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The effects of vitamin B-12 deficiency on lipids and fatty acids of spinal cord myelin were studied in control and vitamin B-12-deficient fruit bats. Very low plasma and brain vitamin B-12 concentrations were present in animals fed the vitamin B-12-free, all fruit diet. Myelin was isolated from the spinal cord of control and vitamin B-12-deficient animals (n = 3 pools) by means of a flotation method in a discontinuous sucrose gradient. The molar concentration of cholineglycerophosphatide was significantly lower in the deficient bats than in the controls. The molar cholesterol: phospholipid ratio in the deficient bats was also significantly lower. The odd-chain fatty acid 15:1 was not detected in cholineglycerophosphatide from the controls, but comprised 1.4% of the total fatty acids in the deficient group. The odd-chain fatty acids 15:0, 17:1 and 19:0 were present in slightly higher amounts in cholineglycerophosphatide of deficient bats. Lipid and fatty acid differences were related to the vitamin B-12-requiring methionine synthetase and methylmalonyl CoA mutase reactions.

Myelin proteins were quantitated in whole tissue and isolated 'myelin fractions' from spinal cord, brainstem and hemispheres of 'shaking pups', a mutation in Springer-Spaniel dogs characterized by hypomyelination of the CNS. The amount of myelin basic protein (MBP) in the brainstem of affected 4-week-old pups was 2.6% of that in age-matched controls, while the levels of 2'3'-cyclic nucleotide phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG) were 10% and 15% of the control levels, respectively. Similar results were obtained in the spinal cord and hemispheres, and the amounts of these proteins in the mutant pups did not change substantially between 4 and 16 weeks of age. The amount of the 21 kDa MBP compared to the 18 kDa MBP was relatively increased in the shaking pups, suggesting that the small amount of myelin formed was immature. The yields of myelin fractions from the mutant pups were very low; e.g., the yield from the brainstems of 4-week-old mutants was only 2.4% of that from age-matched controls and the yield did not increase by 16 weeks. The isolated myelin fractions contained very little MBP (less than 0.5% of total protein) or proteolipid protein, indicating that they were a very immature form of myelin or consisted largely of non-myelin contaminants. MAG in the 'myelin fractions' from the mutant brainstems were 9-15 fold higher and CNP levels were 2-3 fold higher than those in whole homogenates, suggesting that the isolated fractions were enriched in oligodendroglia-derived membranes. Overall, the biochemical results are consistent with a severe hypomyelination of the CNS in which a small amount of immature myelin is formed.

The immunohistochemical localization of two myelin specific proteins-basic protein (BP) and proteolipid protein (PLP)-was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (i) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (ii) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (iii) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance of varicosities can be used as an index of the relative stage of maturation of myelin in an individual fiber. In general, PLP appeared in fibers at a later stage of maturation than did BP based on the above criteria. However, in a relatively small number of fine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (i) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (ii) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (iii) Very small fibers often contain low concentrations of BP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber.

Bovine spinal cord protein from peripheral nerve (BSCP-PN) was detected in the soluble fraction of the initial 0.8 M sucrose homogenate of bovine peripheral nerves by immunodiffusion analyses and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The BSCP-PN in the soluble fraction of the 0.8 M sucrose homogenates was 25% of the BSCP-PN found in the soluble fraction of 0.3 M NaCl homogenates of peripheral nerve. BSCP-PN was also identified in purified bovine peripheral nerve myelin by immunodiffusion analyses and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Densitometry data indicated that the BSCP-PN in myelin decreased from 25% of the total protein to approximately 8% when myelin was extracted with 0.3 M NaCl or 0.05 M HCl. The protein that remained in the BSCP-PN band of the NaCl-extracted myelin was identified as the periodic acid-Schiff II glycoprotein of peripheral myelin. Basic proteins such as BSCP-PN or lysozyme bound to myelin and to NaCl-extracted myelin when they were added to homogenates of myelin in 0.8 M sucrose. Pepsin, an acidic protein, did not bind to myelin under the same conditions. The results suggest that in 0.8 M sucrose, positively charged BSCP-PN released from the cytoplasm by homogenization binds to negatively charged myelin; thereafter, the BSCP-PN-myelin complex remains intact until it is dissociated in media of sufficiently high ionic strength. This interpretation is consistent with the immunohistological studies which demonstrated that BSCP-PN was not in the myelin sheath but was clearly localized in axons and in, or adjacent to, the Schwann cell basement membrane.

Central nervous system myelin isolated from the rainbow trout (Salmo gairdneri) displays a very low median density on zonal gradient centrifugation, banding at approximately 0.32 M sucrose. Its proteins consist of a 36 K (36,000 mol.wt.) component, two Concanavalin A-reactive intermediate proteins IP1 (23,000 mol.wt.) and IP2 (26,200 mol.wt.), and two basic proteins BP1 and BP2, of which the latter co-migrates with rat SBP while BP1 is of slightly smaller size. The trout myelin proteins electrofocus at pH positions similar to those of their mammalian counterparts. Immunoblotting shows that antibodies against rat PNS myelin P0 glycoprotein are bound by IP1 and IP2, but not by 36K. None of the trout myelin proteins react with anti-rat CNS myelin proteolipid protein (PLP) antiserum. The basic proteins BP1 and BP2 bind strongly to antibodies directed against human myelin basic protein. In vivo injection of tritiated fucose or palmitate leads to radiolabeling of IP1 and IP2. Under autolytic in situ conditions the appearance of a glycosylated 20,000 mol.wt. component (IP0) is noted, with parallel reduction of both IP1 and IP2, indicating sequence homologies between IP1 and IP2. The 36K protein is not affected by autolysis.

The lactoperoxidase-catalyzed iodination technique was utilized to incorporate radioactive iodine into membrane proteins which lie on the outer surface of the myelin sheath. An intact, myelinated nerve bundle, the dorsal column of the cat spinal cord, was employed. The enzymatically iodinated proteins were identified by polyacrylamide gel electrophoresis, and the specific radioactivity was determined. Results indicated that several high molecular weight proteins were predominantly labeled by the nonpenetrating lactopreoxidase. Proteolipid protein was also labeled, although to a lesser extent; basic protein was not labeled under these conditions. The data suggest that several high molecular weight proteins are exposed on the outer surface of the myelin sheath. Proteolipid protein is at least partially exposed on the outer surface, although it could be present at both membrane surfaces. Evidence is presented which suggests that the basic protein is located at the inner surface of the membrane, corresponding to the major dense line of myelin.

Gangliosides were isolated from whole spinal cords and cord myelin of human, cat, and rabbit by a revised methodology. The method included the sequential application of DEAE-Sephadex column chromatography, base treatment, Sephadex G-50 column chromatography, and finally Iatrobeads column chromatography. The human whole spinal cord was found to contain about one-tenth of the ganglioside concentration as in cerebral gray matter and about one-third of that in cerebral white matter. Low levels of gangliosides were also found in cat and rabbit whole cords. Only N-acetyl neuraminic acid could be detected in the ganglioside fractions of all three species. The whole cords also possessed unique ganglioside patterns when compared with the patterns of cerebral tissues. The most prominent and consistent features were the reduced concentration of Gd1a and increased amounts of Gm3 and Gd3. Human, but not cat and rabbit, spinal cord also contained Gm4 as one of the major gangliosides. Myelin prepared from the spinal cords of all three species also contained gangliosides. The amounts were only about half of those in the respective cerebral white matter myelin. The cord myelin ganglioside pattern was generally similar to the cerebral white matter myelin within the same species. Gm1 was the most abundant ganglioside in the cord myelin. Gm4 was found to be highly enriched only in myelin prepared from human sources.

Brains and spinal cords of myelin-deficient (md) and littermate control rats were analyzed serially for myelin lipids during the period from 13 to 32 days of age. The glycolipids of md rat brains were severely reduced and remained so during the period of study; brain cholesterol and phospholipids increased moderately but never reached the values for control brains. Deficiency of all three lipid classes was marked in the spinal cord and did not change with age. Among the glycolipids of md rats, deficiency was more severe in cerebrosides than sulfatides. The pronounced reduction of cerebrosides in the early stages of myelination suggests that abnormal synthesis of these glycolipids may be the most important biochemical anomaly responsible for myelin deficiency.--Csiza, C.K. Lipid class analysis of the central nervous system of myelin-deficient Wistar rats.

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance.

CNS myelin was isolated from 24 teleostean fishes and the proteins were analyzed by staining and immunoblotting. All species showed a 36 K protein, two or more glycosylated hydrophobic intermediate protein (IP) components and several myelin basic protein bands (BP). The 36 K protein was specific for teleostean fishes. The IP and BP components displayed substantial variations in their proportions as well as in molecular sizes when comparing the different teleosts. This contrasts with CNS myelin proteins which appear more stable in terrestrial vertebrates.

Myelin isolated from central nervous tissue activates the classic pathway of complement by directly activating C1. Activation of C1 can proceed to form membrane attack complex, C5b-9, in the myelin. Such an interaction between myelin and complement may be important in diseases involving myelin damage, in view of the role of complement in membrane attack and inflammation. To identify the C1-activating protein, myelin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The blots were incubated with C1 or with whole serum complement, followed by immunostaining for C1 or C3, respectively. A duplicate strip was stained with amido black or anti-myelin antibody to visualize the myelin proteins. The results showed that two major protein bands were capable of activating C1. An approximately 56-58-kilodalton band comigrated with the W2 protein and an approximately 45-47-kilodalton band migrated along with, but slightly behind, the W1 Wolfgram doublet.

Border disease (BD) was induced in lambs by inoculation of their dams at 50 days gestation with Border disease virus (BDV) isolate #31. At birth, the clinically affected lambs had diffuse spinal cord hypomyelination, confirmed by immunocytochemical staining for myelin-associated glycoprotein and myelin basic protein. In the BD lambs, large numbers of thyroid follicular epithelial cells and scattered pituitary cells contained BDV antigen by immunofluorescence staining. Double labeling techniques demonstrated the BDV-infected pituitary cells to contain growth hormone, adrenocorticotrophic hormone, prolactin, or luteinizing hormone. Cells containing thyroid stimulating hormone were rare and were not positive for BDV antigen. Infection of the pituitaries and thyroid glands caused no detectable morphologic changes as compared to controls. The BD lambs had statistically significantly (p less than 0.05) lower mean serum concentrations of thyroxine and L-3,3',5-triiodothyronine as compared to age-matched uninfected controls. Similar significant differences in the mean plasma levels of growth hormone and thyroid stimulating hormone were not found. In addition, the BD lambs had a statistically significant (p less than 0.05) lower mean activity of the myelin-associated, thyroid hormone dependent enzyme, 2',3'-cyclic nucleotide-3'-phosphodiesterase in spinal cord tissue. Although not conclusive, these results indicate that the hypomyelination in BD may be due to depressed levels of circulating thyroid gland hormones secondary to a noninflammatory and noncytolytic infection of the thyroid gland by BDV. This is one of the first reports indicating that a virus-induced hormonal alteration may cause a congenital lesion in animals.

Gangliosides were isolated from purified myelin obtained from brain and spinal cord of mature chickens and pigeons. Total concentrations were approximately two- to fivefold greater than for previously reported mammalian species, and their patterns also differed in containing significantly more sialosylgalactosylceramide (GM4). The latter comprised one-third to one-fourth of total myelin ganglioside, approximately equivalent to GM1 (II3NeuNAcGgOse4Cer). As in mammals, GM4 of avian CNS appeared to be localized in myelin. Fatty acids of this ganglioside included both the hydroxy- and unsubstituted types, and long-chain bases were almost entirely C18. Ganglioside GM1 split into two closely migrating bands on TLC, the slower of which resembled mammalian GM1 in having stearate as the main fatty acid with a measurable amount (10%) of C20-sphingosine; the faster band had predominantly longer-chain fatty acids and very little C20-sphingosine.

Myelin, isolated from forebrain and spinal cord of young and adult rats, was distributed by zonal centrifugation on linear (0.32--1.00 M) sucrose gradients in a bell-shaped mode. The peak position of forebrain myelin shifted from the density of 0.58 M sucrose in young animals to that of 0.67 M sucrose in adult rats, while in spinal cord no such pronounced shift was noticed (approximately 0.58 M sucrose). Morphologically, the preparations appeared very similar across the density ranges. Specific activities of acetylcholinesterase were substantially below the total homogenates, while those of 2', 3'-cyclic nucleotide 3'-phosphohydrolase were higher in all fractions, except in the light myelin subfractions from adult spinal cord. Basic proteins decreased from the light to the heavier fractions; higher molecular weight proteins increased, together with proteolipid protein, which in spinal cord reached a plateau and in forebrain decreased towards the heavy side. The ratio of the small basic protein/large basic protein showed higher values in the light myelin subfractions in the regions and ages examined, pointing to a higher degree of maturation.

Rat spinal cord protein (SCP) from peripheral nerves (RSCP-PN) was not detected in purified rat peripheral nerve myelin by sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis or by immunodiffusion analyses using an anti-rat SCP in peripheral nerve (RSCP-PN) serum. The slab gel electrophoretic analyses also revealed that RSCP-PN has an appreciably lower molecular size than the component of rat peripheral myelin that is identified as P2 by its molecular size of 13,600 daltons. Thus, RSCP-PN and rat P2 are unrelated proteins.

The fraction floating on 0.32 M sucrose was isolated from normal mammalian spinal cord and analyzed with regard to protein and lipid composition. Comparisons were made with the myelin fraction isolated from the same spinal cord. A close relationship between the two fractions was indicated by a similar protein banding on SDS-polyacrylamide gel electrophoresis. The relative amounts of various proteins however were different and some high molecular weight proteins appeared unique to the floating fraction. The phospho- and galactolipid patterns, as revealed by thin-layer chromatography, were similar in the floating and the myelin fractions. The proportion of hydrophobic lipids, such as sterols and isoprenyl derivatives, was higher in the floating fraction. Bands co-migrating with cholesterol esters were detected only in the floating fraction from guinea pigs. Marchi-positive material of possible paranodal origin is enriched in the floating fraction. The present findings of a biochemical composition of the floating fraction closely resembling that of myelin is in line with the view that myelin turnover includes a step of degradation localized to the paranodal regions.

Myelin of considerable purity may be isolated from small (minimum 1 mg wet weight) samples of central nervous tissue, using a 4-step centrifugation procedure. The separation of myelin proteins by micro-linear gradient polyacrylamide gel electrophoresis yields similar results to those obtained by macro-scale (homogeneous) gel systems. These techniques have been employed for a preliminary study of the regional composition of myelin fractions from the Quaking mouse.

Normal rabbit spinal cord was homogenized in sucrose and fractionated by centrifugation in a sucrose density gradient system slightly modified after the Norton-Poduslo method for the isolation of myelin. The following fractions were recovered: the fraction floating on 0.32 M sucrose, the myelin fraction at the 0.32 M/0.85 M interface and the pellet. After fixation in glutaraldehyde the fractions were subjected to Marchi staining, a histochemical method used for the demonstration of degenerating myelin. The floating fraction was enriched in Marchi-positive bodies as compared to the homogenate while the myelin fraction and the pellet contained low amounts. No esterified cholesterol was found in the floating fraction. Since histochemical and electron microscopical studies have shown that Marchi-positive myelinoid bodies in the normal CNS are associated with node-paranode regions our results indicate a possibility to isolate and biochemically characterize a presumably closely myelin-related fraction of known anatomical origin. The absence of esterified cholesterol in the floating fraction shows that biochemical or biophysical properties other than a content of esterified cholesterol may give rise to a positive Marchi reaction.

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.

Brain and spinal cord of female mice heterozygous for the jimpy gene were analyzed during development for activity of ceramide galactosyl transferase (CGT) and for levels of myelin basic protein (MBP). CGT activity was low at 13-14 days in brains of heterozygous jimpy females but showed normal levels by 31-36 days, in agreement with our earlier study of this enzyme. In cord, CGT activity was normal or slightly above normal at all ages studied, from 13-14 days into adulthood. In both brain and cord, decreased levels of MBP were observed at 13 days; by 100 days, amounts of MBP approached normal levels. Proven female carriers of the jimpy gene also showed normal levels of CGT activity, MBP, and isolated myelin at 200-250 days of age in both brain and cord. These biochemical findings agree with previous morphologic measurements in cord demonstrating deficits in myelin at early ages but compensation by 100 days. Our results show that compensation occurs earlier in cord than in brain and that levels of MBP show a closer correlation than CGT activity with amounts of myelin, as measured by either morphometric analysis or direct isolation.

Two methylated derivatives of arginine were isolated from the encephalitogenic protein of myelin from the central nervous system. Evidence is presented for the proposed structures, omega-NN'-dimethylarginine and omega-N-monomethylarginine. In the encephalitogenic protein from human brain the proportion 1:6:10 for arginine:monomethylarginine:dimethylarginine residues was found to occur at position 107. Possible roles for the methylated arginine in conformational changes and altered ion-exchange behaviour are discussed.

Amino acid sequences of encephalitogenic proteins from bovine cord and rabbit brain are reported. The bovine protein contains 45 residues. The rabbit protein is identical except for two isopolar substitutions, a dipeptide and amino acid deletion. Analysis of this protein and a 140-residue myelin basic protein indicates that the smaller protein is a portion of the larger encephalitogen. The larger myelin protein contains at least two encephalitogenic sites.