Characterization of phospholipid methylation in rat brain myelin.
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ABSTRACT:
SUBMITTER: Tsvetnitsky V
PROVIDER: S-EPMC1136768 | biostudies-other | 1995 Apr
REPOSITORIES: biostudies-other
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Project description:Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.
| S-EPMC1138732 | biostudies-other
Project description:The synthesis and methylation in vivo of myelin basic protein (MBP) during the mouse brain development has been investigated. When mice ranging in age from 13 to 60 days were injected intracerebrally with L-[methyl-3H]methionine, the incorporation of radioactivity into MBP isolated from youngest brain was found to be the highest and declined progressively in mature brains. This pattern of radioactivity incorporation was inversely correlated with the total amount of MBP in the brains, suggesting a higher ratio of MBP methylation to synthesis in younger brain. To differentiate the relative rate of protein synthesis and methylation, animals were given intracerebral injections of a L-[methyl-3H]methionine and L-[35S]methionine mixture and the ratio of 3H/35S (methylation index) was determined. The ratios in the isolated MBP fractions were higher than those of 'acid extracts' and 'breakthrough' fractions, with a maximal ratio in the youngest brain. This high ratio was well correlated with the higher protein methylase I (PMI) activity in younger brains. The MBP fractions were further separated on SDS/polyacrylamide-gel electrophoresis into several species with apparent Mr ranging from 32,400 to 14,500. The results indicated that each protein species accumulated at a characteristic rate as a function of age. The high-Mr (32,400) species was predominant in younger brain, whereas the smaller MBP was the major species in older brain tissue. The importance of this developmental pattern of MBP synthesis and methylation is discussed in relation to PMI activity.
| S-EPMC1147440 | biostudies-other
Project description:We examined the formation in vivo of molecular subspecies of brain phosphatidylcholine (PC) via the phospholipid-methylation pathway. [3H]Methionine was infused into a lateral cerebral ventricle, and 3H-labelled PC was isolated from brains of rats 0.1-18 h after the infusions. Three major subspecies of this PC, differing in their fatty acid compositions, were separated on silver-impregnated t.l.c. plates, and the proportions of radioactivities in these three PC fractions were determined. The results indicate that newly-formed PC synthesized by methylation of phosphatidylethanolamine at 0.1 h after [3H]methionine contains a significantly higher proportion of polyunsaturated subspecies (i.e. those with six or four double bonds) than does PC obtained later times after injection of [3H]methionine. This change in the composition of 3H-labelled brain PC occurs gradually and is not due to an influx of radioactive PC from the periphery. Our data suggest that polyunsaturated PC (hexaenes and tetraenes) produced in the brain by methylation of phosphatidylethanolamine turns over faster than does that containing more-saturated fatty acids.
| S-EPMC1147994 | biostudies-other
Project description:Myelin fractions with different degrees of compaction were isolated from bovine brain, and post-translational methylation of membrane-associated proteins was studied. When the purified myelin-basic-protein-specific protein methylase I and S-adenosyl-L-[methyl-14C]methionine were added exogenously, the most compact myelin fraction exhibited higher methyl-accepting activity than the less compact dense fractions. The methylated protein was identified as myelin basic protein (18.4 kDa) exclusively among the several myelin proteins from all membrane fractions, by SDS/PAGE/radioautography of methyl-14C-labelled membrane proteins. The methyl-14C-labelled amino acid residue in the basic protein was identified by h.p.l.c. as NG-methylarginine, indicating the high degree of specificity for the arginine residue as well as the myelin basic protein in the intact myelin membranes. The possibility of a charge alteration of myelin basic protein resulting from its arginine methylation was investigated by using the purified component 1 of myelin basic protein. The methylated component was shown to be less cationic than the unmethylated component by Bio-Rex 70 cation-exchange chromatography, since the former preceded the latter. However, in the presence of the denaturant (guanidinium chloride), the two species were co-eluted, indicating that the charge difference between methylated and unmethylated myelin basic protein can only be shown under the renatured condition.
| S-EPMC1150064 | biostudies-other
Project description:In spite of intense interest in how altered epigenetic processes including DNA methylation may contribute to psychiatric and neurodevelopmental disorders, there is a limited understanding of how methylation processes change during early postnatal brain development. The present study used in situ hybridization to assess mRNA expression for the three major DNA methyltranserases (DNMTs)--DNMT1, DNMT3a and DNMT3b--in the developing rat brain at seven developmental timepoints: postnatal days (P) 1, 4, 7, 10, 14, 21, and 75. We also assessed 5-methylcytosine levels (an indicator of global DNA methylation) in selected brain regions during the first three postnatal weeks. DNMT1, DNMT3a and DNMT3b mRNAs are widely expressed throughout the adult and postnatal developing rat brain. Overall, DNMT mRNA levels reached their highest point in the first week of life and gradually decreased over the first three postnatal weeks within the hippocampus, amygdala, striatum, cingulate and lateral septum. Global DNA methylation levels did not follow this developmental pattern; methylation levels gradually increased over the first three postnatal weeks in the hippocampus, and remained stable in the developing amygdala and prefrontal cortex. Our results contribute to a growing understanding of how DNA methylation markers unfold in the developing brain, and highlight how these developmental processes may differ within distinct brain regions.
| S-EPMC3838910 | biostudies-literature
Project description:Protein- and lipid-bound sialic acid was assayed in myelin fractions isolated by four different methods from rat brain homogenates. The extent to which non-myelin cellular membranes contaminate these fractions was assessed by electron microscopy and marker-enzyme assays. Small amounts of sialic acid found in the least contaminated myelin fractions are considered to be constituents of axonal and satellite cell plasma membranes known to be present. The data are discussed with reference to the ultrastructural appearance of myelin.
| S-EPMC1165554 | biostudies-other
Project description:PurposeBrain myelin plays an important role in normal brain function. Demyelination is involved in many degenerative brain diseases, thus quantitative imaging of myelin has been under active investigation. In previous work, we demonstrated the capability of the method known as Relaxation Along a Fictitious Field (RAFF) in the rotating frame of rank n (RAFFn) to provide image contrast between white and gray matter in human and rat brains. Here, we provide evidence pointing to myelin being the major source of this contrast.MethodsRAFFn relaxation time constant (TRAFFn) was mapped in rat brain ex vivo. TRAFFn was quantified in 12 different brain areas. TRAFFn values were compared with multiple other MRI metrics (T1, T2 , continuous wave T1ρ, adiabatic T1ρ and T2ρ, magnetization transfer ratio), and with histologic measurements of cell density, myelin and iron content.ResultsHighest contrast between white and grey matter was obtained with TRAFFn in the rotating frames of ranks n = 4 and 5. TRAFFn values correlated strongly with myelin content, whereas no associations between TRAFFn and iron content or cell density were found.ConclusionTRAFFn with n = 4 or 5 provides a high sensitivity for selective myelin mapping in the rat brain.
| S-EPMC4523459 | biostudies-literature
Project description:BackgroundLipid profiles in the blood are altered in human cocaine users, suggesting that cocaine exposure can induce lipid remodeling.MethodsLipid changes in the brain tissues of rats sensitized to cocaine were determined through shotgun lipidomics using electrospray ionization-mass spectrometry (ESI-MS). We also performed pairwise principal component analysis (PCA) to assess cocaine-induced changes in blood lipid profiles. Alterations in the abundance of phospholipid species were correlated with behavioral changes in the magnitude of either the initial response to the drug or locomotor sensitization.ResultsBehavioral sensitization altered the relative abundance of several phospholipid species in the hippocampus and cerebellum, measured one week following the final exposure to cocaine. In contrast, relatively few effects on phospholipids in either the dorsal or the ventral striatum were observed. PCA analysis demonstrated that cocaine altered the relative abundance of several glycerophospholipid species as compared to saline-injected controls in blood. Subsequent MS/MS analysis identified some of these lipids as phosphatidylethanolamines, phosphatidylserines and phosphatidylcholines. The relative abundance of some of these phospholipid species were well-correlated (R(2) of 0.7 or higher) with either the initial response to cocaine or locomotor sensitization.ConclusionTaken together, these data demonstrate that a cocaine-induced sensitization assay results in the remodeling of specific phospholipids in rat brain tissue in a region-specific manner and also alters the intensities of certain types of phospholipid species in rat blood. These results further suggest that such changes may serve as biomarkers to assess the neuroadaptations occurring following repeated exposure to cocaine.
| S-EPMC4458179 | biostudies-literature
Project description:Recent efforts in biomaterial-assisted brain tissue engineering suggest that match of mechanical properties of biomaterials to those of native brain tissue may be crucial for brain regeneration. In particular, the mechanical properties of native brain tissue vary as a function of age. To date, detailed characterization of age-dependent viscoelastic properties of brain tissue throughout the postnatal development to adulthood is only available at sparse age points in animal studies. To fill this gap, we have characterized the linear viscoelastic properties of the cerebral cortex in rats at well-spaced ages from postnatal day 4 to 4 months old, the age range that is widely used in neural regeneration studies. Using an oscillatory rheometer, the viscoelastic properties of rat cortical slices were measured independently by storage moduli (G') and loss moduli (G″). The data demonstrated increases in both the storage moduli and the loss moduli of cortex tissue over post-natal age in rats. At all ages, the damping factor (G″/G' ratio) remained constant at low oscillatory strain frequencies (<10 rad/s) before it started to decline at medium frequency range (10-100 rad/s). Such changes were not age-dependent. The stress-relaxation response increased over post-natal age, consistent with the increasing tissue stiffness. Taken together, our study demonstrates that age is a crucial factor determining the mechanical properties of the cerebral cortex in rats during early postnatal development. This data may provide the guidelines for age-specific biomechanics study of brain tissue and help to define the mechanical properties of biomaterials for biomaterial-assisted brain tissue regeneration studies.Statement of significanceStudies about age-dependent viscoelastic properties of rat brain tissue throughout the postnatal development to adulthood is sparsely available. To fill up the gap of knowledge, in this study, we have characterized the age-dependent viscoelastic properties and the linear viscoelastic properties of the cerebral cortex throughout the postnatal development stage to adulthood in rats by measuring storage moduli (G'), loss moduli (G″), damping factor (G″/G' ratio) and stress-relaxation response. We have found that age is a crucial factor determining the mechanical properties of the cerebral cortex in rats during early postnatal development. The findings of this study could provide guidelines for age-specific biomechanical study of brain tissue and help to define the mechanical properties of biomaterials for biomaterial-assisted brain tissue regeneration in experimental models in rats.
| S-EPMC9757762 | biostudies-literature
Project description:The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.
| S-EPMC1161735 | biostudies-other