Louis L. Sarliève

Quantitative thin-layer chromatography of glycolipids in animal tissues

Abstract A method is described for quantitative analysis of four glycolipids in brain. The combination of a thin-layer chromatographic system, which makes the previous separation of glycolipids by column chromatography unnecessary, with the orcinol reaction carried out in the presence of silica gel, permitted a rapid and simple determination of cerebrosides, sulphatides and monogalactosyl diglyceride with as little as 50 mg of brain tissue. Somewhat modified procedure was applied for a semi-quantitative determination of glycolipids in liver.

Cerebellar Soluble Lectin Is Responsible for Cell Adhesion and Participates in Myelin Compaction in Cultured Rat Oligodendrocytes

Cultures of rat oligodendrocytes were used to test the possible role of the cerebellar soluble lectin (CSL) in myelin formation. Immunocytochemistry at the ultrastructural level showed that the lectin is present in the cytoplasm of the perikaryon of cultured oligodendrocytes and also on the plasma membrane of the cell body and processes. It is present in compact myelin and in the zones of contacts between different myelin sheaths or oligodendrocyte membranes. Staining of blots of the cultures with iodinated CSL indicated that endogenous glycoprotein ligands for CSL are present in the culture, rendering probable the hypothesis that cell contacts between different oligodendrocytes or between adjacent lamellae in myelin are mediated by lectin-glycoprotein interactions. This hypothesis was demonstrated by two effects of anti-CSL Fab fragments (4 micrograms/ml) on oligodendrocyte cultures: (1) the almost complete detachment of the cell layer from the culture substratum, and (2) the loss of myelin compaction by a separation of lamellae at the intraperiod line. The present findings could explain the complexity of the contacts between cultured oligodendrocyte processes by the formation of CSL bridges between glycoproteins of the membranes of these cells. CSL seems to be a key molecule in adhesion both for intercellular contacts and fixation of cells to the substratum. The small number of glycoprotein subunits found in oligodendrocytes that interact with CSL suggests that CSL-mediated cell adhesion involves a special class of glycoprotein glycans.

Purification and properties of UDP-galactose: Ceramide galactosyltransferase from rat brain microsomes

Abstract UDPgalactose:ceramide galactosyltransferase (EC 2.4.1.62) from rat brain microsomes was solubilized by treatment with a non-ionic detergent, Cemulsol NP-12. A partial purification of the enzyme is described. There was no apparent change in the enzyme solubility after removing the detergent by solvent extraction. The general properties of the solubilized enzyme were essentially the same as in the particulate preparation.

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T4) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP‐galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T4 was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T4 also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T4 and GH may have both independent and complementary actions on the myelin‐associated enzymes during the early postnatal period of brain development.

Brain UDPgalactose: Ceramide galactosyltransferase: Purification of a catalytically active protein obtained after proteolytic digestion

A procedure for the purification of UDPgalactose--2-hydroxyacylsphingosine galactosyltransferase (EC 2.4.1.45) including detergent extraction, ion-excharge chromatography and proteolytic digestion was developed. The active fraction obtained by this procedure had about 100 times higher specific activity than microsomes. Enzymic activity resisted destruction by pronase treatment at 4 degrees C. Agarose gel chromatography indicated the presence of an enzyme-phospholipid-detergent complex with a molecular weight between 400 000 and 500 000. Intact phospholipids seemed to be required for full enzymic activity as evidenced by the drastic loss of activity upon treatment with phospholipase A or C.

Cellular Development and Myelin Production in Primary Cultures of Embryonic Mouse Brain

The development of cell cultures from embryonic mouse cerebral hemispheres has been followed in detail for periods up to 40 days in culture using a variety of approaches. Functionally well differentiated neurons (shown by receptor binding studies, immunocytochemistry and morphological examination) were found to be abundant early in culture and to form cell contacts with oligodendrocytes characterized both immunocytochemically and morphologically. Myelin-like membranes with the periodicity of classical myelin elaborated by oligodendrocytes were detected only after 30 days in culture when neurones were no longer present. These results are discussed with regard to possible mechanisms of initiation of myelin synthesis.

Structure and immunological localization of spleen sulfolipid

By using chemical and immunological techniques, the structure of the spleen sulfolipid was confirmed as being a sulfogalactosylceramide. This lipid was localized in the spleen granulocytes (polymorphonuclear leukocytes).

Investigation of Myelinogenesis in Vitro: Transient Expression of 3,5,3’-Triiodothyronine Nuclear Receptors in Secondary Cultures of Pure Rat Oligodendrocytes

The glial compartment of the central nervous system (CNS) consists of a number of different cell types which interact closely together. It has been recognized for many years that most interfascicular oligodendrocytes of the white matter undergo massive membrane synthesis at myelination, leading to the formation and growth of myelin sheaths around axons in the CNS. Such oligodendrocytes probably subsequently maintain the integrity of myelin throughout life (for a review, see refs. 1,2). On the other hand, “critical periods” of accelerated development characterized by increased sensitivity and influenced by external environmental factors have been identified during braiL development (3). For example, in the rat and mouse, the “critical period” during which hormones influence brain development is associated, among other events, with rapid myelinogenesis occuring in both species between the 10th and 30th day after birth (4). During this period, striking morphological and biochemical changes have been described. The biochemical parameters which best seem to correlate with these temporal changes are the enzymes and compounds most closely associated with myelination. Cerebrosides, galactosyl glycerol lipids, sulfatides, sulfogalactosyl glycerol lipids, and the enzymes catalyzing their synthesis, the myelin basic protein, myelin proteolipid protein (PLP) or a synthetic polypeptide composed of the C-terminal amino acids of the PLP sequence, Wolfgram protein, 2’,3’-cyclic-nucleotide phosphohydrolase (CNP) and pH 7.2 cholesterol ester hydrolase are very useful molecular markers for myelination (for an extensive review, see refs. 5,6).

Multihormonal control of proliferation and cytosolic glycerol phosphate dehydrogenase, lactate dehydrogenase and malic enzyme in glial cells in culture.

We have examined the effect of a physiological concentration of l-triiodothyronine on the activity of cytosolic enzymes in the C6 rat glioma cell line. l-Triiodothyronine decreased glycerol phosphate dehydrogenase activity. This effect seems to be rather specific, since l-triiodothyronine did not change malic enzyme or lactate dehydrogenase activity and did not alter the amount of either cytosolic or total cell protein. Dexamethasone greatly increased glycerol phosphate dehydrogenase and l-triiodothyronine also decreased the response to the glucocorticoid. Noradrenaline or dibutyryl cyclic AMP potentiated the dexamethasone-induced specific activity of this enzyme, and l-triiodothyronine lowered the response to the combined effects of these agents. The effect of l-triiodothyronine is not restricted to the C6 cells, since it also decreased basal glycerol phosphate dehydrogenase activity in primary cultures of cells dissociated from brains of embryonic mice. The results indicate that thyroid hormones have a direct effect on the modulation of cytosolic glycerol phosphate dehydrogenase in cultured cells of glial origin.

The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin-Deficient (mld) Mutant Mice

Abstract: The myelin‐deficient (mld) mutation is an autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mid. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin‐associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low‐molecular‐mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.