Notes: Abstract: Rats fed a diet containing 1.25% elemental tellurium initiated on postnatal day 20 undergo a transient neuropathy characterized by synchronous demyelination of peripheral nerves. In sciatic nerve, the extent of demyelination was maximal after 5 days of tellurium exposure; there was a loss of 25% of the myelin, as assayed by concentration of myelin-specific P0 protein. Tellurium-induced alterations in the metabolic capacity of Schwann cells were examined by measuring the synthesis of myelin lipids in vitro in isolated sciatic nerve segments. Exposure to tellurium resulted in an early marked decrease of ∼50% in overall incorporation of [14C]acetate into lipids, with a preferential depression in synthesis of cerebrosides, cholesterol, and ethanolamine plasmalogens (components enriched in myelin). Most dramatically, within 1 day of initiation of tellurium exposure, there was a profound increase in [14C]acetate-derived radioactivity in squalene; 23% of incorporated label was in this intermediate of cholesterol biosynthesis, compared to 〈 0.5% in controls. In association with the remyelinating phase seen after 5 days of tellurium exposure, synthesis of myelin components gradually returned to normal levels. After 30 days, metabolic and morphologic alterations were no longer apparent. We suggest that the sequence of metabolic events in sciatic nerve following tellurium treatment initially involves inhibition of the conversion of squalene to 2,3-epoxysqualene, and that this block in the cholesterol biosynthesis pathway results, either directly or indirectly, in the inhibition of the synthesis of myelin components and breakdown of myelin.

Notes: The demyelination of peripheral nerves that results from exposure of developing rats to tellurium is due to inhibition of squalene epoxidase, a step in cholesterol biosynthesis. In sciatic nerve, cholesterol synthesis is greatly depressed, whereas in liver, some compensatory mechanism maintains normal levels of cholesterol synthesis. This tissue specificity was further explored by examining, in various tissues, gene expression and enzyme activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Exposure to tellurium resulted in pronounced increases in both message levels and enzyme activity in liver, the expected result consequent to up-regulation of this enzyme in response to decreasing levels of intracellular sterols. In contrast to liver, levels of mRNA and enzyme activity in sciatic nerve were both decreased during the tellurium-induced demyelinating period. The temporal pattern of changes in 3-hydroxy-3-methylglutaryl-CoA reductase message levels in sciatic nerve seen following exposure to tellurium was similar to the down-regulation seen for mRNA specific for PNS myelin proteins. Possible mechanisms for differential control of cholesterol biosynthesis in sciatic nerve and liver are discussed.

Notes: Abstract The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5-mm segments determined. In both control and acrylamide-treated animals, outflow profiles of [35S]methionine-labeled proteins showed a well defined crest which moved down the nerve at a rate of ± 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine-labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide-treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide-treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide-induced increase in the demand for axonally transported material.

Notes: Abstract— Partially purified myelin from brains of 17-day-old rats was separated into 4 subfractions on a discontinuous sucrose gradient by virtue of heterogeneity in density and particle size. The protein composition of each subfraction was determined by densitometry following separation of proteins on polyacrylamide gels in buffers containing sodium dodecyl sulphate. The major proteins studied included two basic proteins, proteolipid protein, the major high molecular weight protein (W) and a group of high molecular weight proteins.The percentage of high molecular weight proteins decreased sequentially from fraction D to A, that of the W protein remained constant, while relative amounts of the two basic proteins increased. Proteolipid protein concentration also increased as a percentage of the total protein from fraction D to B, but the uppermost fraction. A, had a markedly lower amount than fraction B. At 1 h after intracranial injection of [3H]leucine, the specific radioactivity of the basic and proteolipid proteins decreased from fraction D to B, with proteolipid protein in fraction A again anomalous (specific radioactivity higher than expected). These results are consistent with (but do not prove) a precursor-product relationship for individual proteins from denser to lighter subfractions, with the exception of myelin subfraction A.Experiments involving time staggered injections of a [14C] and later a [3H] labelled amino acid gave data which demonstrated that the W and basic proteins were added simultaneously (or with delays of much less than 20 min) to all of the subfractions, while proteolipid protein was added sequentially, from lower to upper fractions on the gradient. This double isotope technique also confirmed our previous observations that proteolipid protein shows a lag in entry into myelin compared to basic protein.

Notes: Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P0), two uncharacterized proteins, and two basic proteins (P1 and P2). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P1 and P2 proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P2 protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P0, P1 and P2 proteins from rabbit sciatic nerve and P0 and P2 proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P1 protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P0 and P2 proteins are unique to the PNS.

Notes: Abstract— Partially purified myelin from the brains of 17-day-old rats was separated into 4 subfractions on a three-step sucrose gradient by virtue of heterogeneity in density and particle size. Precursor-product relationships between different membrane fractions were investigated by determining the specific radioactivity of individual lipids in each subcellular fraction 15 min after intracranial injection of an appropriate precursor. Rats were injected with [2-3H]glycerol. myelin subfractions prepared, and individual lipids separated by TLC. For choline and ethanolamine phospholipids, specific radioactivity was highest in the densest fraction (D), intermediate in the next densest fraction (C), and lowest in the lighter fractions (B and A). Similar results were observed for cerebroside and sulphatide when [3H]galactose was the precursor. These data are consistent with (but do not prove) a precursor-product relationship for individual lipids from the densest to the lightest subfraction.Another experimental design involving time staggered injections of [3H] and [14C] precursors was developed which enables a more definitive result with regard to precursor-product relationships to be obtained. A precursor-product relationship between a given lipid in a dense myelin membrane fraction, and the same lipid in a lighter subfraction, would be indicated by a change in isotope ratio. If there is no precursor-product relationship. Ihe isotope ratio should be constant. Such experiments were done with [3H] and [14C]glycerol. The data indicated that phosphatidyl ethanolamine and its plasmalogen analog were added first to the densest subfraction and then in turn to the lighter subfractions. In contrast, phosphatidyl choline and its plasmalogen analog were added “simultaneously” (i.e. with delays of much less than 15min) to each of the subfractions. Similar experiments with [3H] and [14C]galactose showed that cerebroside, sulphatide and galactosyl diglyceride also entered the subfractions simultaneously rather than in sequential order. Thus the assembly of the myelin sheath involves an obligate order of addition of certain lipids. while other lipids are probably added in a random order.

Notes: 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.

Notes: Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P0, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [32P]orthophosphate or [3H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [32P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [32P]-phosphate into several proteins at the P0 region of polyacrylamide gels. Specific radioactivity of [3H]fucose in P0 protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [32P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.

Notes: Abstract— Seventeen-day-old rats were injected intracranially with [3H]leucine, then sacrificed between 1 and 24 h. Myelin was prepared from the brains on discontinuous sucrose gradients and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulphate. Proteins were stained with acid Fast Green and the distribution was quantitated by densitometry. The gels were then sliced and the radioactivity in each slice was determined. Between 1 and 24 h, the radioactivity in proteolipid protein increased from 18% to 37% of the total radioactivity in the proteins of isolated myelin. During this same period, the per cent distribution of radioactivity in basic and Wolfgram proteins remained constant while that in the remaining high molecular weight proteins decreased. Similar results were also obtained with [3H]glycine as a precursor. The relative specific activity of all of the myelin proteins increased between 1 and 6 h, then remained constant between 6 and 24 h. At 1 h, proteolipid protein reached only 25% of its maximal (6 h) relative specific radioactivity, while the other two proteins reached 50% of maximum. These results indicate a lag in the appearance of labelled amino acids in proteolipid protein relative to the other myelin proteins.

Notes: A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands.Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.