP- nor cytosol-dependent. It accounts for the bulk of the ceramide transfer. As a result during cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus, lipid sorting occurs such that ceramides are largely absent from the transition vesicles and, apparently are delivered to the Golgi apparatus by another mechanism.

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Abstract Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2- 14 C]malonyl-CoA was incorporated into fatty acids of C 16 C 26 . The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C 12 C 24 ) elongation showed that stearoyl-CoA, in the presence of [2- 14 C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C 20 C 26 . [1- 14 C]C 16 CoA and [1- 14 C]C 18 CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1- 14 C]C 18 CoA was a better primer than [1- 14 C]C 16 CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1- 14 C]C 18 CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2- 14 C]malonyl-CoA in the presence of C 18 CoA, on the one hand, and [1- 14 C]C 18 CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.

Solubilization of C18-Co A and C20-CoA elongases from Allium porrum L. epidermal cell microsomes

The effects of n‐octyl‐β‐D‐glucopyranoside, Triton X‐100 and deoxycholate on acyl‐CoA elongation by Allium porrum L. epidermal cell microsomes showed that the Triton X‐100 specifically stimulated the synthesis of C22–C26 acids using C18‐CoA as primer, whereas the fatty acid elongation products of C20‐CoA remained essentially unchanged. n‐Octyl‐β‐D‐glucopyranoside increased the C20 and C22 fatty acid syntheses to the same extent and deoxycholate inhibited C18‐CoA and C20‐CoA elongation. The presence of two different elongation systems, as suggested by these results, has been demonstrated. After solubilization by Triton X‐100, the C18‐CoA and C20‐CoA elongases were separated by sucrose density centrifugation. The fractions corresponding to sucrose concentrations of 0.51 and 0.62 M presented the maximal activities for C18‐CoA and C20‐CoA elongases, respectively. In addition, by gel filtration on a Sephacryl S‐300 column, the C20‐CoA and the C18‐CoA elongases have estimated apparent molecular masses under detergent conditions of 600 and 350 kDa, respectively.

Demonstration of high specificity antibodies against phosphatidylserine

Anti-phosphatidylserine antibodies were raised in rabbits immunized with phosphatidylserine-polyacrylamide gels and with phosphatidylserine-cytochrome c vesicles. A solid-phase immunoenzymatic assay (ELISA) was developed to study the immune serum. The optimal conditions were defined and the technique used to obtain quantitative results. The anti-phosphatidylserine serum diluted to 1/1000 was tested against several phospholipids and was found to be highly specific to phosphatidylserine. This represents the first experimental demonstration of the specificity of antibodies raised against phosphatidylserine.

Assay of long-chain acyl-CoAs in a complex reaction mixture

A method has been developed which allows the quantitative analysis of labeled or unlabeled acyl-CoAs in complex reaction mixtures. The method is based on (a) a quantitative solubilization of acyl-CoAs and lipids, directly in the reaction vessel, by 0.05 M Tris-HCl, pH 7.5/CHCl3/CH3OH (1/3/3, v/v/v); (b) monodimensional TLC of aliquots of the whole reaction mixture, resolving malonyl-CoA, acetyl-CoA, long-chain acyl-CoAs, polar lipids and neutral lipids plus free fatty acids; and (c) quantitation by TLC densitometry and/or TLC radiochromatography. All fractions--and particularly long chain-acyl-CoAs--can then be analyzed for distribution and label of fatty acyl moieties.

The homeotic protein dlk is expressed during peripheral nerve development

To investigate the molecular events controlling myelination of the peripheral nervous system, we compared gene expression of normal mouse sciatic nerves to that of the trembler mouse, whose Schwann cells are blocked in a pre‐myelinating phenotype. Using cDNA array, we assessed expression levels of 1176 genes, and we found that delta‐like protein (dlk), an epidermal growth factor‐like homeotic protein, was expressed in the normal developing nerves, but at a low level in the dysmyelinating mutant trembler. Moreover, dlk expression was down‐regulated when myelin protein expression was up‐regulated, and no expression was observed in the developing brain. These results suggest that dlk expression is required for Schwann cell acquisition of the myelinating phenotype.