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.     

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.     

Expression of glycosphingolipids in serum-free primary cultures of mouse kidney cells: male-female differences and androgen sensitivity

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bg ^j/bg ^j) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cell proliferated for 7 daysin vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The malevs female differences in neutral glycosphingolipids seen in the kidneyin vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10^–5 M testosterone or 5-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growthin vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto-and digalactosylceramides as seenin vivo.     

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.     

The accumulation and metabolism of glycosphingolipids in primary kidney cell cultures from beige mice

In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bg ^J/bg^J mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bg ^J/bg ^J beige mutants were grown in D-valine medium and glycosphingolipids labeled with [³H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [³H]palmitate into glycospingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes. (Mol Cell Biochem 118: 61–66, 1992)     

Soluble gangliosides in cultured neurotumor cells

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [³H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several-fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor-product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t_1/2= 26 h) than membrane-bound GM3 (t_1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane-bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.     

GANGLIOSIDE COMPOSITION OF CHEMICALLY INDUCED RAT NEURAL TUMORS AND CHARACTERIZATION OF HEMATOSIDE FROM NEURINOMAS

Abstract– Experimental rat neural tumors in offspring were induced transplacentally by a single injection of a chemical carcinogen, ethylnitrosourea, 20mg/kg body wt, in the tail vein of the mother. The ganglioside content and pattern in these tumors and the normal tissues from which the tumors originated are described. The ganglioside content in tumors was reduced, on wet tissue weight basis, compared to normal control. However, there was no significant difference of ganglioside content on dry weight or protein basis. Altered ganglioside composition was found in most of the neural tumors. In central nervous system tumors, there was some increase in GM₃ and GT_1b′ (nomenclature according to Svennerholm , 1963), a marked decrease in GM₁ and some decrease in GD_1a, but no apparent loss in GD_1b. Extreme simplification of ganglioside pattern was seen in tumors originated from peripheral nervous system. Large accumulation of GM₃ with concomitant loss of all the higher gangliosides was seen. GM₃ from neurinomas as well as from normal gray matter was isolated and characterized. GM₃ from neurinomas separated into two bands on thin layer chromatographic plates. Both these GM₃ bands had identical sphingosine and carbohydrate composition but differed in their fatty acid composition. The fast moving band had 77% of the total fatty acids as C_20:0 or longer chain while the slow moving band had only 22% of the long chain fatty acids. Normal gray matter GM₃ had one major band containing 82% of and only 17% of the fatty acids as C_20:0 or higher. It is suggested that in the tumor cells either the specificity of the enzyme cytidine monophosphate-N-acetyl neuraminic acid: ceramide dihexoside sialyltransferase for C_18.0 fatty acid containing glycolipid was altered or that the compartmentation of precursor pools for the simpler glycolipids present in normal tissue did not exist in transformed cells.     

The cooperative role of the transformation-sensitive glycoproteins, GP140 and fibronectin, in cell attachment and spreading

The detergent-insoluble matrix of cultured human fibroblasts contains cytoskeletal and nuclear components, as well as two major, noncollagenous glycoproteins, fibronectin and GP140. These glycoproteins are stabilized by extensive intermolecular disulfide bonding. GP140, in contrast to fibronectin, is resistant to digestion with trypsin and is not cross-reactive with antisera prepared against fibronectin (Carter, W. G., and Hakomori, S. (1981) J. Biol. Chem. 256, 6953-6960). GP140 was partially purified, under nonreducing conditions, by differential extraction of trypsinized cells with sodium trichloroacetate. Alternatively, a higher yield of GP140 could be obtained under reducing conditions by extraction with urea-dithiothreitol followed by molecular sieve chromatography in the presence of sodium dodecyl sulfate and dithiothreitol. The purified GP140 contained mannose, galactose, and N-acetylglucosamine residues, totaling 2.7% of the molecule. In addition, mild periodate oxidation of GP140 followed by reduction with NaB3H4 under conditions designed to label sialic acid also labeled the peptide portion of the molecule. Amino acid analysis of GP140 detected periodate-sensitive hydroxylysine residues, as well as hydroxylproline, accounting for the periodate/NaB3H4-induced label in the peptide. These hydroxylated amino acids are major components of collagens and collagen-like proteins. The GP140 isolated under nonreducing conditions was found to induce stable cell attachment and cell spreading when coated on plastic surfaces. The cell attachment could not be inhibited with affinity purified anti-fibronectin antibodies. However, trypsinization of cells under conditions that removed surface fibronectin reduced the ability of the cells to bind to the GP140-coated surface. Metabolic labeling of cells with radioactive glucosamine during 1-h cell attachment experiments incorporated label into both fibronectin and GP140, as well as four other carbohydrate-containing components as part of a stable detergent-insoluble matrix, indicating that the cells rapidly glycosylate both fibronectin and GP140 and incorporate them into the matrix. Long term labeling and chase experiments indicated that fibronectin and GP140 in the matrix are subject to very slow turnover. Neither fibronectin nor GP140 were detectable in the detergent-insoluble matrix of SV40-transformed human fibroblasts by either metabolic or cell surface labeling. These results are consistent with the conclusion that fibronectin and GP140 may function in a cooperative manner in cell adhesion and spreading.     

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.     

Morphological and chromosomal characterization of three new continuous cell lines of Drosophila melanogaster

Three continuous cell lines (GM₁, GM₂ and GM₃) were obtained from embryos of Drosophila melanogaster. Karyotypic analysis revealed characteristics distinguishing each line. Except for some minor variations GM₁ cells had an X and a centric heterochromatic fragment (which is a portion of the Y). GM₂ line was characterized by XO cells showing two new telocentric chromosomes while an autosome of the II pair was missing. GM₃ cells were XY; the Y chromosome, however, was shorter than the normal, having a deletion of the terminal section of the short arm. Several problems concerning the origin of these different genomes are discussed.This work was supported by a grant of the Consiglio Nazionale delle Ricerche, Roma.     

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.     

Prominent immunogenicity of monosialosyl galactosylgloboside, carrying a stage-specific embryonic antigen-4 (SSEA-4) epitope in the ACHN human renal tubular cell line—a simple method for producing monoclonal antibodies against detergent-insoluble microdomains/raft

The binding of Shiga toxin (Stx) to Gb3Cer in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.     

Studies on the substrate specificity of hexosaminidase A and B from liver

β-N-Acetylhexosaminidase A and B were partially purified from normal human liver using DEAE-cellulose column chromatography. Hexosaminidase B was also purified from the livers of patients who had died of Tay-Sachs disease. The hexosaminidase fractions were tested for their ability to hydrolyze the amino sugar moiety of synthetic substrates and of three amino sugar-containing glycolipids, GA₂, globoside, and GM₂.     

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)     

Density dependent inhibition of cell growth in cultures of primary and established lines of cells

The effect of cell crowding on DNA synthesis (incorporation of ³HTdR and ³²PO₄) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells. DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density. Autoradiography after ³HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells.     

Localization of synthetic glycolipids in the cell and the dynamics of their insertion/loss

Modification of the cell surface with synthetic glycolipids opens up a wide range of possibilities for studying the function of glycolipids. Synthetic glycolipids called Function-Spacer-Lipids (FSL; where F is a glycan or label, S is a spacer, and L is dioleoylphosphatidyl ethanolamine) easily and controllably modify the membrane of a living cells. This current study investigates the dynamics and mechanism of the FSL insertion and release/loss. FSL insert into the cell membrane (~1 million molecules per cell) within tens of minutes, almost regardless of the nature of the cells (including the thickness of their glycocalyx) and the size of the FSL glycan. FSLs do not accumulate uniformly, but instead form patches >300 nm in size either entrapped in the glycocalyx, or integrated in the plane of the plasma membrane, but always outside the cell rafts. The natural release (loss) of FSL from the modified cell was two orders of magnitude slower than attachment/insertion and occurred mainly in the form of released microvesicles with a size of 140 ± 5 nm. The accumulation of FSL as patches in the cell membrane is similar to the coalescence of natural glycosphingolipids and supports (along with their long residence time in the membrane) the use of FSL as probes for the study of glycosphingolipid-protein interactions.     

Glycosphingolipids in membrane architecture

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin-labeled derivatives of 2 families of carbohydrate-bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip-flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca²⁺-induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.     

GLYCOSPHINGOLIPIDS IN FETAL TAY-SACHS DISEASE BRAIN AND LUNG CULTURES

Abstract— A study was undertaken of the glycosphingolipids in cell cultures derived from cerebellum of Tay-Sachs disease fetal brain in order to determine the suitability of such cell strains as a model for Tay-Sachs disease. The glycosphingolipids in the Tay-Sachs disease cultured cerebellar cells were compared with those found in normal cultured cerebellar cells, normal and Tay-Sachs cultured lung cells, and normal and Tay-Sachs fetal brain. The glycolipids were separated by TLC, then analyzed by GLC of the trimethylsilyi derivatives of the methylglycosides of the sugar moieties. In the cultured cerebellar lines, the predominant gangliosides were G_M2, G_M3, and G_D3. There was a 4-fold increase of G_M2 in the Tay-Sachs as compared with the normal line. Only G_M3 and G_D3 gangliosides were found in the Tay-Sachs and the normal fetal lung cell cultures. The major neutral glycosphingolipids in all of the cultured cells which were analyzed were glucosylceramide, lactosylceramide, digalactosyl-glucosylceramide, and globoside. When the Tay-Sachs cerebellar cells were labelled with [1-¹⁴C]gluco-samine, some radioactivity was observed in the trihexosylceramide band, indicating the presence of a small amount of a galactosamine-containing trihexosylceramide which may be asialo-G_M2 (G_A2). The trihexosylceramide in Tay-Sachs fetal brain was identified as G_A2 by GLC. Both Tay-Sachs and normal fetal brain gangliosides were more complex than those found in the cultured cells. Long chain fatty acids (C_24:0 and C_24;1) predominated in all of the glycosphingolipids of the Tay-Sachs and the normal cultured cerebellar cells. In contrast, the glycosphingolipids of Tay-Sachs and normal fetal brain contained mainly the shorter chain fatty acids (C₁₆:₀, C₁₈:₀, and C_18:1). The cerebrosides in both the Tay-Sachs and normal fetal brains were mainly glucosylceramide with only small amounts of the galactosylceramide which predominates in infant brain. Cultured cells from the fetal Tay-Sachs disease     

Phosphatidylglucoside exists as a single molecular species with saturated fatty acyl chains in developing astroglial membranes

We previously found that phosphatidylglucoside (PtdGlc), a novel glycolipid expressed in HL60 cells, plays a role in forming signaling microdomains involved in cellular differentiation. Because cells contain minute levels of PtdGlc, pure PtdGlc is very difficult to isolate. Thus, its complete structure has never been assessed. To aid in analyzing PtdGlc, we generated a PtdGlc-specific monoclonal antibody, DIM21, by immunizing mice with detergent-insoluble membranes isolated from HL60 cells [Yamazaki, Y., et al. (2006) J. Immunol. Methods 311, 106-116]. DIM21 immunostaining of murine CNS tissues revealed stage- and cell type-specific localization of the DIM21 antigen during development, with especially high levels of expression in radial glia/astroglia. DIM21 immunostained cultured hippocampal astroglia in a punctate fashion. To characterize the structure of PtdGlc, we isolated DIM21 antigen from fetal brains. Using successive column chromatography, we purified two previously unrecognized glycolipids, PGX-1 and PGX-2, from embryonic day 21 rat brains. DIM21 reacted more strongly to PGX-2 than to PGX-1. Structural analyses with 600 MHz (1)H NMR, FT-ICR mass spectrometry, and GC revealed that PGX-1 is phosphatidyl beta-d-(6-O-acetyl)glucopyranoside and PGX-2 is phosphatidyl beta-d-glucopyranoside. The yields of PGX-1 and PGX-2 were approximately 250 +/- 150 and 440 +/- 270 nmol/g of dried brains, respectively. Surprisingly, both glycolipids were composed exclusively of C18:0 at the C1 position and C20:0 at the C2 position of the glycerol backbone. This saturated fatty acyl chain composition comprising a single molecular species rarely occurs in known mammalian lipids and provides a molecular basis for why PtdGlc resides in raftlike lipid microdomains.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.