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Labeling of animal cells with fluorescent dansyl cerebroside.

A dansyl (diaminoaphthalenesulfonyl)-derivative of cerebroside was prepared which could be effectively incorporated into the plasma membranes of tissue culture cells and erythrocytes. The cells which had assimilated the glycolipid fluoreced intensely and could be observed under a fluorescent microscope. Cells were initially labeled rather homogeneously over the whole surface. With longer incubation time organization of the fluorescent glycolipid took place and patches of the lipid in the membrane were formed. The redistribution and organization of the membrane lipid could be demonstrated most clearly when cells labeled with this fluorescent glycolipid were infected with myxoviruses. After infection of MDBK and BHK cells with fowl plaque virus areas of dense fluorescence appeared at margines of neighboring cells. When BHK cells were infected with Newcastle disease virus fusion of the cells was accompanied by complete redistribution of the glycolipid. Erythrocytes could also easily incorporate dansyl cerebroside. Chicken erythrocytes which contain cytoplsmic and nuclear membranes incorporated the fluorescent glycolipid in both membranes.

Animals↗

Elevated cerebroside antibody levels in human visceral and cutaneous leishmaniasis, Trypanosoma rangeli infection, and chronic Chagas' disease.

A natural cerebroside (antiC) IgM antibody was found at relatively high levels in the serum of every healthy individual studied. The reactivity of the antibody was assessed by using highly purified bovine brain galactocerebroside (galC) or human glucocerebroside (gluC) as antigen. The importance of fatty acid moiety of galC in antigen-antibody reaction was demonstrated by low immunoreactivity using 1-beta-D-galactosyl sphingosine (GS) as antigen and by the absence of absorption to GS-bearing liposomes. The presence of alpha-hydroxy and non-hydroxy fatty acids in galC did not modify its immunoreactivity. Cerebroside antibody binding activity was only partially blocked by 0.5 M galactose or alpha- and beta-methylgalactopyranoside, suggesting poor specificity of antiC for a specific glycosidic residue or linkage. In fact, liposome-bearing gluC absorbed galC. AntiC did not adsorb on rabbit, guinea pig, or human erythrocytes (RBC), but absorbed strongly on rat RBC. Elevated antibody levels were found in 57% of Kala azar patients, 56% of Trypanosoma rangeli-infected patients, 30% of chronic chagasic patients, and 20% of cutaneous leishmaniasis patients, but were not found in 16 other inflammatory or infectious diseases studied. This suggests an association between Kinetoplastida infection and elevated levels of antiC, with parasitic galC acting probably as a highly immunogenic antigen. A possible role of anti-galC in the neuropathological symptoms of Chagas' disease and in the control of parasitemia levels in T. rangeli-infected individuals is discussed.

Animals↗

Isolation and structure determination of Pachybasium cerebrosides which potentiate the antifungal activity of aculeacin.

A set of four cerebrosides was isolated from a Pachybasium species and purified by preparative reversed-phase HPLC. All four products displayed activity in a natural product screen aimed at detecting novel cell wall-active antifungal agents based on synergy with the known glucan synthetase inhibitor, aculeacin. Based on degradation studies, fast atom bombardment mass spectrometry and 13C and high field 1H NMR techniques, the structure of the major cerebroside was determined to be (4E,8E)-N-D-2'-hydroxy-(E)-3'- hexadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4,8-sphingadiene. The other components were found to be the corresponding 2'-hydroxypalmitic acid analog with one less double bond and an analogous pair containing 2'-hydroxystearic acid with and without the 3' double bond.

Antifungal Agents↗

Structure of cerebroside in Aspergillus oryzae.

Structural studies on cerebroside isolated from Aspergillus oryzae were carried out using mainly gas-liquid chromatography-mass spectrometry. The major component fatty acids were 2-hydroxystearic and 2-hydroxy-trans-octadecenoic acid; branched 17?-methyl nonadecasphingadienine isomers were the predominant long-chain bases. The component sugar was only glucose. The structure of the major cerebrosides was assumed to be N-2'-hydroxystearoyl-1-O-glucosyl-17?-methyl sphingadienine and N-2'-hydroxyoctadecenoyl-1-O-glucosyl-17?-methyl sphingadienine.

Aspergillus↗

[Intensity of incorporation of 2-C14-acetate into the cerebrosides and gangliosides of the brain and spinal cord of rabbits in experimental allergic encephalomyelitis].

In rabbits the acute form of experimental allergic encephalomyelitis was caused by inoculation of emulsion of homologous myelin from spinal cord with Freund's adjuvant. In terminal paralytic period of the disease the animals were subcutaneously administered with 2-14C-acetate and a dose 50 micronCi per 100 g of body weight 2 hrs before death. From lumbar and stem sections of the central nervous system purified fractions of cerebrosides and gangliosides were isolated and their specific radioactivity was determined. The intensity of the cerebrosides synthesis was found to be distinctly decreased not only in the more impaired lumbar region but also in the stem section of the central nervous system, where the centers of demyelinization were not observed. The intensity of the gangliosides synthesis was also markedly decreased, in spite of that their content was not altered in the central nervous system under experimental allergic encephalomyelitis.

Acetates↗

Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii.

Major neutral glycosphingolipid components were extracted from Sporothrix schenckii, a dimorphic fungus exhibiting a hyphal saprophytic phase and a yeast parasitic phase responsible for chronic mycotic infections in mammalian hosts. These components, one from the mycelial form and two from the yeast form, were purified and their structures were elucidated by (1)H nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and tandem ESI-MS/MS. All three were characterized as cerebrosides (monohexosylceramides) containing (4E, 8E)-9-methyl-4,8-sphingadienine as the long-chain base attached to N-2'-hydroxyoctadecanoate and N-2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty acyl components. However, while the mycelial form expressed only beta-glucopyranosylceramide, the yeast form expressed both beta-gluco- and beta-galactopyranosylceramides in approximately equal amounts. In addition, while the glucosylceramides of both mycelial and yeast forms had similar proportions of saturated and (E)-Delta(3) unsaturated 2-hydroxy fatty acid, the galactocerebroside of the yeast form had significantly higher levels of (E)-Delta(3) unsaturation. The differences in cerebroside hexose structure represent a novel type of glycosphingolipid dimorphism not previously reported in fungi. Possible implications of these findings with respect to regulation of morphological transitions in S. schenckii and other dimorphic fungi are discussed.

Animals↗

[The inhibition of complement-dependent hemolysis of liposomes containing cerebroside sulfate].

Cerebroside sulfate (CGS) was found to be capable of inhibiting complement-dependent hemolysis. The activity dependence of CGS-containing liposomes on their composition was studied. Mixtures of CGS with phosphatidylethanolamine, phosphatidylserine, sphingomyelin from cattle brain, cerebroside from cattle spinal cord (CG), and egg yolk phosphatidylcholine (ePC) were investigated. In the case of binary CGS/ePC mixtures, the antihemolytic activity varied nonlinearly with an increase in the mass part of CGS: it sharply increased with an increase in the CGS part from 0.3 to 0.5 and decreased by 20-30% of the maximum value with an increase in the CGS part from 0.9 to 1. On the basis of these experiments, the optimum distance between the charged groups of CGS was estimated to be 0.92-1.6 nm. In the ternary compositions of 4:3:3 CGS/ePC/polar lipid, only CG increased the activity of liposomes as compared to that of liposomes from the 4:6 CGS/ePC. The preliminary incubation of CGS-containing liposomes with complement decreased hemolysis more effectively than incubation with other components of the hemolytic system. This suggests that the interaction of CGS-containing liposomes with the complement proteins is responsible for their antihemolytic activity.

Animals↗

Specific tritium labeling of cerebrosides at the 3-positions of erythro-sphingosine and threo-sphingosine.

Cerebrosides containing either threo- or erythro-[3-3-H]sphingosine were synthesized by a new procedure. Glucopyranosyl or galactopyranosyl ceramides were converted to their 3-keto derivatives with 2,3-dichloro-5,6-dicyanobenzoquinone and reduced with 3-H-labeled sodium borohydride. The resulting tritiated cerebrosides, which contained erythro- and threo-sphingosines in the ratio of 84:16, were deacylated with butanol-KOH, and the erythro- and threo-psychosines were separated by silica gel column chromatography and reacylated with lignoceroyl chloride.

Animals↗

[Cerebrospinal fluid phospholipids and cerebrosides in several demyelinating diseases].

The author reports of the achieved results in a quantitative study of general lipids, phospholipids and cerebrosides in the CSF of 37 patients with demyelinating diseases, of 11 patients with vascular brain pathology and 7 with Van Bogart's panencephalitis. The control group consisted of 14 patients without focal lesions of the nervous system, with normal CSF indices. In demyelinating diseases there was a significant increase in the content of kephalines and cerebrosides. In Van Bogart's panencephalitis there was a much higher increase of kephalines, general lipids and phospholipids. In vascular brain disorders there was a moderate increase of all lipids. The possible pathochemical mechanisms of the depicted changes in the content of the lipids in the CSF are discussed.

Adolescent↗

Competitive binding of dynorphin-(1-13) and beta-endorphin to cerebroside sulfate in solution.

Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).

Binding, Competitive↗

Alterations of the cerebroside fraction of human thyroid glycosphingolipids in Graves' disease.

The influence of Graves' disease in human thyroid neutral glycosphingolipids was investigated. The major alteration was in the cerebroside fraction. Although the total amount of cerebroside was not different, the relative proportion of the bands of glucosylceramide separated on borated thin-layer plates was greatly modified in the disease. The band of glucosylceramide containing phytosphingosine and hydroxylated fatty acids decreased strongly, whereas the band with C18 sphingosine and normal fatty acids increased simultaneously. No change was observed in the content of galabiosylceramide. A slight elevation was seen in the amount of globoside at the expense of globotriaosylceramide.

Cerebrosides↗

Carbon 13 NMR spectroscopy of a cerebroside. Proof of the beta-pyranosyl structure of D-glucosylceramide.

The first 13C nuclear magnetic resonance spectrum of a cerebroside is reported. All signals of D-glucosylceramide (I), the cerebroside from human Gaucher's diseased liver, have been assigned by comparison of their chemical shifts with those of model compounds. This data has been used to demonstrate for the first time directly: (a) that the glucosyl moiety must exist in the pyranoside ring form and have a beta anomeric linkage, and (b) that the fatty acyl composition of the aglycon is: alpha-hydroxy, 0%; saturated, 86 +/- 2%; unsaturated long chain, 14 +/- 2%; and unsaturated short chain, less than or equal to 2%. The use of 13C signal shifts in the delta 80 to 120 region (anomeric window) for the assignment of glycon anomeric and ring forms demonstrated herein should be applicable to other glycoconjugates.

Cerebrosides↗

Composition of human cerebrospinal fluid cerebroside.

A technique was developed to isolate sufficient material for compositional analysis of cerebroside from pooled human cerebrospinal fluid. The carbohydrate moiety was principally galactose. The sphingosine base and fatty acid compositions were found to be similar to that of brain cerebroside. The presence of a contaminant in commercial silica gel which chromatographed like the trimethylsilyl derivative of glucose is described.

Amino Alcohols↗

[Changes in the terminal oxidation and active transport of calcium ions in the cerebral mitochondria of white rats following chronic cerebroside administration].

Terminal oxidation and active Ca ion transport have been studied in rat brain mitochondria following intraperitonial administrations of cerebrosides for 30 days. The results obtained have shown that cerebrosides inhibit terminal respiration at its 3rd metabolic state and reduce the rate of phosphorylation. ADP/O and ATP/t coefficients decrease due to the active transport of Ca ions. On increasing ADP and CaCl2 the time necessary for the binding and release of hydrogen ions is prolonged.

Animals↗

Isolation and identification of cerebrosides from the marine sponge Chondrilla nucula.

A cerebroside mixture has been isolated from the marine sponge Chondrilla nucula. Acid-catalyzed methanolysis of this cerebroside mixture afforded methyl glucosides, three long-chain bases, and a mixture of alpha-hydroxy fatty acid methyl esters. The bases were identified as saturated C(17), C(18), and C(19) trihydroxy bases (1,3,4-trihydroxy-2-aminoalkanes) by gas-liquid chromatographic-mass spectrometric analysis of their corresponding trimethylsilyl derivatives. The methyl ester fraction consisted of a mixture of homologous C(16) to C(26) saturated straight-chain alpha-hydroxy esters plus a trace of saturated C(25) iso alpha-hydroxy ester.

Animals↗

Cerebroside galactosidase of brain.

The galactoside bond in cerebroside was found to be cleaved by an enzyme in rat and pig brain. Emulsified stearoyl-(14)C psychosine was used as the substrate and the extent of cleavage was studied by isolating and counting the stearoyl sphingosine (ceramide) formed. The reaction products, ceramide and galactose, were characterized by column and thin-layer chromatography. Cerebroside containing galactose-(3)H was also used to show liberation of galactose. Cholic acid was found to be required for activation of the enzyme, which has a pH optimum of 4.5. Similar cerebrosidase activity was found in spleen, kidney, and lung of rat; liver and heart showed very slight activity. The partially purified enzyme from pig brain also formed ceramide from ceramide lactoside, ceramide glucoside, and cerebronoyl psychosine. The enzyme was active toward o-nitrophenyl galactoside and could be fractionated by Sephadex chromatography into a fraction active toward the nitrophenyl galactoside only and a fraction active toward both this substrate and ceramide galactoside. Human spleen, normal and Gaucher, exhibited cerebrosidase activity.

Animals↗

Some electrical properties of cerebroside and lecithin bilayer membranes.

Electrical potentials of cerebroside and lecithin bilayers in the presence of Na+, Ca2+, and Sr2+ were measured. Calculated membrane specific resistances were about 10(7) ohm cm2. Calculated transference numbers for Na+, Ca2+, and Sr2+ were between 0.5 and 0.6, and were higher in both membranes than in free solution. Thus membranes are slightly cation selective. Specific conductances and transference numbers were then used to calculate ionic flux of membranes. Sodium ions had a much greater ionic flux in both membranes than calcium and strontium ions. The results indicate that the electrical properties of cerebroside and lecithin bilayer membranes are compatible with the structures of those membranes.

Calcium↗

[Purification and characterization of cerebroside sulfatase activator].

Cerebroside sulfatase (CSase) activator was isolated from human liver by acetone precipitation, anion-exchange chromatography, gel filtration and polyacrylamide gel electrophoresis. The CSase activator was a heat-stable protein with an isoelectric point of 4.54. Molecular weight (Mr) of the activator was estimated as 22,000 with the gel permeation and about 8,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native activator is a trimer of a subunit with Mr 8,000. The CSase activator formed a complex with an equimolar amount of cerebroside sulfate (CS), when examined by gel permeation experiments. The activator also bound to galactosylceramide and GM2 ganglioside but scarcely to GM1 ganglioside, and activated to some extent beta-N-acetyl-hexosaminidase A and beta-galactosidase, although the CSase activator could be clearly distinguished from the GM1 beta-galactosidase activator so far known. Though the affinity chromatography using glycolipid ligands, the CSase activator did not recognize sulfate group of CS, but appeared to have a relatively broad specificity for lipid-linked hexose.

Amino Acids↗