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Notes: Abstract: Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both ara-chidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 μM and 5.8 μM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol min−1 mg protein−1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 μM unlabelled arachidonic acid, with a Ki of 7.1 μM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 μM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 μM unlabelled palmitic acid, with a Ki of 5.0 μM and to a much lesser extent by arachidonic acid (Ki, 23 μM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40°C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis. These data together strongly suggest that rat brain microvessels have the capacity to catalyze specifically the formation of acyl-CoA derivatives from several polyunsaturated long-chain fatty acids, including arachidonic acid in the first place. Besides this particular arachidonoyl-CoA synthetase, palmitic acid could be activated with the aid of a second acyl-CoA synthetase.

Notes: Abstract: An ontogenetic survey of the basic protein of myelin, common to both central and peripheral nervous systems, was carried out on normal C57B1 and five dysmyelinating mutant mice. Myelin basic protein (MBP) was quantified by radioimmunoassay in the optic and sciatic nerves of mice from birth to adult stages, giving special attention to the premyelinating and early myeli-nation periods. In the optic nerves of normal mice, MBP was already detectable at birth but the active period of myelin deposition was shown to occur after day 10 postnatal. The timing and rate of accumulation of MBP were normal in Trembler. In contrast, they were abnormal in the other mutants. In the quaking mouse, the active period of MBP deposition was delayed, and its final concentration represented no more than 12% of normal in the adult. No active period of MBP deposition was observed in the other mutants. In the jimpy mouse, a slow accumulation of MBP resulted in a final concentration reaching 2% of the normal value at 25 days. In mid and shiverer mice, the MBP was hardly detectable. In the sciatic nerves of normal mice, the active period of MBP deposition occurred between days 3 and 12 postnatal. No substantial changes occurred in the period of 2 months-2 years. Deposition of MBP was normal in jimpy. In quaking, it was not only delayed but also reduced, leading to a concentration as low as 40% of normal adult. In mid and shiverer PNS, MBP concentrations were very low but noticeably higher than in the optic nerve. In the sciatic nerve of Trembler a myelination-demyelination process was strongly suggested by MBP determination. After a delayed but active period of deposition from days 5–11 postnatal, the MBP content was found to decline within a few days and remained almost undetectable until old age.

Notes: The particulate material of aquaeous homogenate of forebrain was separated by zonal centrifugation in a continuous 0.4-1.2 m-sucrose gradient after sedimentation at 100,000 g to eliminate the soluble material. Based on the absorbance at 280 nm, four major peaks were obtained from adult normal mice corresponding to 1.1 m (A), 0.68 m (B), 0.35 m (C), and 0.12 m (D) sucrose. The two smaller and lighter peaks, C and D, were not present when purified myelin was separated by the same procedure. B consisted of pure compact myelin; A was made of vesicles, sometimes with a double membrane. Throughout development, the myelin peak shifted from 0.58 m in young animals to 0.70 m in very old ones. Moreover, the myelin peak B drastically increased during development, as compared with peak A.-In the Trembler, the profile was close to normal, with a slightly higher yield of myelin which also peaked at a higher density. In the quaking, there were only two shoulders in the myelin density range at 0.68 m and 0.75 m; in the Jimpy only a faint shoulder was seen, at approximatively 0.67 m. In the shiverer, B was absent and only an A peak was present, at approximatively 0.85-0.90 m, which contained non-compact lamellar membranes and myelin figures with an abnormal major dense line. In the mid (an allele of shiverer) the density profile resembled the one obtained in the shiverer (one peak in the 0.88 m region). When considering myelin basic protein (MBP) content in the normal developing animal, it was minimum in fraction A, mainly found in the B myelin peak, but also present in the light fraction (C + D). In young animals this latter peak was prominent, in contrast to the adult. In the Trembler mutant, the profile was close to normal; in the quaking, MBP was mainly found in peak A (0.85 m), and in the Jimpy MBP was very low and nearly constant throughout the gradient (with faint quantities in light C + D fraction). In mid, the content was very low, with a peak in the 0.88 m region, in contrast with its shiverer allele, where MBP is hardly detected. More meaningful myelin studies can be carried out by using zonal centrifugation in continuous sucrose gradient, to determine the density of mutant myelin and the degree of myelin maturation in animals.

Notes: Abstract— Biochemical analysis of the leukodystrophy brain from a case of Pelizaeus-Merzbacher disease, classical type, was performed. A decrease in the amount of solid material present was found. The lyophilized brain weight was reduced to 76% of normal with a slightly greater decrease in the amount of extractable lipid. Total myelin was diminished to 7% of normal. Among specific lipids plasmalogens were present in slightly lowered amounts. Cerebrosides and sulphatides were drastically reduced to 8% of normal, whereas sphingomyelin was less severely affected. Fatty acids from phospholipids were close to normal, only enols being slightly diminished. Analysis of pure cerebrosides and sulphatides revealed that the a-hydroxylated compounds as well as very long chain fatty acids (over C18, especially C23 to C26) were greatly reduced. For chain lengths over C18, the ratio of leukodystrophy fatty acid to normal fatty acid was close to 10%. The defect in very long chain fatty acids is estimated at 99.2% in total brain.Thus, we have found a marked decrease in the amount of very long chain fatty acids and a less marked decrease in sphingolipids. The reduced amount of these acids appears to be partially offset by an increase in the amount of medium-chain fatty acids in sphingolipids. We conclude that one aspect of Pelizaeus-Merzbacher disease may be a defect in the synthesis of myelin very long chain fatty acids (as these acids are far much reduced than any other myelin molecule).

Notes: Intrauterine growth retardation (IUGR) induced by ligation of one uterine artery on day 17 of pregnancy in the rat lead to major abnormalities in the fatty acid content of neurons and oligodendrocytes but not in astrocytes. In neurons from IUGR rats, monounsaturated fatty acids were decreased; in the polyunsaturated series, ω-3 fatty acids were increased and Ω-6 fatty acids were decreased. In oligodendrocytes, monounsaturated fatty acids were also decreased, but the modifications in polyunsaturated fatty acids were the opposite of those in neurons: Ω-3 being decreased and w-6 increased. Although the animals received a normal diet after birth, the alterations were still present in adulthood. In addition, fatty acid composition of brain cells is a very indicative criterion of brain maturation.

Notes: The brains of Quaking and littermate control mice were fractionated by differential and density gradient centrifugation into soluble, microsomal, myelin and related (SN 4) fractions. There were no apparent differences in protein composition between any Quaking and control fraction with the exception of myelin and SN 4. Analysis of CNP activity indicated that in Quaking animals a high proportion of the total activity was localized in microsomal fractions, while in controls a large percentage of activity was found in myelin and SN4; in contrast, there were no marhcd differences in the distribution of AChE activity between Quaking and control fractions.The yield of myelin isolated from Quaking animals was 3.6%, of that from controls by electron microscopy myelin fractions from both Quaking and controls consisted of compact myelin whorls. Zonal centrifugation on continuous sucrose gradients demonstrated that both control and Quaking myelin was distributed in a bell-shaped mode with peak densities at 0.66 0.68 and 0.71-0.75 M-sucrose, respectively. The specific activity of CNP was generally lower in mutant subfractions than in controls. Protein analysis revealed that there were similar qualitative trends between light and heay myelin subfractions from both mutant and control animals, although the levels of proteolipid and small basic proteins were substantially lower in all Quaking fractions. These results indicate that. although all mutant myelin subfractions are compositionally abnormal, the type of particle heterogeneity in Quaking myelin is similar to that observed in controls.

Notes: Abstract— In the ‘Quaking’ mouse, a deficiency in the long chain fatty acid content of galactolipids has been shown to occur.Myelin in the mutant has been compared to myelin in adult and in 12-day-old controls. We have shown that myelin is not only quantitatively reduced but also qualitatively modified, with a higher protein and a lower galactolipid content. Cerebrosides contain only a small amount of kerasin, lacking long chain nonhydroxylated fatty acids in comparison to both controls; the relative percentage of phrenosin is increased.Although many similarities exist between adult Quaking myelin and myelin at 12 days, differences have been shown to occur which may be in relation to a genetic block at an earlier stage of development.

Notes: Abstract: Total polyribosomes were isolated from the brains of 16–20-day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shi/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 ± 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and 〈 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an “assembly” or “synthesis” defect.