Composition and Metabolism of Gangliosides in Rat Peripheral Nervous System During Development

Abstract: Ganglioside composition of rat trigeminal nerve was studied during development in order to understand the changes that occur as a result of cellular differentiation in the nerve. The ganglioside composition of the trigeminal nerve was entirely different from that of brain. The major gangliosides in adult trigeminal nerve were GM₃, GD₃, and LM₁ (sialosyl-lactoneotetraosylceramide or sialosylparagloboside). The structure of LM₁ and other gangliosides was established by enzymatic degradation and by analysis of the products of acid hydrolysis. At 2 days after birth, when the Schwann cells were immature, GM₃ and GD₃ were the major gangliosides in the nerve, 50 and 18 mol %, respectively. As the nerve developed and Schwann cells proliferated and myelinated the axons, the mol % of GM₃ and GD₃ reduced and that of LM₁ steadily increased. Polysialogangliosides did not change drastically with nerve development. The rate of deposition of LM₁ in the nerve with age was very similar to that of myelin marker lipids, cerebrosides, and sulfatides; thus, deposition appears to be localized mainly in the rat nerve myelin. LM₁ also had long-chain fatty acids 22:0 and 24:0, which are not usually found in CNS gangliosides. The ganglioside pattern of the rat trigeminal nerve was very similar to that of rat sciatic nerve, but was different from that of rabbit and chicken sciatic nerve. The activity of the two key enzymes involved in the metabolism of GM₃, viz., CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase and UDP-N-acetylgalactosamine:GM₃-N-acetylgalactosaminyltransferase, was also studied during development of the nerve and brain. The developmental profiles of both enzymes were consistent with the amounts of GM₃ present in the nerve.     

Lipid patterns of embryonal carcinoma cell lines and their derivatives: Changes with differentiation

The lipid composition of several teratocarcinoma cell lines has been examined by biochemical and immunological methods in order to identify properties that might be correlated with the state of cell differentiation. The data indicate qualitative and quantitative changes in the phospholipid, cholesterol, and glycolipid composition. In particular, the ratios of cholesterol/phospholipid and of sphingomyelin/phosphatidylcholine are higher in differentiated cells. Gangliosides with short glycosidic chains (GM₃ and GD₃) are characteristic of undifferentiated, multipotent, embryonal carcinoma cell lines. More complex gangliosides (GM₁ and GD_1a) appear early during the course of differentiation. Each differentiated cell line presents a unique ganglioside map. Results are tentatively correlated with a stabilization of the membrane bilayer in differentiated cell lines, whereas a more fluid state of the membrane in embryonal carcinoma cell lines would allow maximal flexibility. Subtle differences in ganglioside composition among embryonal carcinoma cell lines are discussed in relation with their potentialities, and their developmental age.     

Effects of Cell Density on Lipids of Human Glioma and Fetal Neural Cells

Abstract: Gangliosides, phospholipids, and cholesterol of human glioma (12-18) and fetal neural cells (CH) were analyzed at specified cell densities, from sparse to confluent. Total ganglioside sialic acid, phospholipid phosphorus, and cholesterol increased in the glioma cells on a per cell, mg protein, or mg total lipid basis two- to threefold as cell density increased 25-fold. These same three constituents in the fetal cells increased with cell density on a per cell and mg protein basis but not on a per mg total lipid basis. In glioma cells, the di- and trisialogangliosides (GD₂+ GD_lb+ GT₁) increased from 1–2% of total ganglioside sialic acid at sparse densities to 7–8% at intermediate (logarithmic phase) densities to 10–13% at confluent densities. The set of simpler gangliosides (GM₄+ GM₃+ GM₂) decreased from 50% of total ganglioside sialic acid at sparse glioma cell densities, to 36% at intermediate and 30% at confluent densities. In the fetal neural cells, the set of gangliosides (GM₄+ GM₃+ GM₂) had about 48% of total ganglioside sialic acid in both sparse and confluent preparations. The fetal cells were twofold higher in GM₃ (32.4 ± 2.1%) than the glioma cells (16.8 ± 1.6%), but lower in GM_t (9.1 ± 0.9% versus 18.2 ± 1.8%), cell densities notwithstanding. Confluent cell preparations of both cell lines were consistently higher in ethanolamine plasmalogen than sparse cells. We conclude that in these two neural cell lines quantitative changes in ganglioside and phospholipid species occurred correlatively as cell densities increased. Higher glioma cell densities were associated with greater proportions of complex ganglioside species. These changes in cell membrane constituents during growth may result from cell contact and may indicate a role for them in cell growth regulation and/or differentiation.     

Gangliosides and their cell density-dependent changes in control and chemically transformed C3H/10T1/2 cells

The cloned C3H/10T1/2 mouse embryo cells contained a complex pattern of gangliosides. Two cloned chemical transformants obtained from the C3H/10T1/2 cell line by treatment with 7,12-dimethylbenz(a) anthracene (DMBA-TCL1) and 3-methylcholanthrene (MCA-TCL15) also had complex ganglioside patterns; but the transformants had increased levels of the simplest ganglioside, N-acetylneuraminylgalactosylglucosylceramide (GM₃), and reduced levels of more complex gangliosides. Incorporation of [¹⁴C]glucosamine into gangliosides, as cell-to-cell contact increased in C3H/10T1/2 cells, showed that GM₃ synthesis was decreased and that the synthesis of the more complex ganglioside N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD_1a) was increased. In the two transformants the percentage each individual ganglioside was of total labeled gangliosides was only slightly altered with changing cell density. Turnover of [¹⁴C]glucosamine-labeled gangliosides, as cell density increased, was approximately equal in C3H/10T1/2 cells and MCA-TCL15 cells, but more rapid in the DMBA-TCL1 cells. Most individual gangliosides turned over at about the same rate in the respective cell lines. However, GD_1a increased slightly as a percentage of total labeled gangliosides with increasing cell density in both C3H/10T1/2 cells and transformed cells. The labeling data indicated that the majority of GD_1a synthesis was de novo and only a small part occurred by transfer of sialyl or glycosyl residues to simpler gangliosides or catabolism of more complex gangliosides already present in the outer membrane. Exogenous complex gangliosides added to the medium were more effective inhibitors of DMBA-TCL1 cell growth than of C3H/10T1/2 cell growth. Furthermore, gangliosides added to exponentially growing C3H/10T1/2 and DMBA-TCL1 cells caused both cell lines to incorporate a greater percentage of [¹⁴C]glucosamine into gangliosides more complex than GM₃.     

Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV.

Extracts of cultured skin fibroblasts derived from patients with mucolipidosis IV showed a marked increase and altered distribution of GM₃ and GD₃ gangliosides. GD₃ is elevated 1.5–2 times that of normal whereas GM₃ is elevated to a lesser extent. No abnormalities were found in the neutral glycolipids. These two gangliosides apparently comprise most of the accumulated lipid-like material observed on ultrastructural analysis in this disease.     

Developmental changes in gangliosides during myogenesis of a rat myoblast cell line and its drug resistant variants.

Cloned cells of a myoblast line show the presence of GM₃, GM₂, GM₁ and GD_1a gangliosides. The amount of GM₃, GM₂ and GM₁ gangliosides does not vary significantly during the differentiation of myoblasts to myotubes. However, the concentration of GD_1a transiently increases almost 3-fold just prior to the fusion of myoblasts and returns to the basal levels in the myotubes. Mutant myoblasts selected for 5-azacytidine resistance and unable to fuse produce only GM₃ and traces of GM₂. We conclude that GD_1a probably participates in the fusion process through yet unknown mechanism.     

Ganglioside GM1 beta-galactosidase: studies in human liver and brain

A microcolumn assay for ganglioside GM₁ β-galactosidase (EC 3.2.1.23) has been developed using GM₁ tritiated exclusively in the terminal galactose residue. The reaction is stimulated up to 100-fold by anionic and cationic detergents; this stimulation is inhibited by neutral detergents. 4-Methylumbelliferyl β-d-galactopyranoside is hydrolyzed about seven times more rapidly than GM₁ in human brain (gray matter) and liver. Agarose gel filtration separated two forms of GM₁ β-galactosidase in both brain and liver. The major form (ganglioside GM₁ β-galactosidase A) had a molecular weight of 60–70 × 10³ and the minor form (ganglioside GM₁ β-galactosidase B) 600–800 × 10³. The liver and brain GM₁ β-galactosidases and 4-methylumbelliferyl β-galactosidase A cochromatographed on fractionation. The two forms of the enzyme in liver isolated by gel filtration corresponded to the two major forms found on starch gel electrophoresis and were converted to electrophoretically slower-moving forms after treatment with neuraminidase (EC 3.2.1.8, Cl. perfringens) suggesting that both are sialylated glycoproteins. The activity of GM₁ β-galactosidase in the brain and liver tissue of patients with GM₁ gangliosidosis Types I and II was less than 2% of control values. The mutation in each GM₁ gangliosidosis appears to result in a severe reduction of activity of two ganglioside GM₁ β-galactosidases.     

Gangliosides Are Important for the Preservation of the Structure and Organization of RBL-2H3 Mast Cells

Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. In this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM₁ and the α-galactosyl derivatives of the ganglioside GD_1b. The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell–specific α-galactosyl derivatives of ganglioside GD_1b and GM₁ are important in maintaining normal cell morphology. (J Histochem Cytochem 58:83–93, 2010)     

Evidence of a distribution difference between two gangliosides in bilayer membranes

Freeze-etch electron microscopy, a platinum shadowing technique, has been used to compare the lateral distribution of several gangliosides in bilayer model membranes by directly visualizing bound lectin molecules. In particular, GM₁ and GD_1a, major components of brain ganglioside, were studied in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholines exposed to Ricinus communis agglutinin and wheat germ agglutinin. The distribution of glycolipid showed evidence of microheterogeneity in that bound lectin tended to occur in clusters of several or more molecules. With GD_1a as receptor such clusters were small and very uniformly distributed over the membrane surface. Somewhat larger, irregularly spaced clusters of up to a dozen lectin particles were more typical of membranes bearing GM₁ and, in addition, there were occasional extensive patches of bound lectin coexisting with areas apparently devoid of glycolipid receptor in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholine. Gangliosides in the latter mixtures were not obviously influenced in their lateral distribution by the presence of coexisting fluid and rigid domains. These basic observations seem to extend to bilayer membranes containing mixtures of two gangliosides. The patterns of lectin binding were not grossly affected by incubation time or history of warming and cooling. This study was extended to bilayers of pure dipalmitoylphosphatidylcholine in expectation that the distinctive features characteristic of the P_β′ phase of this lipid might accentuate any behavioural differences between GM₁ and GD_1a.GM₁ was found to exist preferentially in the ‘trough’ regions between P_β′ ripples, while GD_1a showed no apparent preferential arrangement. Given that bound lectins adequately reflect glycolipid distribution in membranes, it would appear that structurally different glycolipids from the same host membrane can assume different distributions on the basis of interactions with defined lipid host matrices.     

THE DISTRIBUTION OF LIPIDS IN THE HUMAN NERVOUS SYSTEM-V. GANGLIOSIDES AND ALLIED NEUTRAL GLYCOLIPIDS OF INFANT BRAIN

—Gangliosides and allied neutral glycosylceramides were isolated from human infant (2-24 months of age) cerebral cortex and white matter. The individual glycolipids were separated quantitatively by a combination of column and thin-layer chromatographic methods on silica gel, DEAE-cellulose and Sephadex G-25. In cerebral cortex G_D1a and G_M1 were the major fractions and constituted more than 70 per cent of the total gangliosides. The concentrations of neutral glycolipids, except for galactosylceramides, were very low: lactosylceramide and glucosylceramide comprised 30 and 5 nmol/g wet weight, respectively. In white matter their concentrations were 10 times higher. The ganglioside concentration was only 50 per cent of that in cerebral cortex: the difference was accounted for mainly by the much lower content of the major di- and trisialogangliosides. Stearic acid was the predominant fatty acid of all brain gangliosides. G_M3, and G_D3 had a considerable content of the very long-chain fatty acids, C₂₂-C₂₄, particularly in the white matter. Glucosylceramide and lactosylceramide had almost identical fatty acid patterns between each other in cerebral cortex and white matter. In the cerebral cortex stearic acid and in the white matter the very long-chain acids predominated. d20:1 Sphingosine comprised more than 20 per cent of total sphingosine in all the gangliosides of the G_l- and G₂-series. G_M3, and G_D3 like lactosylceramide contained significantly less of d20:1 sphingosine. The findings suggest the existence of separate compartments for the biosynthesis of the gangliosides. Glucosylceramides and lactosylceramides of white matter have the same ceramide composition as the galactosylceramides with normal fatty acids and are thus unlikely to be intermediates in the metabolism of the major brain gangliosides which have a completely different fatty acid composition.     

Monomer of the B Subunit of Heat-Labile Enterotoxin from Enterotoxigenic Escherichia coli Has Little Ability to Bind to GM1 Ganglioside Compared to Its Coligenoid

Coligenoid, composed of the B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli, was separated into monomers in the presence of 2% propionic acid containing 6 M urea (pH 3.8). Monomers equilibrated against 0.75% or 0.5% propionic acid containing 3 M urea (pH 3.8) did not reassemble into coligenoid. Complexes of GM₁ ganglioside and coligenoid in these buffers were detected by SDS-polyacrylamide gel electrophoresis, but those of the GM₁ ganglioside and monomers were not. The binding ability of monomer to GM₁ ganglioside in these buffers was about 1% of that of normal coligenoid by GM₁-enzyme-linked immunosorbent assay. Moreover, monomers in these buffers reassembled into coligenoid by buffering against original TEAN buffer, and the binding ability of the resulting coligenoid to GM₁ ganglioside was identical to that of native coligenoid. These data suggest that although coligenoid formation is important for the receptor binding of the B subunit, little binding ability to GM₁ ganglioside remains in monomer of the B subunit.     

Number of Sialic Acid Residues in Ganglioside Headgroup Affects Interactions with Neighboring Lipids

Monolayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and asialo-(GA₁), disialo-(GD_1b) and trisialo-(GT_1b) gangliosides were used to determine the effect of ganglioside headgroup charge and geometry on its interactions with the neighboring zwitterionic lipid. Surface pressure versus molecular area isotherm measurements along with concurrent fluorescence microscopy of the monolayers at the air-water interface were complemented with atomic force microscopy imaging of monolayers deposited on solid substrates. Results were used to further develop a proposed geometric packing model that the complementary geometry of DPPC and monosialoganglioside GM₁ headgroups affects their close molecular packing, inducing condensation of the layer at small mol % of ganglioside. For GA₁, GD_1b, and GT_1b, a similar condensing effect, followed by a fluidizing effect is seen that varies with glycosphingolipid concentration, but results do not directly follow from geometric arguments because less DPPC is needed to condense ganglioside molecules with larger cross-sectional areas. The variations in critical packing mole ratios can be explained by global effects of headgroup charge and resultant dipole moments within the monolayer. Atomic force microscopy micrographs further support the model of ganglioside-induced DPPC condensation with condensed domains composed of a striped phase of condensed DPPC and DPPC/ganglioside geometrically packed complexes at low concentrations.     

Number of Sialic Acid Residues in Ganglioside Headgroup Affects Interactions with Neighboring Lipids

Monolayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and asialo-(GA₁), disialo-(GD_1b) and trisialo-(GT_1b) gangliosides were used to determine the effect of ganglioside headgroup charge and geometry on its interactions with the neighboring zwitterionic lipid. Surface pressure versus molecular area isotherm measurements along with concurrent fluorescence microscopy of the monolayers at the air-water interface were complemented with atomic force microscopy imaging of monolayers deposited on solid substrates. Results were used to further develop a proposed geometric packing model that the complementary geometry of DPPC and monosialoganglioside GM₁ headgroups affects their close molecular packing, inducing condensation of the layer at small mol % of ganglioside. For GA₁, GD_1b, and GT_1b, a similar condensing effect, followed by a fluidizing effect is seen that varies with glycosphingolipid concentration, but results do not directly follow from geometric arguments because less DPPC is needed to condense ganglioside molecules with larger cross-sectional areas. The variations in critical packing mole ratios can be explained by global effects of headgroup charge and resultant dipole moments within the monolayer. Atomic force microscopy micrographs further support the model of ganglioside-induced DPPC condensation with condensed domains composed of a striped phase of condensed DPPC and DPPC/ganglioside geometrically packed complexes at low concentrations.     

The characterization of sphingolipids from neurons and astroglia of immature rat brain

Abstract— Isolated neuronal cell bodies and astroglia of young (15–20-day-old) rat brains were both found to contain small concentrations of a variety of glycosphingolipids, including glucosylceramide, galactosylceramide, sulphatide, dihexosylceramide and gangliosides. These sphingolipids, plus sphingomyelin, were isolated, quantitated and their fatty acid and long chain base patterns determined. These data were compared to similar data obtained on these lipids isolated from whole brain and myelin of rats of the same age range. Glucosylceramide was found in an amount equal to galactosylceramide in neurons, and accounted for 35 per cent of the total monohexosylceramide in astroglia. Dihexosylceramide was present in nearly the same amount as sulphatide in both cell types. The sphingolipids of each cell type had characteristic fatty acid patterns. Generally the whole brain fatty acid patterns resembled those of astroglial lipids rather than neuronal lipids. In no case did the cell sphingolipid fatty acids resemble those of myelin. However, the galactosylceramide and sulphatides of both cells had unsubstituted and α-hydroxy acids, both of which had appreciable quantities of C₂₄ acids. The ganglioside fatty acids of each cell type were similar and not unusual, but were quite different from those of glucosylceramide and dihexosylceramide; the latter having appreciable quantities of 16:0 and acids longer than 18:0. The ganglioside patterns of these cells were similar and only slightly different from that of whole brain. Long chain bases of sphingolipids were mainly C₁₈-sphingosine in both cell types, and those of ganglioside and sphingomyelin contained small amounts of C₂₀-sphingosine.     

Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, ¹²⁵I-FGF-2 binds to micelles formed by gangliosides GT_1b, GD_1b, or GM₁. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT_1b > GD_1b > GM₁ = GM₂ = sulfatide > GM₃ = galactosyl-ceramide, whereas asialo-GM₁, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM₁ to immobilized FGF-2 indicates that FGF–2/GM₁ interaction occurs with a K_d equal to 6 μM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112–129) and, to a lesser extent, FGF-2(130–155), whereas peptides FGF-2(10–33), FGF-2(39–59), FGF-2(86–96), and the basic peptide HIV-1 Tat(41–60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of ¹²⁵I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT_1b > GD_1b > GM₁ > sulfatide a = sialo-GM₁. Accordingly, GT_1b,GD_1b, GM₁, and GM₂, but not GM₃ and asialo-GM₁, prevent the binding of ¹²⁵I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM₁ to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT_1b was the most effective among the gangliosides tested while asialo-GM₁, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.     

GM3 ganglioside induces hamster fibroblast growth inhibition in chemically-defined medium: ganglioside may regulate growth factor receptor function

GM₃ or GM₁ ganglioside exogenously added in chemically-defined medium incorporate equally into cells. However, only GM₃ showed a significant growth inhibition to hamster fibroblasts (BHK). The GM₃-fed cells became refractory to growth stimulation by fibroblast growth factor (FGF) in chemically-defined media. Radiolabeled FGF accumulated on GM₃-fed cells, but not on GM₁-fed cells. Both GM₃ and GM₁ did not directly interact with FGF. These data suggest that GM₃ may regulate the function of the receptors for growth hormones.     

FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE SUDANOPHILIC LEUCODYSTROPHY: THE JIMPY MUTANT

The fatty acid composition of cerebrosides, sulphatides and ceramides was determined at 15-16 days post partum in the brain of the Jimpy mutant and in littermate controls. There was a marked deficit in the long chain fatty acids (C₂₂-C₂₄) of cerebrosides and sulphatides of Jimpy brain, with the unsubstituted fatty acids affected more than the alpha-hydroxy fatty acids. A decrease of long chain normal fatty acids was also found in the ceramides of Jimpy brain. The deficit of long chain fatty acids in these sphingolipids of the Jimpy brain was more severe than that found in the Quaking mutant which has a less extensive disorder of myelin formation.     

Lipid composition of activated sludge in a pilot plant for anaerobic ammonium oxidation

The lipid composition of the microbial community inhabiting activated sludge in a pilot reactor for the anaerobic oxidation of ammonium (anammox) at the Kur’yanovo Treatment Plant (Moscow) has been studied. The fatty acid composition is mostly based on common fatty acids C₁₄–C₁₈ (95%) with both normal and isomeric structures. The biomass of activated sludge was found to contain lipids with the so-called ladderane substances (ladder alcohols and fatty acids) that are common for anammox bacteria: C₂₀-[3]-lad-derane and C₂₀-[5]-ladderane alcohols and C₁₈- and C₂₀-[3]-ladderane and C₁₈- and C₂₀-[5]-ladderane acids. In addition, the native extract contained both simple and compound ethers of the above-mentioned substances with residues of phosphocholine, phosphoethanolamine, and phosphoglycerine. The spectra of the electron impact and tandem mass spectrometry of certain substances have been obtained and published for the first time.     

SIALYLTRANSFERASES IN RAT BRAIN: REACTION KINETICS, PRODUCT ANALYSES, AND MULTIPLICITIES OF ENZYME SPECIES

Abstract— The incorporation of NeuNAc from CMP-NeuNAc into endogenous glycolipids and glyco-proteins, and exogenously added GM_1a (monosialoganglioside) and desialylated fetuin (DS-fetuin) was studied with particulate preparations from 11 to 15 day old rat cerebra. The apparent +K++_m values of the enzyme systems for the different substrates, assayed with 0.5 mg enzyme protein, were: CMP-NeuNAc, 0.13 mm (same with endogenous and exogenous glycolipid and glycoprotein substrates); GM_1a, 0.20 mm ; DS-fetuin, 0.15 mm (or 1.2 mm in terms of acceptor sites). The activities, expressed as nmoles NeuNAc incorporated per 0.5 mg enzyme protein per 30 min incubation at 37°C and pH 6.3, were 0.094, 0.039, 0.17 and 0.64 with the endogenous glycolipids, endogenous glycoproteins, exogenous GM_1a and exogenous DS-fetuin, respectively. Incorporation into endogenous glycolipids was mainly in GM₃, while exogenously added GM_1a was converted to GD_1a. Incorporation into endogenous glycoproteins yields about 20 sialoglycopolypeptides on SDS-polyacrylamide gel electrophoresis. Neura-minidase pretreatment of the particulate enzyme preparation decreased sialylation of the higher molecule weight polypeptides but increased sialylation of the lower molecule weight species. The sialyltransferase activity with the endogenous glycolipid substrates was more heat resistant than the activities with exogenous GM_1a. Since more than 60% of the endogenous glycolipid activity was due to the conversion of lactosylceramide to GM₃, the sialyltransferase responsible for this reaction appears to be different from the one that acts on GM_1a. This was supported by the observation that exogenously added GM_1a did not diminish the incorporation of NeuNAc into endogenous lactosylceramide. These two glycolipid sialyltransferase activities were distinguishable from the glycoprotein sialyltransferase activity since exogenous DS-fetuin did not compete with either the endogenous or the exogenous glycolipids for CMP-NeuNAc.