Human brain gangliosides: developmental changes from early fetal stage to advanced age

The developmental profiles of the four major brain gangliosides, GM1, GD1a, GD1b, and GT1b, were examined in human frontal lobe covering the period from 10 fetal weeks to 80 years of age. The ganglioside concentration increased approx. 3-fold from the 10th gestational week to the age of about 5 years. Gangliosides GM1 and GD1a increased 12-15-fold during the same period. The most rapid increase of GM1 and GD1a occurred around term, during the period for dendrite arborization, outgrowth of axons and synaptogenesis. GT1b showed a quite different developmental curve. It was the major ganglioside during the 3rd to 5th gestational month, whereafter its concentration dropped rapidly to term, from which time the concentration then increased up to 50 years of age. Similar curves were found for the other gangliosides of the b-series, GD3, GD2, GD1b and GQ1b. Ganglioside 3'-isoLM1 was a characteristic early fetal ganglioside which dropped rapidly to the 5th gestational month, reached a small peak around term and then disappeared during adulthood. The concentration of gangliosides of the neolacto series was larger than that of the lacto series during the whole developmental period. In the beginning of the second trimester, 3'-LM1 constituted 2% and LD1 10% of total ganglioside sialic acid. The new findings demonstrate more dynamic changes of the ganglioside patterns during development than noted in previous studies.     

Sialosyllactotetraosylceramide, 3''-isoLM1, a Ganglioside of the Lactotetraose Series Isolated from Normal Human Infant Brain

A ganglioside antigen was detected in infant human brains by the monoclonal antibody C-50. Structural analysis of the isolated ganglioside antigen showed it to be 3'-isoLM1, sialosyllactotetraosylceramide. The concentration of this ganglioside in human infant brain was 0.5 nmol/g.     

Ganglioside GM1 metabolism in living human fibroblasts with β-galactosidase deficiency

Summary The uptake and catabolism of [³H-ceramide]-G_M1 was followed in living fibroblasts from patient with different forms of -galactosidase deficiency. Gangliosides are identified according to the nomenclature of Svennerholm (1963). A total inability to metabolize the ingested substrate was found in infantile G_M1-gangliosidosis whereas cells from an adult G_M1-gangliosidosis variant showed a slower rate of degradation, compared with controls. Morquio B fibroblasts had a comparable catabolism of G_M1 as controls. Fibroblasts from different types of galactosialidosis, a recessive disease associated with a coexistent -galactosidase/neuraminidase deficiency all showed degradation of ingested G_M1. In view of the molecular defect in this disease, this catabolism must be due to the 10–20% of monomeric -galactosidase molecules present in the lysosomes. Unexpectedly, in these cells an impaired metabolism of G_M3 was found. The same finding was observed when cells with an isolated neuraminidase deficiency (mucolipidosis I) were loated with G_M1. A hypothesis is presented to explain these results.     

Receptor-specific large-scale purification of cholera toxin on silica beads derivatized with lysoGM1 ganglioside

1. A receptor-specific affinity chromatographic method for large-scale purification of cholera toxin is described. The receptor ganglioside for cholera toxin, GM1, is hydrolysed to lysoGM1 which is then covalently coupled, via stabilized Schiff's bases, to porous silica beads (Spherosil) onto which a layer of DEAE-dextran has been adsorbed and cross-linked before coupling. Columns of these Spherosil-DEAE-dextran-lysoGM1 beads, in contrast to particles derivatized with lysoGA1, bound the cholera toxin of Vibrio cholerae culture filtrates, after which the toxin could be eluted with the aid of an acid citrate buffer (pH 2.8). 2. The toxin-binding capacity was directly proportional to the amount of lysoGM1 in the column: 2.3 mg/mu mol lysoGM1. The yield of purified toxin after acid elution and pH neutralization was essentially quantitative (83-107%). 3. The affinity-purified toxin contained less than 5% impurities, but consisted of a mixture of predominantly intact holotoxin and B subunit protomer which could readily be separated by gel filtration on Sephadex G-100. 4. Scaling up of the technique was possible: a 1 kg column enabled us to treat 1000-1 cultures of V. cholerae and thus to isolate 20 g of cholera toxin per cycle.     

Composition of gangliosides and phospholipids of neuronal and glial cell enriched fractions

Abstract— Fractions enriched in neuronal cell bodies and in glial cells were isolated from rabbit cerebral cortex by discontinuous gradient centrifugation. The ratio of total lipid to protein was approx. 50 per cent higher in the glial fraction than in the neuronal fraction. The fatty acid composition for the major phosphoglycerides was with few exceptions, similar for neurons and glia. The ganglioside concentration was very low for both cell types, but was approx. twice as high in the glial cells as in the neurons. The pattern of individual gangliosides was, however, very similar for the glial and neuronal fractions and did not differ from that of unfractionated cerebral cortex, synaptosomes and mitochondria. The latter results are discussed in relation to the estimated amounts of plasma membrane in the neuronal and glial fractions.     

DEVELOPMENTAL PROFILES OF GANGLIOSIDES IN HUMAN AND RAT BRAIN

Abstract— The developmental profiles of individual gangliosides of human brain were compared with those of rat brain. Interest was focused mainly on the pre- and early postnatal development. Human frontal lobe cortex covering the period from 10 foetal weeks to adult age and the cerebrum of rat from birth to 21 days were analysed. Lipid-NANA and lipid-P were followed; in the rat, also protein and brain weight. A limited number of samples of human cerebral white matter and cerebellar cortex were also studied. The following major results were obtained:

• 1 The ganglioside concentration increased approximately three-fold within a short period: in rat cerebrum, from birth to the 17th day; in human cerebral cortex, from the 15th foetal week to the age of about 6 months. The largest increase in the rat brain occurred by the 11th to the 13th day; in human brain by term. The relative increase of gangliosides during this period was more rapid than that of phospholipids.
• 2 A hitherto unknown distinct early period of ganglioside and phospholipid formation in rat occurred by the second to fourth day.
• 3 The changes in brain ganglioside pattern, characteristic of the developmental stages of the rat, were found to be equally pronounced in the human brain.
• 4 Regional developmental differences in the ganglioside pattern were demonstrated in human brain. A characteristic white matter pattern, rich in monosialogangliosides, had developed by the age of 1 year. The increase in ganglioside concentration and the formation of the definitive ganglioside pattern of cerebellar cortex occurred later than in cerebral cortex. This cerebellar pattern was characterized by a very large trisialoganglioside fraction.
• 5 The two periods of rapid ganglioside metabolism in rat brain preceded the two periods of rapid protein biosynthesis.

     

Distribution and fatty acid composition of phosphoglycerides in normal human brain

A thin-layer chromatographic procedure for the isolation of tissue phospholipids and their subsequent analysis is described. The method has been applied to the determination of the fatty acids of phosphoglycerides in human brain from the early fetal stage to old age. The study shows changes in the distribution and fatty acid composition of each phosphoglyceride in normal brain, although they are quite small after early childhood. A lipid-specific fatty acid pattern for each of the four major phosphoglycerides was found. Besides this, the pronounced differences between fatty acids of the lipids from the cerebral cortex and from the adjacent white matter justify speaking of a tissue-specific fatty acid pattern for brain phosphoglycerides. The phospholipids of cerebral white matter contained more monoenoic acid but much less polyunsaturated fatty acid than those of cerebral cortex. The brain phosphoglycerides also showed an age-dependent fatty acid pattern. With increasing age the concentration of the fatty acids of the linoleate family diminished while that of the linolenate family increased. Brain inositol phosphoglycerides, the fatty acid composition of which has not been studied systematically before, were characterized by a large concentration of arachidonate which was nearly as high for white as for gray matter and showed only small changes with age.     

Membrane lipids of human peripheral nerve and spinal cord.

Major membrane lipids were determined in specimens of human peripheral nerve (cauda equina) and spinal cord of 10 subjects aged 20-70 years. The same lipids were also assayed in myelin from the same tissues isolated with two different procedures and in myelin of cauda equina from 3 subjects aged 17-91 years isolated with a third method. The concentrations (mean and standard deviation) of phospholipids were 90 +/- 11 and 96 +/- 9 nmol/g fresh weight; of cholesterol 70 +/- 15 and 101 +/- 16; of cerebroside 19 +/- 3 and 41 +/- 7; of sulfatide 10 +/- 1 and 11 +/- l; and of gangliosides 0.80 +/- 0.08 and 0.40 +/- 0.05 N in cauda equina and spinal cord, respectively. The proportion of ethanolamine phosphoglyceride was lower and that of sphingomyelin higher in cauda equina than in spinal cord. The myelin of peripheral nerve and spinal cord contained almost the same proportions of lipids as the whole tissue. The protein-bound sialic acid content was 3-fold higher than the lipid-bound sialic acid content in cauda myelin. The fatty acid patterns of choline, ethanolamine, inositol and serine phosphoglycerides of spinal cord and its myelin, were very similar to those of cerebral white matter, while the phosphoglycerides of cauda equina had higher proportions of monoenoic acids and lower proportions of polyunsaturated fatty acids. The fatty acid patterns of sphingomyelin, cerebroside and sulfatide of spinal cord were similar to those of cerebral white matter, while those of cauda equina contained significantly more saturated fatty acids. This suggests that the lipid and fatty acid compositions of peripheral nerve are particularly suitable for the formation of a tightly packed myelin membrane which can be a powerful shield against infections and other injuries.     

Synthesis on nontoxic, antibody-binding Escherichia coli heat-stable enterotoxin (STa) peptides

Abstract Modified Escherichia coli heat-stable enterotoxin (ST_a) peptides have been synthesized to identify structures that retain the immunological properties of native ST_a but lack toxicity. Two synthetic peptides, corresponding to the 15 C-terminal amino acid residues of ST_a (ST_h) except for the replacement of one or two Cys residues by Ala, had ≥ 35 000-fold reduced toxicity in infant mice despite almost intact activity as compared to native ST_a to inhibit monoclonal anti-ST antibody binding to solid phase ST_a. Three shorter peptides of 6–8 amino acid residues also did not manifest any toxic activity and showed significant but much reduced reactivity with anti-ST_a antibodies.