Harish C. Agrawal

PARTIAL CHARACTERIZATION OF A NEW MYELIN PROTEIN COMPONENT

A new protein component has been demonstrated in myelin isolated from rat whole brain and from white matter dissected from bovine, dog and rabbit brain. It is also present in rabbit optic nerve. It does not appear to be present in other subcellular fractions of rat brain. It has a molecular weight of 20,540 ± 490(S.D.), as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. A rapid procedure for the isolation of myelin is also described.

POSTNATAL CHANGES IN FREE AMINO ACID POOL OF RAT BRAIN

THE broad objective of the present investigation was to study the distribution and changes in easily extractable (free) amino acids and other ninhydrin-positive substances in the rat brain during development. These data are needed to provide basic information on the metabolic factors operative during structural and functional maturation of the brain and to lead to a better understanding of the correlation between the changes in amino acid metabolism and specific structural features a t various developmental stages. The rat is particularly useful for such an investigation since its brain is unmyelinated at birth (WAELSCH, SPERRY and STOYANOFF, 1941). Myelination begins at about 12 days and is completed at about 40 days in this species. With the development of a more refined technique of column chromatography (SPACKMAN, STEIN and MOORE, 1958) for the separation and identification of amino acids and their peptides, it has become possible to determine with precision and accuracy their concentration in biological extracts. Column chromatographic studies of TALLAN, MOORE and STEIN (1954) showed the presence of a large number of ninhydrin-positive compounds in the cat brain. FRIEDBERG and GREENBERG (1947) reported that rat brain has a high concentration of free amino acids, second only to that in kidney. Although the physiological significance of the high amino acid content in the brain is not yet clearly understood, studies of GMTONDE and RICHTER (1955, 1956) with isotopically labelled amino acids revealed that free amino acids are rapidly incorporated into the brain proteins. Recent studies Of SHAW and HIENE(I965) established the presence of fifty-eight ninhydrin positive substances, including nine unknown peaks in four different areas of the rat brain with the column chromatographic methods of PIEZ and MORRIS (1960) and of HAMILTON (1963). In the present study the changes in free amino acid pool of the rat brain have been investigated during ontogeny. An attempt was made to correlate the variation in amino acid pool with the chemical and physiological maturation of the brain.

DEVELOPMENTAL CHANGES IN MOUSE BRAIN: WEIGHT, WATER CONTENT AND FREE AMINO ACIDS

Abstract— —Glutamic acid, glutamine, GABA and aspartic acid exhibited postnatal increases in the developing mouse brain at the same time that the other amino acids, both essential and non‐essential, of the pool decreased. The most significant decreases were observed in the concentrations of taurine, phosphoethanolamine, glycine and alanine. The period of rapid accumulation of the members of the glutamic acid family in the mouse brain was concurrent with dramatic increases and decreases in brain weight and water content, respectively.

Expression of Myelin Proteolipid Protein and Basic Protein in Normal and Dysmyelinating Mutant Mice

Abstract: Expression of myelin proteins was studied in the brains of 21‐day‐old normal mice and three dysmyelinating mutants—jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell‐free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21‐day‐old quaking brain homogenates showed a reduction in the steady‐state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21‐day jimpy brain homogenates revealed no appreciable steady‐state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.

Purification and immunohistochemical localization of rat brain myelin proteolipid protein.

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N‐terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X‐100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10‐day‐old rats.

d‐β‐HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D‐β‐hydroxybutyrate (β‐OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3‐14C]β‐OHB or [2‐14C]glucose was injected subcu‐taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3‐14C]β‐OHB rapidly labelled brain amino acids at all ages whereas [2‐14C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3‐14C]β‐OHB from birth‐15 days of age whereas brain alanine was more heavily labelled by [2‐14C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β‐OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [14C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2‐,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3‐14C]β‐OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [14C]label from [2‐14C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [14C]label into the brain amino acids of the 15‐day‐old [3‐14C]β‐OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?‐OHB at 6 days was 19 8 min and at 15 days was 12‐2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3‐14C]β‐OHB injected rats compared to the [2‐14C]glucose injected rats despite a heavier labelling of brain glutamate in the [3‐14C]β‐OHB injected group. We interpreted these data to mean that β‐OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Abstract: Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti‐large basic protein serum was shown by the immunoblot technique to cross‐react with all five forms of basic protein present in the myelin of 8‐day‐old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.

Anti-neurofilament protein antibodies in opsoclonus-myoclonus

Opsoclonus-myoclonus (OM) is a neurological disorder usually occurring in infancy, clinically manifested by various involuntary movements. The pathogenesis of OM is unknown, but since the disease often is associated with viral infection or with neuroblastoma, an immunologic basis for OM has been postulated. We have studied two children with OM whose serum contained antibodies directed against the 210 kDa neurofilament protein; these antibodies were not seen in the serum of 21 children with other neurological disorders. Neurofilament proteins, which are found only in neurons, may be of prime importance in neuronal function, especially during development of the nervous system. Our findings suggest that generation of antibodies to the neurofilament proteins can occur in patients with opsoclonus-myoclonus; the role of the anti-NF210K antibodies in the pathogenesis of OM, however, is uncertain.

BIOCHEMICAL MATURATION OF HUMAN CENTRAL NERVOUS SYSTEM MYELIN

—A developmental study of the lipid and protein composition of human CNS myelin was undertaken. The relative concentrations of the major lipid classes, cholesterol, glycolipids and phospholipids exhibited little change except for a modest decrease in the concentration of the phospholipids. In contrast to the total phospholipids, marked variations in the relative concentrations of individual phospholipids were found. Sphingomyelin increased over two‐fold, and phosphatidyl choline decreased to almost half its original concentration.

Effect of N,N,N′,N′-tetramethylethylenediamine on the migration of proteins in SDS polyacrylamide gels

Abstract Changing the concentration of TEMED in SDS polyacrylamide gels was found to affect the migration of proteins. Elevation of TEMED levels caused a generalized decrease in mobility with some proteins being affected more than others. With various brain protein preparations this differential effect could be used to improve the separation of adjacent protein bands. In additon, it was found that a change in the TEMED concentration affected the results of molecular weight determinations. The effect of TEMED was also observed in one non-SDS system.