N. M. Neskovic

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.

SUBCELLULAR AND SUBMICROSOMAL DISTRIBUTION OF GLYCOLIPID-SYNTHESIZING TRANSFERASES IN YOUNG RAT BRAIN

The subcellular and submicrosomal distributions of four glycolipid‐synthesizing transferases were studied in young rat brains.

Biosynthesis of glycolipids in myelin deficient mutants: Brain glycosyl transferases in Jimpy and Quaking mice

Abstract Two glycosyl transferases involved in the biosynthesis of glycolipids were investigated in the brain of myelin deficient mutants, Jimpy and Quaking mice. The first, UDP-galactose-ceramide galactosyl transferase, which catalyzes the synthesis of cerebroside from UDP-galactose and ceramide, was markedly reduced in Jimpy mouse and its activity did not change during the myelination period. In Quaking mouse the activity of this enzyme was also reduced but showed characteristic developmental changes, similar to those observed in normal mouse brain. The second glycosyl transferase, UDP-glucose-ceramide glucosyl transferase, involved in the biosynthesis of gangliosides, had a different pattern of developmental changes, and normal levels of activity were found in mutant brain. These data, as well as others discussed in the paper, indicate the existence in the postnatal brain development of two different glycosyl transferase systems, presumably with independent genetic controls.

Paps-cerebroside sulphotransferase activity in brain and kidney of neurological mutants.

Three recessive mutations in mice result in grossly defective mye~ination in the central nervous system (CNS) while myelination of peripheral nerves is normal. Two of these mutants, the Jimpy (jp) and a myelin synthesis deficient mutation (msd) are sex-linked and may be alleles [ 1,2] , whereas the third one, the Quaking (qk) is autosomai recessive. Brain cerebrosides and sulphatides are greatly reduced in all three mutants f2-51 . Moreover, a deficiency in the glycosyl-transferases involved in the biosynthesis of cerebrosides has now been shown in the Jimpy [6,7 ] , Quaking [7,8] and msd ]9] mice. Sulphatide synthesis also appeared to be altered since there was reduced incorporation of ‘“C-galactose into this lipid 17, IO]. However this type of study does not distinguish between the possibility that sulphatide biosynthesis is decreased because the precursor cerebrosides are greatly reduced, and the possibility that the activity of the 3’-phosphoadenosine-5’.phosphosulphatecerebroside suiphotransferase (PAPS-CST), responsible for synthesizing sulphatides from cerebrosides, is depressed. The results presented show that in the three mutants, when compared to normal animals, the PAPS-CST activities in the brains were greatly reduced while they were unchanged in the kidneys.

Enzymatic synthesis of lactosylceramide by a galactosyltransferase from developing chicken retina

Abstract The activity of UDP-galactose: glucosylceramide galactosyltransferase was studied during chick retina ontogenesis. This galactosyltransferase catalyses the formation of lactosylceramide, a reaction which is considered to be one of the initial steps of ganglioside biosynthesis. The activity was detected in 7 day-old embryos and was present in adult animals. The highest specific activity was found at the 11th day of embryonic life. The radioactive product was identified as lactosylceramide by comparison with standard glycolipids, by radiochromatography, by thin layer chromatography of the o -methyl-glycoside derivatives and by gas-liquid chromatography. The enzyme required the presence of Mn 2+ , had a pH optimum of about 6·7 and was stimulated by Triton X-100.

THE FATTY ACID COMPOSITION OF PHOSPHOLIPIDS AND GLYCOLIPIDS IN JIMPY MOUSE BRAIN

Abstract— Phospholipids and sphingolipids from brains of normal and Jimpy mice were isolated in a pure form by thin‐layer chromatographic procedures. The fatty acid composition of the major phospholipids, i.e. ethanolamine glycerophospholipids, serine glycerophospholipids, choline glycerophospholipids and inositol glycerophospholipids, as well as sphingomyelin, cerebrosides and sulphatides was determined by gas‐liquid chromatography. A specific fatty acid pattern for each of the four glycerophospholipids was found. The fatty acid composition of inositol glycerophospholipid, which has not previously been studied in mouse brain, was characterized by a high concentration of arachidonic acid. After 16 days of age, fatty acid analysis showed definite differences between the phospholipids from normal and mutant brains. A small increase of polyunsaturated fatty acids in glycerophospholipids of ethanolamine, serine and choline from the Jimpy central nervous system was found, which has been explained by the myelin deficiency. Sphingomyelin, cerebrosides and sulphatide analyses showed a wide distribution of saturated and mono‐unsaturated fatty acids in both normal and mutant mice. A reduction in the amount of long‐chain fatty acids was demonstrated in mutant brain sphingolipids; in sulphatides and cerebrosides, the amount of non‐hydroxy fatty acids was reduced to a greater extent than in sphingomyelin.

Regulation of myelinogenesis

From the investigation of the myelin deficient mutants (Jimpy and Quaking) some information could be obtained concerning normal myelination. We have: 1. (1) Evidence in favor of the psychosine and psychosine sulphate pathways in vivo. 2. (2) Evidence for the presence of the ganglioside G4 (GM1) in myelin. 3. (3) Evidence that myelination is a coordinated phenomenon at the molecular level. 4. (4) Demonstrated a marker of myelin and of oligodendroglial membranes: 2′,3′-cyclic AMP 3′-phosphodyrolase. We observed in the mutants the following phenomena: 1. (1) A decrease in the rate of synthesis of: UDP-galactose sphingosine galactosyl transferase; UDP-galactose ceramide galactosyl transferase; PAPS sphingosine sulphotransferase; PAPS cerebroside sulphotransferase; 2′,3′-cyclic AMP 3′-phosphohydrolase. 2. (2) A decrease in the synthesis of long-chain fatty acids. 3. (3) A decrease in the synthesis of ganglioside G4 (GM1). 4. (4) A striking decrease in myelin proteolipid proteins; a less-marked decrease in myelin basic proteins. We suggest the following possible primary causes of the defects: 1. (1) A defect in the maturation of oligodendrocytes. 2. (2) A mutation of a regulatory gene or the binding site of a depressor of the transcription of the mRNA of enzymes involved in myelination. 3. (3) A mutation of a factor involved in the interaction between oligodendroglia and axons. 4. (4) A mutation of an enzyme whose products subsequently induce the later stages of transcription of the enzymes involved in myelination. In addition, it should be pointed out that since the Jimpy and MSD mutations are sex-linked and the Quaking mutation is autosomal, recessive myelination is controlled by two different chromosomes.

Enzymatic synthesis of psychosine in “Jimpy” mice brain

The previous studies [l-4] have provided the basic data on a biochemical disorder in the central nervous system (CNS) of “Jimpy” mouse, a neurological mutant characterized by a severe myelin deficiency. The most marked feature of “Jimpy” brain was a striking reduction of cerebrosides and sulfatides. It was suggested that a disturbance of galactolipid synthesis may be the primary cause of cerebroside and sulphatide reduction leading to a defective myelin formation [2,3]. Experiments conducted in vitro [S-7] with brain tissue indicated that UDP-galactose is the precursor of the galactose moiety of cerebrosides, and that cerebrosides are formed from sphingosine via psychosine. Although the findings suggesting that cerebrosides may be formed directly from ceramide have been reported [8], psychosine seems to be more likely precursor. The study described in this communication was undertaken in order to elucidate whether the psychosine pathway is involved in the metabolic error of “Jimpy” mice. The results presented show that the incorporation of radioactivity into psychosine following incubation of UDP-galactose-14C and sphingo sine with brain homogenate is significantly decreased in “Jimpy” brain. UDP-galactose-14C (uniformly labelled in the galactose moiety) was obtained from New England Nuclear Corp. (Boston). Natural sphingosine was prepared by

PHOSPHOLIPID METABOLISM IN LIGHT AND DARK ADAPTED EXCISED RETINA

Abstract— The phospholipid composition of, and the incorporation of labelled phosphorus into the different phospholipids of rat and calf retina have been studied. The influence of various conditions, such as dark and light adaptation, during the preparation of retina, lipid extraction and incubation of retina with radioactive phosphorus was investigated.

Enzymatic deficiency in neurological mutants. Brain uridine diphosphate galactose: Ceramide galactosyl transferase in jimpy mouse

There is a substantial amount of data supporting the view that the myelination defect in the Jimpy mutation is accompanied by a disturbance of glycolipid biosynthesis in the central nervous system (CNS). Cerebrosides and sulphatides, important glycolipid components of the myelin sheath, are significantly reduced in Jimpy brain [l-3] . The incorporation of galactose-14C in uivo into cerebrosides and sulphatides is much lower in Jimpy brain than in normal controls [4-61. Further, there is a marked decrease of a galactosyltransferase catalyzing the transfer of galactose from UDPgalactose to sphingosine to give galactosyl-sphingosine @sychosine) [S, 71. The activity of this enzyme was also decreased in Quaking mouse, another mutant with defective myelination [8]. This finding was of particular interest since psychosine is considered as an intermediate in the postulated pathway of cerebroside biosynthesis [9, lo] . Ceramide was active as the acceptor of both glucose and galactose from their nucleotide derivatives in the presence of glycosyltransferases from embryonic chicken brain [ 1 l] . The presence of a galactosylceramide transferase (UDP-galactose:ceramide galactosyl transferase) has also been reported in mouse brain [ 121 . The enzyme was active only with ceramide containing hydroxy fatty acids. The following reaction catalyzed by this enzyme was proposed as a possible route of cerebroside synthesis in brain [12] :