OLIGOSACCHARIDE STORAGE IN BRAINS FROM PATIENTS WITH FUCOSIDOSIS,GM1-GANGLIOSIDOSIS AND GM2-GANGLIOSIDOSIS (SANDHOFF'S DISEASE)1

Abstract— Analysis of whole autopsy brain from a patient with fucosidosis (α-fucosidase deficiency) revealed minor storage of H-antigen glycolipid [Fuc (α, 1→2) Gal-GlcNAc-Gal-Glc-Ceramide] and a slightly abnormal ganglioside composition in the form of a two-fold elevation of G_M1 and the presence of a fucose-containing glycolipid (a minor component) which co-migrated with G_D1a. The major storage materials in fucosidosis brain were an oligosaccharide (Fuc-Gal-GlcNAc-Man[Fuc-Gal-GlcNAc-Man]-ManGlcNAc) and a disaccharide [Fuc(α, 1→6)-GlcNAc] in the approximate ratio of 5:1. Lesser amounts of a related oligosaccharide (Gal-GlcNAc-Man[Gal-GlcNAc-Man]-Man-GlcNAc) were isolated from the brain of patients with G_M1-gangliosidosis (Types I and II) where the major storage material is known to be G_M1-ganglioside (Gal (β, 1→3)GalNAc(β, 1→4) [NeuNAcf(α, 2→3) Gal(β, 1→4)Glc-Ceramide). Similarly, a related oligosaccharide (GlcNAc-Man [GlcNAc-Man]-Man-GlcNAc) was isolated from the brain of a patient with a total deficiency of N-acetyl-β-d -hexosaminidase (Sandhoff variant of G_M2-gangliosidosis) where the major storage products are known to be G_M2-ganglioside (GalNAc (β 1→4) [NeuNAc (α, 2→3)Gal(β, 1→4)Glc-Ceramine) and its asialo derivative. These studies indicate that glycoproteins containing at least 2 mol of l -fucose per oligosaccharide unit are normally catabolized in human brain. Further, it appears that such glycoproteins are initially catabolized by an endo-N-acetylglucosaminidase to release an oligosaccharide which is then degraded by the sequential action of exo-glycosidases.     

Programmed cell death in neurotumour cells involves the generation of ceramide

Ceramide has been typically thought of as the membrane anchor for the carbohydrate in glycosphingolipids but many studies have suggested that it may cause apoptosis. Apoptosis or programmed cell death (PCD) is thought to be responsible for the death of one-half of neurons surviving the development of the nervous system. The potential involvement of the sphingomyelin-ceramide signaling process as an integral part of PCD was therefore examined in several neurotumour cell lines. We show that synthetic C2-ceramide (N-acetylsphingosine), a soluble ceramide analogue, can rapidly trigger PCD in these cells, characterized by: 1) classic DNA laddering on agarose gels; 2) DNA fragmentation as determined by Hoechst Dye; and 3) cell viability (mitochondrial function and intact nuclei) assays. We report that staurosporine can both activate PCD (by all three criteria above) in neurotumour cells and increase both the formation of ceramide and ceramide mass. Both ceramide formation and the induction of PCD were further enhanced by the co-addition of a ceramidase inhibitor oleoylethanolamine (25 µM). Staurosporine and oleoylethanolamine were similarly effective in inducing ceramide formation and PCD in immortalized hippocampal neurons (HN-2) and immortalized dorsal root ganglion cells (F-11). Our data suggests that formation of ceramide is a key event in the induction of PCD in neuronally derived neurotumour cells.Abbreviations PCD programmed cell death - PKC protein kinase C - HPTLC high-performance thin-layer chromatography - DETAPAC diethylenetriaminepentaacetic acid - DMEM Dubelco's modified Eagle's medium - FCS fetal calf serum - PBS phosphate-buffered saline - DAG diacylglycerol - DDI distilled-deionized - Cer ceramide - SM sphingomyelin Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Novel structural components of the ventral disc and lateral crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment.     

Procedures for manufacturing double-barrelled ion-sensitive microelectrodes employing liquid sensors

Liquid ion-sensitive/selective sensors are available for most inorganic ions of physiological and biochemical importance. In order to measure intracellular ionic activities in relatively small cells, it is advisable to manufacture and use double-barrelled microelectrodes. Procedures for making two types of double-barrelled ion-sensitive microelectrode are described in detail. Such microelectrodes have been used successfully to measure intracellular K+, Cl- and Na+ activities in retinal horizontal cells of fish and body-wall muscles of insect larvae.     

LIPID AND PROTEIN CHANGES IN SCIATIC NERVE DURING WALLERIAN DEGENERATION

Lipid and protein changes have been measured and correlated during early Wallerian degeneration in the same rat sciatic nerve. The major structural glycoprotein disappears at a steady rate and little remains after 8 days. There is a concomitant appearance of a protein with the same molecular size as basic protein B₂ and which is probably formed from the glycoprotein. Basic protein (B₁) is slowly lost, but Wolfgram protein tends to increase possibly because of glial cell proliferation. Cholesterol ester has appeared 3 days after sectioning, while cholesterol and probably cerebroside are reduced. Triglyceride levels show considerable variation, but biphasic increases tended to occur at 3-4 and 10-14 days. Loss of phospholipids is a later event. The changes in lipid and protein are discussed in relation to the stability of the peripheral nerve myelin membrane.     

Synthesis of myelin glycosphingolipids (galactosylceramide and galactosyl(3-O-sulfate)ceramide (sulfatide)) by cloned cell lines derived from mouse neurotumors

Clonal cell lines derived from both spontaneous and chemically induced rat and mouse brain tumors were screened for their ability to incorporate H232SO4 into galactosyl(3-O-sulfate)ceramide (sulfatide). High levels of 35SO4 incorporation into sulfatide were found only in two of the mouse cell lines studied (G26-20 and -24). Tumors produced by subcutaneous injection of these cell lines into C57BL/6 mice were also unique in that they contained high levels of both sulfatide and galactosylceramide. The synthesis of large amounts of sulfatide and galactosylceramide by a clonal cell line of neurological origin suggests that the original tumor was of oligodendrocyte or Schwann cell origin. In common with a large number of mouse and rat astrocyte cell strains and their derived tumors, these glial cells lacked the ability to synthesize gangliosides such as monosialotetraglycosylceramide and disialotetraglycosylceramide (as judged by analytical and [3H]GlcNH2 incorporation studies). This appears to be a unique characteristic of neuroblastoma-derived cell strains such as N18, NB2a, and NB41A.