THE COMPOSITION OF MYELIN FROM THE MUTANT MOUSE ''QUAKING''

Abstract— Myelin was isolated from the brains of adult Quaking mice, a mutant showing a deficiency of myelin in the central nervous system, and normal controls. The mutant myelin was found to have a higher flotation density than that of the control and showed marked differences in lipid composition. The myelin from Quaking mice was found to be deficient in cerebroside and ethanolamine phospholipid. Acrylamide gel electrophoresis of total myelin protein demonstrated a pronounced deficiency of proteolipid protein. The activity of cyclic 2',3'-AMP phosphohydrolase was normal.     

CHANGES IN THE PROTEIN COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Abstract— Myelin was isolated from the brains of mice at various ages by a procedure involving a final purification on a continuous CsCl gradient. Myelin protein accumulated throughout development, increasing from 0.25 mg of protein/brain at 8 days of postnatal age to 3.5 mg of protein/brain at 300 days, although the rate of accumulation was greatest at about 21 days of age. Quantitative studies of the protein composition of these samples were carried out, utilizing discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Mouse brain myelin, contained (in order of increasing molecular weight) two basic proteins, an uncharacterized doublet, proteolipid protein, and a group of high molecular weight proteins. There were marked changes in the quantitative distribution of these proteins with increasing postnatal age. The basic protein fraction of total myelin protein increased from about 18 per cent at 8 days to 30 per cent at 300 days of age. Proteolipid protein increased even more dramatically, from 7 to 27 per cent in the same time interval. These chemical studies were correlated with ultrastructural investigations, both of the developing myelin sheath in situ and the isolated myelin obtained from mice of various ages. A hypothesis, relating the observed changes in protein composition of myelin during development to its mode of formation, is developed. Another subcellular fraction, separated from myelin, by virtue of its greater density in a CsCl gradient, was also studied. It was a vesicular, membranous fraction present at a level of 0.35 mg of protein/brain at all ages and was related to myelin in terms of protein composition.     

GANGLIOSIDES IN DEVELOPING MOUSE BRAIN MYELIN

Abstract— Gangliosides were isolated from myelin prepared from mouse brains of different ages (23 to 490 days). Quantitative estimation of lipid-bound sialic acid levels indicated a gradual increase from 560 μg/g of myelin at 23 days to about 1200 μg/g of myelin at older ages. The major ganglioside in all myelin preparations was the monosialoganglioside G₄ (G_M1). However, considerable amounts of di- and trisialo species also were found in myelin from young animals. In contrast to human myelin in which the monosialoganglioside, sialosylgalactosylceramide (G₇) was highly enriched (L edeen et al. , 1973), a much smaller enrichment of this ganglioside was noticed in mouse brain myelin. Ganglioside G₇ was not detectable in myelin until the animals were 35 days old, and showed a slight increase with increasing age after that. The results strongly indicated that the concentration of G₇ in myelin is species specific and age dependent. The study also demonstrated that the ganglioside accretion in developing mouse brain myelin was attributable to the enrichment of monosialogangliosides G₄ (G_M1), G₅ (G_M2) and G₇ at the expense of polysialogangliosides.     

IN VITRO SYNTHESIS OF THE MYELIN BASIC PROTEINS IN THE DEVELOPING MOUSE BRAIN: PROPERTIES OF A HOMOGENATE SYSTEM

—An in vitro system using mouse brain homogenates has been developed to study the synthesis of the myelin basic proteins. Incorporation of [³H]leucine into protein in this system did not require additional energy sources. The system was slightly stimulated by glucose and strongly inhibited by puromycin. Myelin basic proteins were isolated from incubation mixtures by conventional techniques of solvent extraction and column chromatography, and finally separated into the large and small components by polyacrylamide gel electrophoresis in an acetic acid-urea system. Gels were stained, sliced, dissolved, and counted, and relative rates of incorporation of label into the two basic proteins were determined at several ages. The ratio of radioactivity incorporated into the small (S) and large (L) basic proteins, over a 30 min incubation period, was found to increase from 0.97 at 10 days to 1.59 at 21 days and decline thereafter. These data generally agree with earlier studies on the in vivo synthesis of the myelin basic proteins in mice. An interesting feature of the time course was that incorporation of [³H]leucine into the purified myelin basic proteins relative to incorporation into total protein in the homogenate increased almost 2-fold during the course of the 30-min incubation. This suggested that post-translational processing of at least one of the two basic proteins was occurring. To examine this possibility further, experiments were conducted in which incorporation was allowed to proceed for 2–5 min, before being inhibited with puromycin; the incubation was then continued for up to 25 min longer. Although total incorporation was inhibited immediately after puromycin addition, label was found to continue to accumulate in the basic proteins to the extent of 30–100% above controls. These data support the notion that the MBPs are synthesized as precursors and then processed to yield authentic myelin basic proteins and that this processing can occur in vitro.     

COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Myelin was isolated from the brains of mice at ages of 14, 24, 41, 44, 47, and 182 days and the contents of lipid phosphorus, cholesterol, lipid galactose, alkenyl groups, ethanolamine phosphoglycerides, choline phosphoglycerides, sphingomyelin, and serine and inositol phosphoglycerides were determined. Significant differences in the composition relative to total lipid phosphorus were found in the myelin. At 14 days of age, the myelin had lower relative amounts of cholesterol, galactolipids, alkenyl groups, and ethanolamine phosphoglycerides and a higher relative amount of choline phosphoglycerides.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

FATTY ACID COMPOSITION OF CEREBROSIDES IN MICROSOMES AND MYELIN OF MOUSE BRAIN

Abstract— The fatty acid composition of cerebrosides isolated from myelin and from light and heavy microsomes of adult mouse brain was determined. 2-Hydroxy fatty acids represented 80 per cent of the fatty acids in myelin cerebrosides and approximately 55 per cent of the fatty acids in both light and heavy microsomes. In myelin, the majority of the fatty acids, both normal and hydroxy, were of chain length > C-20; in microsomes, shorter chain acids (C-16 to C-20) predominated.     

A TECHNIQUE FOR THE STUDY OF ACETYLCHOLINE TURNOVER IN MOUSE BRAIN IN VIVO

Abstract— —A method to measure the rate of acetylcholine turnover in mouse brain in vivo has been developed. It is based on the formation of labelled acetylcholine from intravenously injected labelled choline. The isotopic dilution of choline in the brain has been measured by assaying endogenous choline in the brain by an enzymatic method using tritium-labelled acetyl-CoA and purified choline acetyltransferase.
The rate of acetylcholine turnover in the brain could be calculated at 50 n-moles acetylcholine/g/min in conscious mice. In anaesthetized mice and in mice treated with oxotremorine, a decrease of acetylcholine turnover to about 10 n-moles/g/min was found.     

THE SYNTHESIS OF MYELIN IN DEVELOPING RAT BRAIN

Abstract— —The synthesis of myelin proteins has been studied in the grey and white matter slices of developing rat brain by measuring the incorporation of [³H]lysine and [¹⁴C]arginine into polypeptide. The incorporation was sensitive to cycloheximide and puromycin at 1 mM concentration. Developing rat optic nerve slices, free of retinal ganglion cells, were able to synthesize myelin basic and proteolipid proteins, but rat retinal preparation failed to synthesize myelin basic protein. Rabbit retinae were able to synthesize myelin basic and proteolipid proteins. Significant activity of the myelin marker enzyme 2',3'-cyclic nucleotide-2'-phosphodiesterase has been found in the rabbit retina but not in rat retina. The results presented in this communication suggest that myelin proteins in the rat CNS are synthesized by the oligodendroglial cells and that neurons probably do not participate.     

OCCURRENCE OF ALKANES IN BRAIN MYELIN. COMPARISON BETWEEN NORMAL AND QUAKING MOUSE

Alkanes, a new class of neurolipid, were found in mouse brain, the level being reduced in the Quaking mutant. These hydrocarbons are concentrated in myelin; minor amounts being found in microsomes, mitochondria and synaptosomes. The average recovery is 7.1 μg/mg in normal myelin, 2.2 in the Quaking myelin. The distribution pattern of these alkanes was determined by gas liquid chromatography and was found to differ in normal and Quaking myelin; the hydrocarbons consist mainly of n-alkanes ranging from C₂₁ to C₃₂ with even and odd aliphatic chains.     

THE SURFACE PROPERTIES OF ISOLATED RAT BRAIN MYELIN: A MICROELECTROPHORETIC STUDY

Abstract— The surface properties of isolated rat brain myelin have been investigated by the method of particle microelectrophoresis. The mobility has been measured as a function of ionic strength and composition of the buffer, the results suggesting the presence of areas of non-ionogenic material. Antisera against ganglioside and myelin produce a fall in mobility whereas antiproteolipid apoprotein produces a slight increase but antibasic protein and anticerebroside sera had no effect on mobility. Of the lipases studied only phospholipase D produced a clear increase in mobility. From this preliminary study some indications of the asymmetric disposition of components in the myelin sheath may be derived. The ganglioside appears to be wholly located at intraperiod line interface, along with some fraction of the galactocerebroside and phosphatidylcholine. There appears to be few amino groups, but SH groups are present.     

EFFECTS OF ESSENTIAL FATTY ACID DEFICIENCY ON MYELIN AND VARIOUS SUBCELLULAR STRUCTURES IN RAT BRAIN

Abstract— Essential fatty acid deficiency initiated in rats prior to birth and continued for 140 days after birth affects the fatty acid composition of ethanolamine phosphoglyceride of brain subcellular fractions (myelin, microsomes, mitochondria and synaptosomes). It was confirmed that the fatty acid composition of the same phospholipid class differs considerably among the various subcellular fractions: myelin has the highest concentration of long-chain monoenes, while in the other fractions saturates and polyenes predominate. In EFA deficiency it was found that (1) trienes are elevated and tetraenes decreased in all fractions, (2) the triene/tetraene ratio, which is considered a biochemical index of essential fatty acid deficiency, is highest in myelin and lowest in the synaptosomal fraction, and (3) in all fractions there is a shift towards more unsaturated members of the same fatty acid family.     

MYELIN BASIC PROTEIN MICROHETEROGENEITY IN SUBFRACTIONS OF RAT BRAIN MYELIN

Abstract— Myelin subfractions were prepared from adult rat brain by discontinuous sucrose gradient ultracentrifugation. Gel electrophoretic studies at pH 10.6 in the presence of urea revealed differences in basic protein microheterogeneity among subfractions. With increasing myelin density there was a decrease in the most positively charged components of both large BP and small BP. Since these components are the least modified by deamidation and phosphorylation, it seems likely that the heavier myelin subfractions are enriched in the more modified components of the microheterogeneous population of BP. These observed differences may be related to the regulatory processes controlling biosynthesis, organization, and catabolism of BP in CNS myelin.     

CHANGES IN THE OSMIOPHILIA OF MYELIN AND LIPID CONTENT IN THE KITTEN OPTIC NERVE

Myelin osmiophilia has been shown to develop significantly later than myelin staining by Luxol fast blue and Sudan black, in the developing kitten optic nerve. These histological changes are accompanied by alterations in the lipid composition of the optic nerve. Although myelination commences at about 10 days post partum in the nerve the appearance of cerebrosides is unexpectedly delayed. Changes in fatty acid chain length and lipid composition of optic nerve are consistent with the suggestion that ‘early’ myelin may be unchanged glial plasma membrane.     

SUBCELLULAR DISTRIBUTION AND STRUCTURAL POLYMORPHISM OF MYELIN BASIC PROTEIN IN NORMAL AND JIMPY MOUSE BRAIN

Abstract— Brains from 20 day old normal and 20 day old Jimpy mice were fractionated by a modification of the procedure described by Eichberg et al. (1964). Each of the fractions obtained was subjected to radioimmunoassay (RIA) for myelin basic protein (MBP). From both the normal brain and the Jimpy brain MBP was recovered in three separate membrane fractions designated P1A. P2A. and P3A. which differed in their sedimentation properties but which had similar densities (less than 1.08 g'ml). In the Jimpy brain compared to normal brain the amounts of P1A and P2A were greatly reduced but the amount of P3A was increased. During development in the normal brain the amount of MBP in the PIA fraction increased in parallel with the accumulation of myelin. The amount of MBP in P2A increased gradually during active myelination and decreased slightly in the adult. The amount of MBP in P3A increased sharply during the period of most active myelination and decreased approx 10-fold as the rate of myelination in the brain declined. Electron microscopic examination revealed that the P1A and P2A fractions from normal brain contained myelin fragments while the P1A and P2A fractions from Jimpy brain contained numerous vesicular membranous structures with little if any identifiable myelin. The P3A fraction from both normal and Jimpy brain contained small vesicles of uniform size, some with polyribosomes attached. Each of the fractions was analyzed by a technique combining sodium dodecyl sulfate polyacrylamide gel electrophoresis with RIA for MBP in order to identify and quantitate the four different forms of MBP with molecular weights of 21.5 K. 18.5 K. 17 K and 14 K dalton. The proportions of the four MBPs were characteristic for each fraction. The relative proportions of the four proteins were 14 K > 18.5 K > 17 K > 21.5 K daltons in all the fractions except P1A Jimpy in which 21.5 K dalton protein was the predominant form of MBP present. The cellular origin of the MBP containing fractions from normal and Jimpy brain is discussed.     

INFLUENCE OF LIPIDS ON THE ACTIVITY OF CEREBROSIDE-SULPHOTRANSFERASE IN MOUSE BRAIN: A COMPARATIVE STUDY OF JIMPY AND NORMAL MOUSE BRAINS 1

Abstract— The effect of lipids other than the substrate cerebroside on the activity of cerebroside-sulphotransferase (CST) in Jimpy and normal mouse brain was investigated. The enzyme activity of an acetone-treated microsomal preparation can be stimulated in the presence of the extracted lipids either with or without addition of exogenous cerebroside as a substrate. The CST activity in the Jimpy mutant compared to that in normal animals differs from 18% in homogenate to approx 80% in solubilized or acetone-extracted microsomes. An addition of total lipid from normal mouse brain to microsomal preparations from which lipid has been removed by acetone results in a stimulation of Jimpy CST activity up to a value of 80% of normal mouse brain microsomes. Both Jimpy and normal mouse brain CST can be also stimulated by the addition of single lipid components such as cholesterol and lecithin by 50% in normal and 100% in Jimpy brain microsomes. These findings lead to the hypothesis that there is a lipid requirement for CST activity other than the substrate cerebroside.     

A MORPHOGENETIC STUDY OF LANCEOLATE,A LEAF-SHAPE MUTANT IN THE TOMATO

Mathan , D. S., and J. A. Jenkins . (U. California, Berkeley.) A morphogenetic study of lanceolate, a leaf-shape mutant in the tomato . Amer. Jour. Bot. 49(5): 504–514. Illus. 1962.—The single-gene mutant, lanceolate (La/La⁺), which has simple, entire leaves rather than the oddpinnately compound leaves of the normal tomato (Lycopersicon esculentum), differs from normal (La⁺/La⁺) in many characters which can be related to fewer and larger cells in meristematic regions. The homozygous lanceolate (La/La) is sometimes lethal, but is usually expressed as reduced, which consists of a hypocotyl without cotyledons, or occasionally either by modified, in which there is a single fused cotyledon and a bud, or by narrow, in which the bud produces a shoot with simple leaves even smaller than those of lanceolate and a tendril-like inflorescence without flowers. In an attempt to overcome the effect produced by the lanceolate gene, the mutant was treated with a number of substances. Embryos without cotyledons cultured in White's medium plus adenine or tyrosine or both gave a higher frequency of narrow plants. With added gibberellic acid, no narrow plants developed from embryos without cotyledons. When tyrosine was sprayed on young narrow plants, they developed larger leaves and morphologically normal but sterile flowers. On the other hand, gibberellic acid sprayed on young lanceolate plants altered their development in the direction of narrow.