Isolation Of Myelin Research Articles

Isolation, Purification and Verification of Peripheral Nerve Myelin Derived from Bovine Cauda Equina

Background: Animal models of peripheral neuroinflammation are essential to understanding the pathogenesis of, and deducing new treatments for demyelinating polyneuropathies. This paper updates and describes adapted methods for the isolation, purification and verification of bovine peripheral nerve myelin used to reliably induce peripheral neuritis in susceptible mice. Methods: Myelin was isolated via discontinuous sucrose and continuous cesium chloride density gradient ultracentrifugation from frozen adult bovine cauda equina. Negatively stained electron microscopy was employed to confirm myelin structure. Phase contrast and routine light microscopy, gravimetric analyses, gel electrophoresis and lens culinaris fluorescent western blotting, analytical colorimetric assays and indirect immunohistochemistry were performed to analyze whole myelin or its protein and lipid subcomponents. Myelin isolates were used to induce experimental autoimmune neuritis in 8-12 Weeks old female Swedish Jim Lambert/ Jackson mice. Results: Negatively-stained electron microscopy demonstrated the two-dimensional repetitive lamella structure of myelin and a lack of organelle contamination. Lipids accounted for ~72% of the dry mass of bovine peripheral nerve myelin, while ~25% was protein/ proteolipid and <4% insoluble residue based on gravimetric analyses. Glycoproteins accounted for the majority of isolated myelin proteins, with myelin protein zero (~28 KDa) being the most prevalent protein. Phospholipids accounted for ~60% of the total dry weight of bovine peripheral nerve myelin. Surface expression of galactocerebroside and sulfatide, two major lipid subcomponents present in myelin, was detected by indirect fluorescent immunohistochemistry. Bovine peripheral nerve myelin reliably induced a severe macrophagepredominant demyelinating polyneuropathy with associated axonal loss in female mice, consistent with experimental autoimmune neuritis. Conclusions: These updated and adapted methods using readily available resources can be applied to rapidly isolate, purify and verify peripheral nerve myelin from animal sources, and determine its protein and lipid composition prior to use in peripheral neuritis animal models.

Isolation of Myelin

The methods used to prepare myelin involve homogenization of the tissue in isotonic sucrose solution, followed by the isolation of myelin membranes by a series of steps that include density gradient centrifugation and differential centrifugation. Homogenization of nervous tissue in isotonic sucrose causes the myelin sheath to peel from the axon and form relatively large myelin vesicles. The large size of the myelin vesicles, together with the fact that myelin membrane has a lower density than other biological membranes, make differential centrifugation and density gradient centrifugation the main tools for the isolation of this membrane. Three protocols are outlined in this unit: isolation of a highly-purified myelin fraction from the central nervous system (CNS); separation of a highly-purified CNS myelin fraction into subfractions of different densities; and isolation of myelin from the peripheral nervous system (PNS).

A Unique Role for Fyn in CNS Myelination

We analyzed the role of Fyn tyrosine kinase in CNS myelination by using fyn(-/-) null mutant mice, which express no Fyn protein. We found a severe myelin deficit in forebrain at all ages from 14 d to 1 year. The deficit was maximal at 1 month of age and was similar regardless of mouse strain background or whether it was determined by bulk isolation of myelin or by quantitation of myelin basic protein. To determine the cellular basis of the myelin deficit, we counted oligodendrocytes in tissue sections of mice expressing oligodendrocyte-targeted beta-galactosidase, and we used light and electron microscopy to examine the number and morphology of myelinated fibers and size of myelinated CNS structures. All of these parameters were reduced in fyn(-/-) mice. Unexpectedly, there were regional differences in the myelin deficit; in contrast to forebrain, fyn(-/-) cervical spinal cord exhibited no reduction in myelin content, number of oligodendrocytes, or number of myelinated fibers, nor was myelination delayed developmentally. We found that oligodendrocytes express Src, but there was no significant reduction of myelin content in null mutants lacking the Fyn-related kinases Src, Yes, or Lyn. Finally, we investigated the molecular features of Fyn that are required for myelination and found that a single amino acid substitution, which abolishes the tyrosine kinase activity of Fyn, resulted in a myelin deficit as great as that observed in the complete absence of Fyn protein. These results demonstrate that Fyn plays a unique role in myelination, one that requires its kinase activity.

Amyloid precursor protein is enriched in axolemma and periaxolemmal-myelin and associated clathrin-coated vesicles.

The amyloid precursor protein (APP) is widely distributed within the CNS, where it is expressed in both neurons and glia. We have isolated axolemma and periaxolemmal-myelin from rat brain and have determined by Western blot that APPs, Mr 100-110 kDa, are major constituents of these membrane. Isolation of axolemma, periaxolemmal-myelin, and compact myelin show that while APP represents 1 and 0.6% of the proteins of these respective membranes, it is absent from compact myelin. These results indicate that APP transported down the axon is deposited at sites in the axolemma as well as the synapse, and that within the myelin complex, APP is targeted to the periaxolemmal domain. Both axolemma and periaxolemmal-myelin contained a 10.5 kDa APP peptide which, based on reactivity with anti-C-terminal APP antibodies but not with anti-N-terminal antibody, appears to be a membrane-associated C-terminal fragment. Western blots with antibodies to Alzheimer precursor-like proteins (APLP) indicate that APP immune reactivity is not a result of cross reactivity with APLPs. Isolation of axolemma from human autopsy material showed nearly identical results with a clear enrichment, relative to homogenate, of APP Mr 100-110 and the 10.5 kDa C-terminal peptide. The demonstration of APP in axolemma and periaxolemmal-myelin was replicated in membrane isolated from bovine brain. Bovine studies were extended to analysis of white matter clathrin-coated vesicles; these data show that coated vesicles isolated from white matter, under conditions that previous studies indicate are largely endocytic vesicles, contain levels of APP comparable to that found in axolemma and periaxolemmal-myelin. In addition, these vesicles contain cysteinyl and aspartyl proteases. Incubation of axolemma with cathepsin B at pH 6.0 caused a rapid loss in the immune reactivity of APP Mr 100-110 and Mr 10.5 when analyzed with antibodies to APP672-695. This appears to be the result of hydrolysis within the epitope and not proteolysis of APP or the C-terminal peptide, since no loss of reactivity was observed when analyzed with antibodies to sites more distal to the C-terminus. Thus, cathepsin B hydrolyses membrane bound APP close to the C-terminus and may be a useful tool for altering C-terminal APP function.

Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37 degrees C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.

Isolation and characterization of bovine brain myelin distribution of 5'-nucleotidase.

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2',3'-cyclic nucleotide 3'-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5'-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH:cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5'-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.

Myelin composition and activities of CNPase and Na+,K+-ATPase in hypomyelinated "pt" mutant rabbit.

A disorder of CNS myelination was found in paralytic tremor ("pt") rabbits. The condition is inherited in a sex-linked recessive mode. Ultrastructurally, an obvious myelin deficiency with aberration of myelin sheath formation is observed. The yield of myelin isolation was reduced to 20-30% of control. Myelin isolated from 4-week-old "pt" rabbits contained reduced amounts of galactosphingolipids and of several myelin protein markers. Moreover, myelin basic protein, analyzed by two-dimensional gel electrophoresis, showed a deficit in its more basic components. All these facts suggest a delay in myelin maturation. Ganglioside content was increased as well as Na+,K+-ATPase specific activity. 2',3'-Cyclic nucleotide phosphodiesterase (CNPase) specific activity was the same in "pt" as in control myelin but differed by having greater sensitivity to detergent activation.

Isolation of myelinoid Marchi-positive bodies from normal rabbit spinal cord

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: (1) the fraction floating on 0.32 M sucrose, (2) the myelin fraction at the 0.32 M/0.85 M interface and (3) 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.

Further evidence for an intrinsic neuraminidase in CNS myelin.

An intrinsic neuraminidase activity in rat brain CNS myelin has been demonstrated and compared with the neuraminidase activity in rat brain microsomes. With use of ganglioside GM3 as a substrate, the myelin-associated neuraminidase exhibited a shallow pH curve with an optimum at pH 4.8 whereas the microsomal activity had a marked optimum at pH 4-4.3. Neuraminidase activity in both fractions was optimized in 0.3% Triton CF-54 but activation was much greater in the microsomes. When the neuraminidase activities were examined at 60 degrees C, the myelin neuraminidase activity was more than sevenfold of that observed at 37 degrees C and was linear for at least 2 h; the microsomal activity increased only fivefold initially and exhibited a continual loss in activity. Addition of excess microsomes to the total homogenate prior to myelin isolation resulted in no change in myelin neuraminidase activity. When the two membrane fractions were examined at equivalent protein concentrations in the presence of additional cations or EDTA (1 mM), similar but not identical effects on neuraminidase activity were seen. The microsomal neuraminidase was considerably more susceptible to inhibition by divalent copper ion. Activity in both fractions was markedly inhibited by Hg2+ and Ag+ whereas EDTA had no effect on either activity. The myelin-associated neuraminidase activity was the highest in cerebral hemispheres, followed by brainstem, cerebellum, and spinal cord and was extremely low in sciatic nerve. In fact, the myelin neuraminidase activity was higher than the microsomal enzyme activity in the cerebral hemispheres.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelin in the developing human cerebrum

In order to ascertain the true composition of human myelin at the earliest stages of development and to shed some light on the controversial results published to date, the lipid and protein composition of myelin from unfrozen cerebrum of very young infants was compared to the values obtained later in development and to those obtained by other investigators using frozen human brain. The effect of 3 different procedures of myelin isolation, as well as varying periods of storing the brain in frozen state, were tested on the final composition of myelin. All myelin preparations were of a very high degree of purity, and the lipid composition changed very little either with age or by varying the method or the time of storage of the brain. Protein composition, on the other hand, changed drastically after freezing the brain for increasing periods, and the percentage of myelin basic protein (MBP) decreased to one-third of its original value after about a year of brain storage. However, when myelin was isolated from unfrozen brain immediately after autopsy, very high values of MBP were obtained, even in newborn cerebral myelin, which was quite mature from a morphological as well as from a biochemical point of view. These data reveal that the developmental changes of human myelin are much more subtle than has previously been thought.

Early adrenalectomy increases myelin content of the rat brain

Rats were adrenalectomized (ADX) or sham-operated (SHAM) on the 11th day of life and killed on days 35–36, 63, or 151–153 for the isolation of cerebral myelin from each animal. Despite having lower overall body weights. ADX rats had heavier cerebra than SHAM control rats at all ages. Mean cerebral weight increases were 10.0% at day 35–36, 15.3% at day 63, and 16.7% at day 151–153. Recovered myelin dry weights were even more elevated in the ADX rats, but only at day 63 (41.7% increase) and 151–153 (42.1% increase). At both of these ages, there was a clear linear relationship between cerebral wet weights and the amount of myelin recovered from the cerebra. Analysis of the day-63 myelin samples showed no group differences in total cholesterol or protein concentration or in the specific activity of the myelin marker enzyme 2′:3′-cyclic-nucleotide 3′-phosphodiesterase (CNP). However, myelin isolated from the ADX rats appeared to be deficient in both galactolipid and phospholipid. Optic nerve myelination was assessed in all animals by measuring CNP activity in homogenates prepared from this tissue. No difference between ADX and SHAM rats was observed at any age. These results indicate that early adrenalectomy stimulates myelin deposition in the rat brain as part of a more general, long-lasting enhancement of brain growth. Myelin from the brains of ADX animals may be slightly abnormal in its lipid composition. Finally, the optic nerve data may mean that myelination is not affected equally in all areas of the CNS by the loss of adrenal glands.

Lipid composition of brain myelin from normal and hyperphenylalaninemic chick embryos

The influence of hyperphenylalaninemia on the lipid composition of brain myelin has been investigated in 19-day-old chick embryos. CNP-ase activity was used as myelin marker enzyme for myelin isolation. CNP-ase activity was significantly lower in hyperphenylalaninemic myelin when compared with control. No significant differences were observed after experimental treatment in the total lipid content of myelin as well as in the proportion of cholesterol:phospholipid:galactolipid. Nevertheless, a clear increase in the percentage of esterified cholesterol was found. No appreciable alterations were observed in the phospholipid composition of brain myelin from both control and hyperphenylalaninemic chick embryos. However, the ratio of unsaturated to saturated fatty acids in serine plasmalogen and sphingomyelin was considerably increased by this treatment. This ratio in choline and ethanolamine phosphatides from treated embryos did not differ from that of controls.

Characterization of tubulin in mouse brain myelin.

Analysis of mouse brain myelin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that in the high-molecular-weight range it contained, besides the Wolfgram protein doublet, proteins comigrating with actin and with both subunits of tubulin. The occurrence of these alpha and beta subunits was confirmed by peptide mapping in myelin analyzed by two-dimensional electrophoresis. This tubulin did not arise from an artifactual binding of soluble brain tubulin to the myelin fraction: addition of exogenously labeled tubulin to brain homogenates proved that during myelin isolation by the procedure of Norton and Poduslo (1973) the contaminating tubulin was washed out. On the other hand, the distribution of tubulin isoforms in myelin was investigated by isoelectric focusing and compared with the distribution of the 21 isoforms listed for the whole brain soluble tubulin. It was shown that many isoforms were found in myelin (three isoforms for the alpha subunit and nine for the beta subunit), and that some isoforms were represented both in myelin and in soluble tubulin, but in different relative proportions.

Effects of Monensin on Assembly of Po Protein into Peripheral Nerve Myelin

Abstract: The ionophore monensin has been used in a variety of systems to block secretion of glycoproteins or assembly of glycoproteins into membranes. We examined the effects of monensin on assembly of the Po glycoprotein into PNS myelin, and compared this agent with the glycosylation inhibitor tunicamycin in our system. Sciatic nerves from 9‐day‐old rat pups were sliced and incubated in vitro. Electron microscopy of the Schwann cells in slices incubated with monensin revealed extensive swelling of the Golgi complex. Incubation with 10−7M monensin inhibited total protein synthesis by about 20% and fucose incorporation into protein about 35%. Following isolation of myelin, proteins were separated by sodium dodecyl sulfate gel electrophoresis. Monensin inhibited the appearance of Po in myelin, while causing its accumulation in a denser membrane fraction. In addition, a slightly faster‐migrating species of Po labeled with both [3H]fucose and [14C]glycine was observed in all fractions. Assembly of basic proteins into myelin was not affected. Preincubation with 10 μg/ml tunicamycin for 30 min prior to incubation with [3H]fucose and [14C]glycine for 2 h resulted in a 65% decrease in [3H]fucose incorporation into Po, and the appearance of a new [14C]glycine‐labeled peak that migrated in the region of the 23K protein reported by Smith and Sternberger. [3H]Fucose incorporation was inhibited earlier, and to a greater extent, than protein synthesis. Our results show that processing of the Po glycoprotein is sensitive to both monensin and tunicamycin, and that monensin partially blocks assembly of Po into myelin.

Study of myelin purity in relation to axonal contaminants.

Axonal remnants are considered a probable source of contamination of isolated myelin in view of the relatively tight axon-glial intercellular junction. Using the rabbit optic system to label specifically axonal components, we have found the levels of such contaminants to depend on the myelin isolation procedure, the tissue source, and the nature of the contaminant. A procedure employing repetitive treatments with EGTA was found to be highly effective in removing proline-labeled axonal proteins, the estimated upper limit of such contamination being approximately 0.6-1.2% of the myelin protein. The standard isolation procedure of Norton and Poduslo, supplemented with an additional discontinuous gradient step, proved equally effective in removing rapidly transported proteins from myelin isolated from the superior colliculus or lateral geniculate body. When the optic tract was the source, however, the EGTA procedure proved more effective in removing both rapidly and slowly transported proteins. Axonal gangliosides labeled with N-[3H] acetylmannosamine were efficiently removed by both procedures, adding support to the proposition that gangliosides detected in isolated myelin are intrinsic to that membrane.

Experimental hyperphenylalaninemia: Effect on central nervous system myelin subfractions

We examined the effects of experimental hyperphenylalaninemia on the formation of subfractions of myelin in the central nervous system of 18- to 110-day-old rats. Hyperphenylalaninemia was induced by giving rats daily subcutaneous injections of α-methylphenylalanine and phenylalanine from the 3rd to the 15th postnatal day. A mixture of [ 3H]glycine and [ 14C]glucose was injected intraventricularly into 17-, 20-, and 24-day-old control and experimental rats 18 h prior to the isolation and subfractionation of CNS myelin. Nonradioactive myelin subfractions were isolated from 43- and 110-day-old rats. Animals that had been hyperphenylalaninemic between the 3rd and 15th postnatal day exhibited a deficiency of total myelin protein throughout the study. With the exception of one age, these rats had a deficit of the classical, mature light myelin. The light and medium myelin subfractions from 18- to 43-day-old experimental rats and heavy myelin from the 25- and 43-day-old experimental rats had a deficiency of myelin basic proteins and an excess of the high molecular weight proteins. Nonetheless, previously hyperphenylalaninemic rats incorporated more [ 3H]glycine into the basic proteins of all myelin subfractions. The proportion of total brain [ 3H] glycine and [ 14C]glucose that was incorporated into combined (total) myelin subfractions was near normal in rats that were hyperphenylalaninemic between the 3rd and 15th postnatal day. However, these rats demonstrated decreased 3H and 14C incorporation into light myelin at 21 and 25 days, increased 3H and 14C incorporation into medium myelin at 25 days, and increased incorporation into heavy myelin at 21 days.

A rapid and simple microprocedure for myelin isolation.

In order to conduct enzymatic studies on myelin from discrete regions of brain or spinal cord, we required a method capable of yielding purified myelin from tissue samples weighing 50 mg or less. Microprocedures that have been reported such as that of Detering and Wells (1976) or Fagg et al. (1978) were not suitable for rapid and routine myelin preparations. In this communication, the results of experiments in which we were able to isolate myelin from as little as 5 mg of whole brain are reported. Data are presented to show that the myelin isolated by this procedure is comparable to the myelin isolated by the macro‐scale procedure of Norton and Poduslo (1973a).

Isolation and characterization of myelin-related membranes.

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M‐sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W1 and W2) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac‐tions of W1 and W2 were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) was up to 2‐fold higher than that of myelin in the heavier subfractions of W1 and W2. The major myelin‐associated glycoprotein was also increased in these subfractions as determined by periodic acid‐Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W1 and W2 subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome‐depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.

The effect of tityustoxin from scorpion venom on the release of acetylcholine from subcellular fractions of rat brain cortex

TITti~usroxtr., a potent neurotoxin purified from the venom of the scorpion Tityus serrulatus increases the release of cellular acetylcholine (GOMEZ el a1., 1971, 1973). The effect of the toxin is dependent on several factors and is blocked by tetrodotoxin and absence of Na+ or Cat+ in the incubation medium. Acetylcholine occurs in subcellular fractions of brain tissue in three forms : free form obtained front the high speed supernatant ; bound form present in the synaptic vesicles and a labile bound form, present in the cytoplasm of synaptosomes (CHAKRIN and WHTITAKER, 1969). Since tityustoxin reduces bound acetylcholine from incubated slices of brain tissue, we decided to investigate on which of the above subcellular fractions it acts . Tityustoxin was purified as previously described by a combination of extraction and chromatographic techniques using Sephadex G-25 and CM-cellulose (GontEZ and DtNtz, 1966) . The preparation of brain slices, incubation procedure and acetylcholine assay was performed as described (GOMEZ et a1., 1973, 1975) . Acetylcholine was extracted from each fraction according to the procedure of McLErttvAx and ELhIOT (1950) . The subcellular fractionation was conducted according to the procedure described by DE ROBERTIS et al., (1962) . The fraction that sedimented at 11,5008 containing myelin, nerve endings, mitochondria and lysosomes was termed the crude mitochondrral fraction . The microsomal fraction sedimented at 100,0008 for 30 min aad the supernatant was the high speed supernatant . Synaptic vesicle membranes (M=fraction) were prepared according to the method of DE RoBEttns er al., (1963) . The procedure allowed the isolation of myelin, mitochondria, nerve ending ghosts (Ml), synaptic vesicles and membranes (M~ and soluble material (M,) . Radioactivity experiments : brain slices were incubated in the medium described with dusopropylphosphofluoridate in the presence of acetylcholine (methyl-taC) chloride (AmershamJSearle ; 54 mCi/mM). After 60 min, the slices were rinsed with gold incubation medium, digested with Nuclear Chicago Solubiliur or again incubated in the absence (control) or presence of tityustoxin. After 15 min of incubation, they wen homogeniud and the subcellular fractions Ml, M=and M, prepared according to the procedure previously described .

Biochemical and pathological studies of myelin in hexachlorophene intoxication

Adult female rats were fed a diet containing 500 ppm hexachlorophene (HCP). Morphological study of brains from these animals showed vacuolation of the myelin sheaths due to separation of myelin lamellae at the minor dense line. However, myelin could be isolated from the brains of these animals in normal yield. The myelin isolated from HCP-fed animals had normal lipid and protein compositions as shown by analyses of the individual lipids and by disc gel electrophoresis of the proteins. Assay of the myelin-specific enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase, showed normal specific activity in myelin obtained from HCP-fed rats. Brains of HCP-fed rats showed an increase in wet weight and a decrease in dry weight, with the chloroform-methanol insoluble fraction showing the greatest weight loss. During isolation of myelin from HCP-fed rats material was found floating over 0.32 M sucrose. This "floating fraction" contained a higher ratio of lipid to protein but the same relative proportions of the individual lipids as are found in myelin. The yield of "floating fraction" from each HCP-fed rat was less than 10 percent of the yield of myelin. Disc gel electrophoresis demonstrated the presence of the usual myelin proteins in this fraction, but with a slight increase in the relative amount of the low molecular weight basic protein. The data were compared to reports on the biochemistry of triethyltin poisoning, and it was concluded that vacuolation of myelin in HCP poisoning is probably due to increased permeability of myelin lamellae to water and electrolytes.