Tandem Mass Spectrometry Multiplex Analysis of Glucosylceramide and Galactosylceramide Isoforms in Brain Tissues at Different Stages of Parkinson Disease.

Previous studies demonstrated that Parkinson disease (PD) is associated with a decreased activity of the glucocerebrosidase (GCase) enzyme in brain tissues. The objective of this study was to determine if GCase deficiency is associated with the accumulation of its glucosylceramide (GluCer) substrate in PD brain tissues. An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed, optimized, and validated for the multiplex analysis of GluCer isoforms (C18:0, C20:0, C22:0, C24:1, and C24:0) in brain tissue samples. These molecules were chromatographically separated from their isobaric galactosylceramide (GalCer) counterparts using normal phase chromatography. The analysis was performed by tandem mass spectrometry in the multiple reaction monitoring (MRM) acquisition mode. Limits of detection ranging from 0.4 to 1.1 nmol/g brain tissue were established for the different GluCer isoforms analyzed. For the first time, GluCer isoform levels were analyzed in temporal cortex brain tissue samples from 26 PD patients who were divided into three PD disease stages (IIa, III, and IV) according to the Unified Staging System for Lewy Body Disorders. These specimens were compared with brain tissue samples from 12 controls and 6 patients with Incidental Lewy Body Disease. No significant GluCer concentration differences were observed between the 5 sample groups. The GluCer isoform levels were also normalized with their matching GalCer isoforms. The normalized results showed a trend for GluCer levels which increased with PD severity. However, the differences observed between the groups were not significant, owing likely to the high standard deviations measured.

Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae.

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and (1)H-NMR. We identified them as ß-glucosylceramide and ß-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of ß-galactosylceramide in A. oryzae.

MALDI imaging mass spectrometry of lipids by adding lithium salts to the matrix solution.

Mass spectrometry imaging of lipids using MALDI-TOF/TOF mass spectrometers is of growing interest for chemical mapping of organic compounds at the surface of tissue sections. Many efforts have been devoted to the best matrix choice and deposition technique. Nevertheless, the identification of lipid species desorbed from tissue sections remains problematic. It is now well-known that protonated, sodium- and potassium-cationized lipids are detected from biological samples, thus complicating the data analysis. A new sample preparation method is proposed, involving the use of lithium salts in the matrix solution in order to simplify the mass spectra with only lithium-cationized molecules instead of a mixture of various cationized species. Five different lithium salts were tested. Among them, lithium trifluoroacetate and lithium iodide merged the different lipid adducts into one single lithium-cationized species. An optimized sample preparation protocol demonstrated that the lithium trifluoroacetate salt slightly increased desorption of phosphatidylcholines. Mass spectrometry images acquired on rat brain tissue sections by adding lithium trifluoroacetate showed the best results in terms of image contrast. Moreover, more structurally relevant fragments were generated by tandem mass spectrometry when analyzing lithium-cationized species.

Novel myelin penta- and hexa-acetyl-galactosyl-ceramides: structural characterization and immunoreactivity in cerebrospinal fluid.

Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O-acetylation of the 2-hydroxy-fatty acid. The immuno-reactivity in human cerebrospinal fluid (CSF) to these acetylated glycolipids was examined in central nervous system (CNS) infectious disease, noninflammatory disorders, and multiple sclerosis (MS). Screening for lipid binding in MS and other neurological disease groups revealed that the greatest anti-hydrophobic FMC reactivity was observed in the inflammatory CNS diseases (meningitis, meningo-encephalitis, and subacute sclerosing panencephalitis). Some MS patients had increased reactivity with the hydrophobic FMCs and with glycoglycerophospholipid MfGL-II from Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked.

Identification of sixteen new galactocerebrosides from the starfish Protoreaster nodosus.

Twenty-one galactocerebrosides (1-21), including sixteen new compounds (3-7, 9-17, 19, 21), were identified from a cerebroside molecular species obtained from the chloroform/methanol extract of pyloric caeca dissected from the starfish Protoreaster nodosus. The structures of these galactocerebrosides were determined on the basis of chemical and spectroscopic evidences. Especially, one-pot GC-MS analysis following methanolysis and periodate oxidation of these galactocerebrosides gave efficient structural information of ceramide moiety rapidly in minute amounts.

Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase.

We report here a method of simultaneously quantifying glucosylceramide (GlcCer) and galactosylceramide (GalCer) by normal-phase HPLC using O-phtalaldehyde derivatives. Treatment with sphingolipid ceramide N-deacylase which converts the cerebrosides in the sample to their lyso-forms was followed by the quantitative labeling of free NH(2) groups of the lyso-cerebrosides with O-phtalaldehyde. Using this method, 14.1 pmol of GlcCer and 10.4 pmol of GalCer, and 108.1 pmol of GlcCer and 191.1 pmol of GalCer were detected in zebrafish embryos and RPMI 1864 cells, respectively, while 22.2 pmol of GlcCer but no GalCer was detected in CHOP cells using cell lysate containing 100 microg of protein. Linearity for the determination of each cerebroside was observed from 50 to 400 microg of protein under the conditions used, which corresponds to approximately 10(3) to 10(5) RPMI cells and 5 to 80 zebrafish embryos. The present method clearly revealed that the treatment of RPMI cells with a GlcCer synthase inhibitor P4 resulted in a marked decrease in GlcCer but not GalCer, concomitantly with a significant decrease in the GlcCer synthase activity. On the other hand, GlcCer but not GalCer increased 2-fold when an acid glucocerebrosidase inhibitor CBE was injected into zebrafish embryos. Interestingly, the treatment of CHOP cells with ciclosporin A increased GlcCer possibly due to the inhibition of LacCer synthase. A significant increase in levels of GlcCer in fibroblasts from patients with Gaucher disease was clearly shown by the method. The proposed method is useful for the determination of GlcCer and GalCer levels in various biological samples.

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte.

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.

Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells.

During animal development, cells become progressively more restricted in the cell types to which they can give rise. In the central nervous system (CNS), for example, multipotential stem cells produce various kinds of specified precursors that divide a limited number of times before they terminally differentiate into either neurons or glial cells. We show here that certain extracellular signals can induce oligodendrocyte precursor cells to revert to multipotential neural stem cells, which can self-renew and give rise to neurons and astrocytes, as well as to oligodendrocytes. Thus, these precursor cells have greater developmental potential than previously thought.

Guillain-Barré syndrome and optic neuritis after Mycoplasma pneumoniae infection.

We report the case of a 12-year-old girl who developed Guillain-Barré syndrome (GBS) and optic neuritis (ON) following Mycoplasma pneumoniae infection. Her symptoms, including bilateral vision impairment and tingling in her hands and right foot, were resolved after methylprednisolone pulse therapy. Serum anti-galactocerebroside (Gal-C) IgM antibodies were detected in our patient. This is the first report of a child with GBS and ON associated with M. pneumoniae infection.

Expression of HIV receptors, alternate receptors and co-receptors on tonsillar epithelium: implications for HIV binding and primary oral infection.

BACKGROUND: Primary HIV infection can develop from exposure to HIV in the oral cavity. In previous studies, we have documented rapid and extensive binding of HIV virions in seminal plasma to intact mucosal surfaces of the palatine tonsil and also found that virions readily penetrated beneath the tissue surfaces. As one approach to understand the molecular interactions that support HIV virion binding to human mucosal surfaces, we have examined the distribution of the primary HIV receptor CD4, the alternate HIV receptors heparan sulfate proteoglycan (HS) and galactosyl ceramide (GalCer) and the co-receptors CXCR4 and CCR5 in palatine tonsil. RESULTS: Only HS was widely expressed on the surface of stratified squamous epithelium. In contrast, HS, GalCer, CXCR4 and CCR5 were all expressed on the reticulated epithelium lining the tonsillar crypts. We have observed extensive variability, both across tissue sections from any tonsil and between tonsils, in the distribution of epithelial cells expressing either CXCR4 or CCR5 in the basal and suprabasal layers of stratified epithelium. The general expression patterns of CXCR4, CCR5 and HS were similar in palatine tonsil from children and adults (age range 3-20). We have also noted the presence of small clusters of lymphocytes, including CD4+ T cells within stratified epithelium and located precisely at the mucosal surfaces. CD4+ T cells in these locations would be immediately accessible to HIV virions. CONCLUSION: In total, the likelihood of oral HIV transmission will be determined by macro and micro tissue architecture, cell surface expression patterns of key molecules that may bind HIV and the specific properties of the infectious inoculum.

The properties of brain galactocerebroside monolayers.

Using a Langmuir film balance we have compared the properties of films of the brain galactocerebrosides at 37 degrees C. There are two types of cerebroside in brain, those with an alpha-hydroxy substituent on the acyl chain (HFA) and those without (NFA). At equivalent pressures the areas of both cerebroside films are significantly less than the areas of films of the brain glycerolipids, the choline and ethanolamine phosphatides. The isotherm of NFA galactocerebrosides has two discontinuities, one at low and one at high film pressure, while the isotherm of HFA galactocerebrosides is a smooth curve at all film pressures. Below the high-pressure transition the area of the NFA film is significantly larger than the area of the HFA film. When compressed beyond the high-pressure transition there is a marked hysteresis between compression and expansion isotherms of the NFA galactocerebrosides. The pressures of both films continue to rise steeply when they are compressed into areas which are too small for them to exist as simple monolayers. We conclude that under compression cerebroside films form bilayer structures; that bilayer formation starts at low pressure and occurs progressively as the HFA cerebroside monolayer is compressed, but occurs more abruptly in the NFA cerebroside monolayer at the high-pressure-transition region of the isotherm. A study of pure cerebrosides with a single defined acyl chain shows that there is a correlation between the relative volumes of the hydrophobic and hydrophilic parts of the molecule and the ease of bilayer formation. The larger the relative volume of the hydrophilic group the more readily the cerebroside forms a bilayer film. Other brain lipids added to cerebroside monolayers have sharply differing effects on their areas. The areas of films containing cholesterol are less than the areas calculated by adding the areas of the pure components multiplied by their mole fractions. On the other hand, the area of phosphatidylcholine-containing films is much larger than calculated.

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers.

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).

A rapid procedure for the preparation of oligodendrocyte-enriched cultures from rat spinal cord.

Spinal cords and cerebra from 7-day-old rat pups were compared as tissue sources for the isolation of oligodendrocytes and for studies on the development of these cells in culture. After 1 day in culture the serum-containing medium was replaced by a chemically-defined medium, which contained a cocktail of hormones that stimulated oligodendrocyte development. The cultures were characterized with various immunocytochemical markers; monoclonal A2B5 for bipotential glial progenitor cells, anti-galactocerebroside (GC) serum for oligodendrocytes, and anti-glial fibrillary acidic protein (GFAP) serum for astrocytes. The number of positive cells was counted and expressed as a percentage of total cells. At 1 day in culture the cell cultures from spinal cord contained 30% GC+ cells, increasing to 90% after 7 days in culture. In cultures derived from cerebra the percentage of GC+ cells was always lower than in cultures from spinal cord. In cerebral cultures GFAP+ cells increased from 15% at 1 day in culture to 30% at 7 days in culture, whereas it remained low in spinal cord cultures. The activity of oligodendroglial marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase was followed during development in culture. The specific activity increased rapidly in both types of culture but was more than threefold higher in cultures derived from spinal cord. This procedure yields, within one week and without subculture, primary glial cultures from rat spinal cord, that are highly enriched in oligodendrocytes (greater than or equal to 90%; 3.10(5) oligodendrocytes per rat pup).

Glycosphingolipids of guinea pig gastric mucosa.

Glycosphingolipids have beenn isolated from guinea pig gastric mucosa and their composition and content determined. The neutral glycospingolipids were found to consist of mono-, di-, tri- and pentaglycosylceramide. The acidic glycosphingolipids wee represented by galactosyl and lactosyl sulfatides, and GM4, GM3 and GD3 gangliosides. None of the analyzed glycolipids contained N-acetylglucosamine and fucose.

Gangliosides and neutral glycolipids of human adrenal medulla.

Glycolipids were isolated from human adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The lipid-bound sialic acid was about 234 microgram/g fresh tissue. The glanglioside fraction contained two major gangliosides which accounted for 93% of the total lipid-bound sialic acid. They were identified as GM3, N-acetylneuraminylgalactosylglucosylceramide and GD3, N-acetylneuraminyl N-acetylneuraminylgalactosylglucosylceramide on the basis of cochromatography with authentic standards, sugar composition analysis, and neuraminidase digestion. GM3, N-acetylneuraminylgalactosylglucosylceramide and GD3, N-acetylneuraminyl N-acetylneuraminylgalactosylglucosylceramide occurred in a ratio of approximately 3 : 2, and the ratio seemed to be rather constant irrelevant of age and sex differences. The neutral glycolipid fraction consisted of GL1a, glucosylceramide (18%), GL1b, galactosylceramide (23%), GL2a, lactosylceramide (27%), GL3, digalactosylglucosylceramide (20%), and GL4, globoside (12%). The major fatty acids of all these glycolipids were 16 : 0, 18 : 0, 22 : 0, 24 : 0 and 24 :1.

Divalent cations cooperatively stabilize close membrane contacts in myelin.

Intact nerve myelin compacts to a dehydrated structure of closely apposed membranes when exposed to isotonic solutions at least 10 mM in calcium or tetracaine. The repeat period of the membrane pair in the compacted structure measured by X-ray diffraction is about 126 A in both central and peripheral mammalian nerve myelins whereas the normal periods are about 158 and 178 A, respectively. The electron density profile of compacted myelin shows an asymmetric membrane unit with thickness similar to that of the symmetric bilayer of flocculated myelin lipids. The centrosymmetrically averaged myelin membrane profile is similar to that of the lipid bilayer except at the surface where residual protein is concentrated. Dispersions of extracted total myelin lipids flocculate under similar conditions to those causing myelin compaction, indicating that similar forces act in both processes. Compaction is always accompanied by lateral segregation of intramembrane particles out of the close-packed domains. Lateral displacement of intramembrane proteins form compacted domains can be driven by the attraction of the lipid surfaces for each other. Rates of compaction vary with compacting reagent, concentration, tissue, and temperature, and probably reflect the permeability of the tissue. Extensive compaction by calcium or tetracaine leads to disruption and vesiculation of the spirally wrapped myelin membranes.

Asymmetric extraction of membrane lipids by CHAPS.

We have characterized and quantitated the lipids which are cosolubilized with serotonin 5-HT1A sites from sheep brain using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Dialysis of the CHAPS extract produced a [3H]8-hydroxy(2-di-n-propylamino)tetralin [( 3H]8-OH-DPAT) binding vesicular preparation of the protein. Quantitative analysis of the lipids present in the CHAPS extract by HPTLC and transmittance-densitometry revealed extraction of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidyl serine (PS) and phosphatidic acid (PA) in striking preference over cholesterol, galactosylceramides, sulfatides and sphingomyelin. All lipids present in the clear CHAPS-extract were coeluted with the [3H]8-OH-DPAT binding preparation were separated by centrifugation, 95-100% of the [3H]8-OH-DPAT binding protein was retained in the vesicle-containing pellet. The supernatant contained small amounts of cholesterol, PE and PC, but virtually no PS, PI, or PA, whereas the vesicular pellet contained all the lipids mentioned, indicating that PS, PI and PA are more tightly bound to the vesicles than PE, PC and cholesterol. SDS-PAGE analysis of the pellet revealed two major protein bands, at 58 kDa and 33.5 kDa, respectively. Our report outlines a simple and improved densitometric assay used for the first detailed analysis of lipids cosolubilized with an active, membrane protein, and also, a simple assay for CHAPS.

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth.

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3',4'-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

Distribution and immunoreactivity of glia in the retina of the rabbit.

Glial markers, namely antibodies to glial fibrillary acidic protein (GFAP), vimentin, galactocerebroside (GC), and 08 antigen, were used to study the occurrence and location of neuroglial cells in adult rabbit retinae. Müllerian glia were vimentin-positive, lacked detectable amounts of GFAP, and were found in all parts of the rabbit retina. The neuronal A-type horizontal cells were labeled by vimentin antibody only in the superior retina and at the medullary rays but not in the inferior retina. They lacked GFAP in all regions. Astroglia showing GFAP and vimentin immunoreactivity were absent from most of the superior and inferior retina, being found only in the myelinated area of the ganglion cell axon bundles, the medullary rays. Thus the rabbit retina differs from the retinae of all mammals studied to date by this restriction of astroglia to just one area. The medullary rays, which are known to be myelinated, were labeled by the antibodies to GC and 08 antigen. Boycott and Hopkins ('84) found, using whole-mounted rabbit retinae stained by the reduced silver method of Richardson, that all neurons in the ganglion cell layer of the rabbit retina have a cilium, while cells that have only a diplosome are either neuroglia or microglia. By using this criterion as a basis to differentiate between neurons and glia, the absence of neuroglia from the nerve fiber layer outside the medullary rays was confirmed in the same silver-stained material. Thus, the data obtained from immunocytochemistry and conventional silver staining agree closely. It has been concluded that, at least in the adult, significant lengths of ganglion cell axons extend without astroglial sheaths.

Aging, senile dementia, and the intralaminar microchemistry of cerebral cortex.

We compared the microchemical architecture or right frontal isocortex from patients with senile dementia and age-matched and younger controls. Neuronal connectivity within deep lamina of the cortical column (Brodmann area 9) tended to decline in normal aging and was profoundly depleted in senile dementia. In both aging and senile dementia, there was a significant 20% loss of total cells (neurons and glia) in cortical layers III to VI. In senile dementia, marked diminution of total ganglioside sialic acid per neuron and galactocerebroside per cell in the lower lamina far exceeded alterations associated with aging itself. This structural loss may imply deafferentation of the cortex, owing to loss of projections from subcortical areas such as nucleus basalis. Selective vulnerability of axodendritic arborization of neurons in lower lamina may be correlated to the impaired cognitive functions of senile dementia.