POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

Differential distribution of immunoreactive angiotensinogen in the hind-brain and spinal cord of neonatal and adult rats

The present communication deals with the cytochemical localization of angiotensinogen (ATG) immunoactivity in the hind-brain and spinal cord of neonatal (1-day-old) and adult (3-month-old pregnant) female rats. In the neonatal hind-brain, the immunoreactive cells were more numerous than in that of adult rats. In the adult rat hind-brain, the number of ATG-positive cells was quite limited in each nucleus. Further, in some nuclei, only neurons or neuroglia were positive, while in others the immunoactivity was observed in both the components. Spinal cords of neonatal rats showed a few undifferentiated ATG-positive cells in the grey matter. Contrary to this, the spinal cord of adult animals contained numerous immunoreactive glial cells in the grey matter, fasciculus cuneatus and fasciculus gracilis. Immunoactivity in the neurons was localized in the Nissl bodies.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

STUDIES ON THE MECHANISM OF DEMYELINATION: TRIETHYL TIN-INDUCED DEMYELINATION

The metabolism of myelin undergoing breakdown as a result of edema induced by chronic administration of triethyl tin (TET) dissolved in the drinking water (10 mg/l.) was examined. The spinal cord showed more edema and loss of myelin than the brain. Uptake in vitro of [1-¹⁴C]acetate into myelin lipids of slices of brain or spinal cord from TET-treated rats was depressed until 4–5 weeks after the beginning of the regime, then rose to above normal levels. The uptake of [l-¹⁴C]leucine into myelin protein rose within several weeks of TET treatment to levels averaging over 300 per cent of normal and remained high even after the TET was removed. The high levels of [l-¹⁴C]leucine incorporation were inhibited by cycloheximide and were not explained by an increase in the size of the free amino acid pool. The three classes of myelin proteins, basic, proteolipid protein, and Wolfgram protein shared in the increased incorporation. Spinal cord myelin showed the greatest metabolic response, brain stem myelin less, and myelin from the forebrain was minimally affected by the TET treatment. Myelin prelabelled by intracisternal injection of [l-¹⁴C]acetate and [l-¹⁴C]leucine before the onset of TET administration showed faster turnover in myelin proteins in relation to the myelin lipids than the control in the most severely affected animals, but not in others less affected. A ‘floating fraction’ was observed floating on 10.5% (w/v) sucrose during the myelin purification. This fraction showed metabolic characteristics typical of myelin, and myelin-labelling studies at various stages of the animal's development showed it to be derived from recently synthesized myelin. The floating fraction from the brain contained less cerebroside and more lecithin than myelin, while the spinal cord floating fraction composition was much like that of myelin. The floating fractions contained less protein typical of myelin (basic and proteolipid protein) and more highmolecular-weight protein which may have been derived from contaminating microsomes. The floating fraction was presumed to be partially deproteinated myelin. The use of TET-treatment as model for demyelination as a result of edema and proceeding in the absence of macrophages is discussed.     

Implantation of embryonic neocortex and spinal cord into injured peripheral nerve of adult rats

Spinal cord and cerebral cortex of 14-day-old embryos of Wistar rats were implanted into the sciatic nerve of mature rats in order to study dynamics of the development of neuronal and neuroglial elements in ectopic sites. By means of light and electron microscopy it has been stated that the implanted nerve cells of the cortex and spinal cord survive during 5 month and differentiate from neuroepithelial cells and neuroblasts up to young and mature neurons. It was found that thirty days after operation the spinal cord implants contained myelinated nerve fibers and numerous synapses. The data obtained suggest that the implants of fetal spinal cord are more favorable for regeneration of the injured nervous stems than the cerebral cortex.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

CAPACITIES FOR BINDING AMINO ACIDS BY tRNAs FROM RAT BRAIN AND THEIR CHANGES DURING DEVELOPMENT

–The total tRNA and some specific tRNAs from the 100,000g soluble fraction of rat brain were measured during development (postnatal ages 4–55 days). For determination of specific tRNAs we developed a method that measured their capacities to bind specific amino acids. Levels of total tRNA were decreased in the soluble fraction from the brains of 55-day-old rats in comparison to those for the 4-day-old rats. The aminoacylation capacities of tRNAs for phenylalanine, lysine, proline, valine, leucine, alanine and isoleucine were diminished in the 55-day-old rats in comparison to those for 4-day-old rats when expressed per unit wet weight of brain. When the 4- to 55-day changes in aminoacylation capacity of each specific tRNA was expressed relative to that of the total tRNA, tRNA^Phe and tRNALys^Lys were diminished; tRNA^Pro, tRNA^Vel, tRNA^GIY and tRNA^Leu showed no significant changes; and tRNA^A1a and tRNA^Ile were increased. Incorporation of amino acids into a material insoluble in hot TCA (probably proteins) in a ribosome-free system occurred in the brain preparations. Out of ten different amino acids studied, arginine and tyrosine exhibited the highest values for this type of transfer.     

DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

Studies on the protein-synthesizing activity of the ribosomes of rat liver. The activity of free polysomes

1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.     

The effect of 4-dimethylamino-3′-methylazobenzene on 14C-labelled amino acid incorporation by rat-liver polysome preparations

1. The incorporation of (14)C-labelled amino acids into polysomal protein was studied in a system consisting of polysomes and pH5 enzyme obtained 4 and 40hr. after a single intraperitoneal injection of 4-dimethylamino-3'-methylazobenzene. Labelling of the polysome fraction of preparations of both the 4hr.-treated and 40hr.-treated rats was considerably higher than in the normal control. 2. In further experiments on protein synthesis by polysomes from azo-dye-treated rats, the effect of replacing pH5 enzyme with cell sap was studied. Incorporation of (14)C-labelled amino acids into polysomal protein was similar to that of the control. 3. Aggregate size of polysomes obtained from rats injected previously with 4-dimethylamino-3'-methylazobenzene was studied by sucrose-gradient centrifugation. Polysomes prepared at 4hr. after azo-dye administration contained a considerable amount of large aggregates (approx. 700s), whereas at 40hr. after administration of the azo-dye the amount of large aggregates was less than in the control. 4. Determination of the ultraviolet spectra of polysome preparations from both normal and azo-dye-treated rats revealed no difference between the preparations. On the other hand, the ultraviolet spectra of cell-sap fractions from the different preparations showed that there is a definite shift in the absorption maximum from 272mmu (normal) to 267mmu, 40hr. after treatment, with an intermediate value of 270mmu for the preparation from 4hr.-treated rats. The absorption minimum changes from 250mmu (normal) to 245mmu for the preparation from 40hr.-treated rats.     

Protein synthesis and aging: studies with cell-free mammalian systems.

A cell-free system devoid of polysomes, which translates natural mRNA, has been prepared from rat liver. It contains ribosomal subunits, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and protein factors necessary for translation. Protein synthesis required an energy-generating system, mRNA, and 3 mM Mg2+ concentration, and it was inhibited by 7-methylguanylic acid. The total extent and the rate of protein synthesis were approximately 30% greater when the translating system was prepared from livers of 3-month-old rats, as compared to 30-month-old rats. A ribosome-free fraction containing the protein factors required for translation was also prepared from 3-month-old and 30-month-old rat livers and brains, by extraction with 0.5 M KCl. The high-salt extracts were analyzed for elongation factors EF-1 and EF-2 in a poly(U) translating system. Although the activity of EF-2 was similar in preparations from young and old rats, the EF-1 activity in the 3-month-old rat livers and brains was 30 to 40% greater than in 30-month-old animals. The protein synthesizing activity of high salt-washed ribosomes stripped of endogenous peptidyl-tRNA and mRNA, from livers and brains of young and old animals, was the same.     

Age-related changes of the GABA-B receptor in the lumbar spinal cord of male rats and penile erection

Dorsal horn neurons of lumbosacral spinal cord innervate penile vasculature and regulate penile erection. GABAergic system is involved in the regulation of male sexual behavior. Because aging is frequently accompanied by a progressive decline in erectile function, the aim of this work was to examine age-related changes of the GABA-B receptor in the lumbar spinal cord. Sprague-Dawley rats of 10 and 21 days old, 3, 9 and 20 months old were used. GABA-B receptors were evaluated by quantitative autoradiography using [3H]-Baclofen as ligand with or without GABA (10 microM) to determine the non-specific binding. Ten days after birth a homogeneous neuroanatomical distribution pattern was found in the gray matter, however at 20-day-old adult distribution emerged becoming heterogeneous with the highest binding values at layers II-III and X. In dorsal layers a significant decrease was observed in 9-month-old rats while layer X showed an earlier decrease (21-day-old). GABA-B receptor affinity showed significant age-dependent and regional increase. The GABA-B receptor decrease in aged rats seems not to be related to this receptor inhibitory function in penile erection. Moreover the changes found in GABA-B receptor binding anatomical distribution may indicate its role in the morphological development of the lumbar spinal cord rather than in the decline of the erectile function.     

Morphine tolerance in arthritic rats and serotonergic system

To understand whether chronic inflammation alters the development of morphine tolerance, the tail-flick test was used to evaluate the analgesic effect of morphine (75 mg tablet, s.c.) in the arthritic rats at the day 9-12 after the inoculation with Freund's adjuvant. Spinal cord monoamines and amino acid neurotransmitters were concomitantly measured. Chronic inflammation attenuated the antinociceptive effect of morphine as tolerance developed faster in the arthritic rats compared to the vehicle-treated controls. In addition, ratio of 5-hydroxyindole-3-acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) increased in the lumbar spinal cord of arthritic rats without any change in the concentrations of norepinephrine, glutamate, aspartate or GABA. Interestingly, increased serotonin turnover in the spinal cord was observed in both control and arthritic rats 24 hours after morphine treatment. Overall, the results suggest a significant role of serotonin up-regulation in the spinal cord during chronic pain and the development of morphine tolerance.     

Comparison of lectin-binding patterns between young adult and older rat glycoproteins in the brain

Glycoproteins in the soluble fraction and in the membrane fraction of various portions of brains and spinal cords, obtained from 9-week-old rats and 29-month-old rats, were comparatively analyzed by SDS-polyacrylamide gel electrophoresis and lectin staining. The glycoprotein patterns of each brain part showed marked differences by the age of donors. The most prominent evidence in the soluble fractions of white matter, basal ganglia, and spinal cord detected by WGA is that the glycoproteins with an apparent molecular weight of 123K and 115K have increased in the aged rats. In addition, the reactivity of 115K with Con A and PNA has also increased in the aged rats. On the other hand, reactivity of an apparent molecular weight of 115K with WGA has increased in the membrane fractions of white matter, basal ganglia, hippocampus, cerebellum, and spinal cord from the aged rats. In contrast, by MAM, which is specific for Sia2®3Gal linkage, an apparent molecular weight of 115K has been detected only in the membrane fraction of cerebellum and it has decreased in the aged rats. Reactivity of an apparent molecular weight of 133K and 125K in the membrane fractions of white matter and basal ganglia with LCA has decreased in the aged rats. In contrast, reactivity of the front band with LCA and AAL has increased and that of 130K with AAL has decreased in spinal cord from the aged rats, respectively. These results indicate that the glycosylation state of the protein in the brain changes during aging.     

Analysis and Comparison of In Vitro Synthesized Glial Fibrillary Acidic Protein with Rat CNS Intermediate Filament Proteins

Intermediate filament (IF) proteins from rat spinal cord were analyzed by two-dimensional gel electrophoresis and compared with the in vitro translation products of a messenger RNA-dependent reticulocyte lysate system stimulated with 16-day-old rat brain polysomes. In two dimensions, the molecular weight 49,000 to 50,000 band of the IF preparation resolved to seven spots, whereas antiserum to glial fibrillary acidic (GFA) protein precipitated only two immediately adjacent radiolabeled in vitro synthesized products, with molecular weights of 49,000 to 50,000. Autoradiographs of two-dimensional gels of extracted IF proteins incubated with iodinated IgG fraction of GFA protein antiserum showed that all seven spots were recognized by the antiserum. These observations suggest that the primary gene product of GFA protein is modified either by post-translational processing or experimental artifact.     

The spinal cord supports of vertebrae in the crown‐group salamanders (Caudata,Urodela)

The development of spinal cord supports (bony thickenings which extend into the vertebral canal of vertebrae) in primitive (Salamandrella keyserlingii) and derived (Lissotriton vulgaris) salamanders were described. The spinal cord supports develop as the protuberances of periostal bone of the neural arches in the anteroproximal part of the septal collagenous fibers which connect a transverse myoseptum with the notochord and spinal cord, in the septal bundle inside the vertebral canal. Spinal cord supports were also found in some teleostean (Salmo salar, Oncorhynchus mykiss) and dipnoan (Protopterus sp.) fishes. The absence of the spinal cord supports in vertebrates with cartilaginous vertebrae (lampreys, chondrichthyan, and chondrostean fishes) corresponds to the fact that the spinal cord supports are bone structures. The absence of the spinal cord supports in frogs correlates with the lack of the well developed septal bundles inside the vertebral canal. The spinal cord supports are, presumably, a synapomorphic character for salamanders which originated independently of those observed in teleostean and dipnoan fishes. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Evidence for the presence of major peripheral myelin glycoprotein P0 in mammalian spinal cord and a change of its glycosylation state during aging.

Glycoproteins, which react with Lens culinaris agglutinin, in the membrane preparation of various portions of brains and spinal cords, obtained from 9-week-old rats and 29-month-old rats, were comparatively analyzed by SDS-polyacrylamide gel electrophoresis. In contrast to the samples from brain, which showed similar staining patterns in the two different age groups, the glycoprotein patterns of spinal cords showed marked differences by the age of donors. The most prominent evidence is that a glycoprotein with an apparent molecular weight of 30 kDa (gp30) was detected in the aged rats, but not in the young adult rats. Based on the amino acid sequence data around the glycosylation site, the gp30 was identified as P0, which is a member of immunoglobulin superfamily and a major structural component of mammalian peripheral nerve myelin. This is the first report indicating that P0, which has been considered as a peripheral nerve-specific glycoprotein, occurs also in the spinal cord of mammals. In addition, nonglycosylated P0 molecule could be detected in the spinal cord of young adult rats by anti-P0 polyclonal antibody. These results indicate that the glycosylation state of the P0 molecule in the spinal cord changes during aging.     

STUDIES ON THE MECHANISM OF DEMYELINATION: MYELIN AUTOLYSIS IN NORMAL AND EDEMATOUS CNS TISSUE

The effects on myelin of autolysis in situ after death and after purification were studied in normal brains and spinal cords and in those made edematous as a result of chronic triethyl tin (TET) feeding. Myelin prepared from normal and edematous brains and spinal cords autolyzed for 12 h at 4°C contained only slightly less basic protein than that prepared from freshly killed animals. The amounts of a light lipid-protein fraction (dissociated myelin) usually obtained during purification of myelin from edematous CNS were about the same in tissue from freshly killed rats and those autolyzed for 12 h at 4°C. Autolysis for 12 h at room temperature resulted in formation of large amounts of dissociated myelin and loss of basic protein, but more dissociation and basic protein loss occurred in CNS from edematous brains and spinal cords than from the normal. Purified myelin prepared from freshly-killed normal and TET-fed rats was incubated at 37°C in media of several ionic strengths. In Krebs-Ringer bicarbonate (physiological extracellular fluid) extensive dissociation of myelin occurred with much less in 0.04 M-Tris buffer, pH 7.2, and only small amounts were formed in 0.01 M-Tris. In all cases myelin from edematous CNS formed more dissociated fraction than did the normal myelin. Basic protein loss was also proportional to the ionic strength of the media, but there was no difference in loss between normal and TET-myelin. Two different factors, proteolysis and physical extraction of basic protein by salt solutions, may be contributing to myelin dissociation and loss of basic protein.