Marilynn K. Rumley

CHANGES IN THE COMPOSITION OF THE DEVELOPING MOUSE BRAIN DURING EARLY MYELINATION

RECENT interest in changes in the composition of the mammalian brain during the early maturation stage undoubtedly results from the recognition that the study of such chemical changes, when combined with a study of morphogenesis, may suggest the localization (or function) of chemical components of brain. On the other hand, the relatively static compositional and histological picture of the adult brain offers no such promise. Earlier efforts were hampered by the inadequacy of analytical methods available (KOCH, 1904; KOCH and CARR, 1909; KOCH and KOCH, 1913; DONALDSON, 1916; MACARTHUR and DOBY, 1919) and by the fact that the chemical development of brain was studied only with respect to a few constituents (DONALDSON, 1916; MAC ARTHUR and DOISY, 1919). The paucity of parameters and the lack of a significant frame of reference rendered interpretation of such results difficult. The greatest amount of work done in this respect has naturally focused on the changes in the patterns of brain lipids because a superficial parallelism between increase in lipid content, increase of myelination, and general maturation of brain function, in terms of motor co-ordination and behavioural patterns, was evident. This parallelism appeared to justify intensive work on changes of known brain lipids. This approach, though sporadically taken up during the past 30 years, suffered not only from analytical shortcomings, but also from lack of information as to what all the lipid constituents of brain were. Perhaps the most significant information on the rapidity with which compositional changes occur in the developing brain was provided by the early studies of WAELSCH ei al. (1941) which showed the high rate of incorporation of labelled fatty acids into myelin lipids during early myelination. The efforts to date have recently been critically reviewed (SPERRY, 1955: FOLCH-PI, 1955; LEBARON and FOLCH, 1956). More recent work (FOLCH-PI, 1955; SPERRY, 1955) has investigated the nature of the changes in the lipids of the developing brain, and the information thus gained has superseded the extensive survey of BRANTE published in 1949. It is thus generally conceded that the maximal content of cerebrosides and sphingomyelins corresponds to that phase in the development of mammalian brain in which histologically detectable myelination reaches its maximum level (KOCH and KOCH, 1913 ; FOLCH-PI, 1955 ; BRANTE, 1949), and that the appearance of gangliosides (strandin) and phosphatides antedates this phase (FOLCH-PI, 1955). The major unproven assumptions in these tentative conclusions are (a) that increase in a lipid component

Neuraminic Acid as a Constituent of Human Cerebrospinal Fluid.

Conclusions 1. The neuraminic acid (NA) content of 49 samples of human cerebrospinal fluid (CSF) was studied. In all instances the quantitative relationship of neuraminic acid to the cerebrospinal fluid protein was far in excess of that prevalent in serum. In 26 samples in which the total protein was 30 mg % or less, the neuraminic acid was 4-18 times in excess of that expected if it were derived from serum and bound to protein, as is the case in blood. Unlike its state in serum, most of the CSF neuraminic acid is in freely dialyzable form (60-80%). That moiety of CSF neuraminic acid which is derived from serum is in protein-bound, non-dialyzable form. 2. The origin of the dialyzable NA is unknown, but is not related to ingress of serum proteins into the CSF. The implications of these findings are discussed.

The substrate specificity of mouse brain peptidase activity.

RECENT studies have revealed the existence of peptides in free form in mammalian brain (UZMAN, 1958~) at all ages of post-natal development as well as in invertebrate nerve (DEFFNER and HAFTER, 1959). Studies on human brain have also indicated that some peptides may play a copper-binding role in hepatolenticular degeneration (UZMAN, 19586) and thus bring about the characteristic deposition of copper within glia cells in this disease. It seemed therefore desirable to obtain more information concerning the efficacy and specificity with which peptides with different amino acid compositions and sequences can be split by the mammalian brain. As an initial step towards this end we have undertaken a survey of the peptidase activity of whole mouse brain towards a wide spectrum of peptides representing different structural arrangements of constituent amino acid residues. The choice of mouse cerebral enzyme activity was dictated by the detailed information and experience already available to us on this species both in terms of developmental histology and of chemical composition (FOLCH-PI, 1955; UZMAN and RUMLEY, 1958; UZMAN, 1958~). The only information available to us in connection with cerebral peptidase activity has been the earlier meticulous studies of POPE on the enzymic hydrolysis of a single dipeptide, DL-alanyl-glycine, by the different layers of rat (POPE, 1952) and human cortex (POPE, 1959) which indicated the presence of dipeptidase activity in neuronal and glial cell bodies and processes. The present report concerns itself with our observations on the striking quantitative differences in the peptidase activity of mouse brain, under in ritro conditions and without addition of co-factors or activators, towards known peptides with different compositions, and is a detailed extension of our earlier brief preliminary report on the N-glycyl-dipeptidase activity of mouse brain (UZMAN eta/ . , 1960).

THE INHIBITION OF CEREBRAL DIGLYCINASE BY α‐AMINO‐, α‐KETO‐ AND α‐HYDROXY ACIDS

THE SUBSTRATE specificity which the peptidase activity of the brain exhibits ( UZMAN et al., 1960; 1961) is especially remarkable with respect to substrates which are normally not hydrolysed by cerebral peptidase extracts or whole brain slices. Among those substrates which are only minimally attacked by brain peptidases is glycylglycine, which has been shown to be wholly split if a specific diglycinase is activated by cobalt (VAN DEN NWRT and UZMAN, 1961). Furthermore, it has also been shown that triglycine, which is split by a separate tripeptidase of brain into the N-terminal glycine and diglycine (UZMAN et al., 1961; 1962) will be totally split into 3 moles of glycine if cobalt ions are added to activate a separate diglycinase (VAN DEN NWRT and UZMAN, 1961; UZMAN et al., 1962). In this respect the tripeptidase activity of brain resembles the tripeptidase activity of calf-thymus tripeptidase (ELLIS and FRUTON, 1951). These authors showed that in addition to the total hydrolysis of triglycine in the presence of cobalt, these tripeptidase preparations would also split L-leucyl-diglycine and L-valyl-diglycine completely by the combined activities of tripeptidase and cobalt-activated diglycinase. However, in the course of our studies on the hydrolysis by brain of L-leucyl-diglycine, L-valyl-diglycine, and DL-alanyldiglycine, the N-terminal amino acid was hydrolyzed without addition of any cofactors, but contrary to expectation, the addition of cobalt ions failed to promote the hydrolysis of the remaining diglycine residue (UZMAN et al., 1962). The present study was undertaken to clarify this inhibition of the total hydrolysis of tripeptides containing the diglycine residue even when cobalt is added to the system. This report is concerned with the demonstration of the specificity of cerebral diglycinase and the potent inhibitory effect that some amino acids, a-keto acids, and a-hydroxy acids have on the cobalt-activated diglycinase system of brain.

The lipophilic peptides and proteins of brain. III. Difference between brain and peripheral nerve.

Abstract The respective neurosclerin contents of mouse, monkey, and cat brain were determined and compared with the amounts obtained from the sciatic nerve of the corresponding species. These values were related to the cerebroside content of the tissues. In all three species the neurosclerin content of brain exceeds that of the sciatic several fold, leading to widely divergent neurosclerin to cerebroside and neurosclerin to cholesterol ratios. In lobster leg nerves which lack myelin, the presence of a fraction homologous to the sciatic neurosclerin fraction was shown. The relationship of these findings to the proteolipids is discussed. It is suggested that neurosclerin probably represents a highly lipophilic membrane protein whose relationship to myelin proteins is as yet obscure.