Large Alterations in Ganglioside and Neutral Glycosphingolipid Patterns in Brains from Cases with Infantile Neuronal Ceroid Lipofuscinosis/Polyunsaturated Fatty Acid Lipidosis

Lipid composition was studied on cerebral tissue from nine children who had died of a progressive encephalopathy called the infantile form of neuronal ceroid lipofuscinosis (INCL) or polyunsaturated fatty acid lipidosis (PFAL). In the terminal stage of the disease, the concentrations of all lipid classes were found to be significantly reduced in the cerebral and cerebellar cortex and white matter. The concentration of gangliosides of the cerebral cortex was 15% and that of cerebrosides (galactosylceramide) in white matter 0.2-5% of the normal values for the children's ages. The reduction of gangliosides mainly affected those of the gangliotetraose series, particularly GD1a. The fatty acids of the linolenic acid series were strongly reduced in ethanolamine and serine phosphoglycerides. A very large increase up to 100-fold of oligoglycosphingolipids of the globo series and two fucose-containing lipids of the neolacto series was found in the forebrain of the three advanced cases examined. The brain tissue also contained very high concentrations of mono-, di-, and trisialogangliosides of the lacto and neolacto series, gangliosides with type 1 chain dominating. The structures of the gangliosides were tentatively identified by gas chromatography-mass spectrometry and monoclonal antibodies with carefully determined epitope specificity. The gangliosides and neutral glycosphingolipids had very similar fatty acid composition, consisting of about 40% stearic acid and 40% C24-acids.     

Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro.

Eighteen linker insertion mutants with mutations in the adenovirus precursor to terminal protein (pTP), which were originally constructed and tested in virions by Freimuth and Ginsberg (Proc. Natl. Acad. Sci. USA 83:7816-7820, 1986), were transferred to expression plasmids for assay of the various functions of the isolated pTP. Function was measured by the ability of individual pTP mutant proteins to participate in the initiation of replication from an adenovirus DNA end, by their activity in assays of DNA elongation, and by the intracellular distribution of pTP demonstrated by indirect immunofluorescence. Ten of the 11 mutants that were active in virion formation were also functional in DNA replication reactions in extracts, while 1 had reduced function. Four mutants with mutations that were lethal to virus production were also inactive in DNA replication reactions. These four mutations are probably located at sites required for the function of pTP in DNA synthesis. Three pTP mutants with mutations that were lethal or partially defective with respect to virion formation were active in reactions requiring pTP for initiation and elongation in extracts. All three of these mutant pTPs targeted normally to the nucleus, suggesting a defect after this step in replication. Since pTP has been reported to bind the nuclear matrix, these pTP mutants may have mutations that define sites necessary for binding to this structure. Several mutants with mutations that lie outside the putative nuclear targeting region were aberrantly localized, suggesting either that additional domains are important in nuclear localization or that there are alterations in protein structure that affect nuclear transport for some pTP mutants.     

Binding specificity of monoclonal antibodies to ganglioside, Fuc-GM1

The binding specificity of thirteen mouse monoclonal antibodies reacting with Fuc-GM1, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)-Gal beta 1-4Glc beta 1-1Cer, a ganglioside found to be associated with small cell lung carcinoma (O. Nilsson et al. (1984) Glycoconjugate J. 1, 43-49) was studied. The results are based upon radioimmunodetection of their binding to structurally related glycolipids adsorbed to microtiter plates or chromatographed on thin-layer plates. Four of thirteen antibodies reacted only with Fuc-GM1 and both the fucose and the sialic residues were necessary for binding. Optimal binding was obtained when the sialic acid was N-acetylneuraminic acid. When this sialic acid residue was substituted with N-glycoloylneuraminic acid the binding activity was reduced and up to 10-times more Fuc-GM1 was needed for detection. The ceramide composition did not influence the binding. The other nine monoclonal antibodies cross-reacted with glycolipids containing structures closely related to Fuc-GM1 and differed from the specific ones by recognizing a smaller portion of the carbohydrate moiety in Fuc-GM1. These results indicate that anticarbohydrate monoclonal antibodies, recognizing structures involving a large proportion of the sugar in the glycolipid, possess a high specificity and might be useful for detection of tumor-associated ganglioside antigen.     

Expression of the Ganglioside GD3 in Human Meningiomas Is Associated with Monosomy of Chromosome 22

The ganglioside composition of 59 meningiomas has been compared with a molecular genetic analysis of chromosome 22 in the same specimens. Major gangliosides were GM3 (II3NeuAc-LacCer) and/or GD3 [II3(NeuAc)2-LacCer]. In specimens with no or partial deletions of chromosome 22, the GM3 ganglioside predominated, and the mean value for GM3, 61% of total sialic acid, was around four times higher than that for GD3. A loss of chromosome 22, found in 56% of the specimens, was shown to be associated with an increase in the proportion of ganglioside GD3, with the ratio between mean values of GM3 and GD3 being approximately 1:1. Logistic regression revealed that the probability of predicting monosomy of chromosome 22 by the GD3 proportion was 66%.     

A sulfated glucosylceramide from rat kidney

A novel sulfated glycosphingolipid containing a sulfated glucosyl residue was isolated from rat kidney and purified to homogeneity by column chromatographies with DEAE-Sephadex and silica beads. By compositional analyses, permethylation studies, one- and two-dimensional proton magnetic resonance spectroscopy, infrared spectroscopy, negative secondary ion mass spectrometry, solvolysis, and immunostaining on thin layer chromatogram, the structure of this glycolipid was proposed to be HSO3-3Glc beta 1-1Cer (where Cer is ceramide). The ceramide portion consisted of 4-D-hydroxysphinganine as the sole long chain base, and the fatty acid consisted of predominantly tetracosanoic acid, deduced from both composition analysis and negative secondary ion mass spectrometry. The yield of glucosyl sulfatide was about 5 nmol/g of tissue, being about three times as much as that of lactosylceramide sulfate.     

Application of a panel of antiganglioside monoclonal antibodies to cytologic specimens.

The cytologic evaluation of poorly differentiated tumors frequently poses a diagnostic dilemma as to the tissue of origin. To assess the diagnostic utility of monoclonal antibodies (MAbs) in these situations, we applied a panel of three highly purified MAbs specific for tumor-associated ganglioside epitopes to a diverse series of cytologic specimens. The panel was composed of DMAb-3, reactive with the epitope GalNAc beta 1-4 (NeuAc alpha 2-3)Gal- of GM2; DMAb-7, reactive with the epitope (NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4(Glc or GlcNAc)- of GD3 and 3'8'-LD1; and DMAb-20, reactive with the epitope GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal- of GD2. The cytologic material consisted of air-dried Cytospin preparations prepared predominantly from fine needle aspirates and stained with the ABC immunohistochemical method. Positive reactivity was recognized when greater than 5% of tumor cells stained with the antibody; lesser reactivity was called negative. DMAb-3 stained 9/14 (64%) glial tumors, 4/13 (31%) nonglial central nervous system tumors, 1/21 (5%) melanomas, 7/38 (18%) non-small cell carcinomas (NSCC), 1/15 (7%) small cell carcinomas (SCC), 0/9 (0%) lymphomas/leukemias, 2/10 (20%) sarcomas, 1/7 (14%) miscellaneous tumors and 2/2 (100%) reactive fluids. DMAb-7 recognized 14/14 (100%) glial tumors, 9/13 (69%) non-glial central nervous system tumors, 19/22 (86%) melanomas, 19/43 (44%) NSCC, 5/15 (33%) SCC, 2/9 (22%) lymphomas/leukemias, 6/10 (60%) sarcomas, 1/7 (14%) miscellaneous tumors and 4/4 (100%) reactive fluids. DMAb-20 stained 6/14 (43%) glial tumors, 2/13 (15%) nonglial central nervous system tumors, 1/21 (5%) melanomas, 4/38 (10%) NSCC, 0/15 (0%) SCC, 0/9 (0%) lymphomas/leukemias, 1/10 (10%) sarcomas, 1/7 (14%) miscellaneous tumors and 1/3 (33%) reactive fluids. The GD3-reactive DMAb-7 recognized a large portion of many tumor types and thus is not diagnostically useful alone. DMAb-3 and DMAb-20 were more selective and showed the strongest reactivity for glial tumors and minimal reactivity for melanomas, small cell carcinomas, and lymphomas or leukemias. DMAb-3 and DMAb-20 may be useful as components of a larger panel of MAbs in distinguishing between poorly differentiated tumors in samples derived from the central nervous system.     

Gangliosides in the human glioma cell line U-118 MG grown in culture or as xenografts in nude rats

This study was undertaken to characterize gangliosides in the human glioma cell line U-118 MG. The cell line was grown both in cell culture and as xenografts in nude rats. A common finding in both culture and xenograft cells was the high proportion of the lactoseries ganglioside 3'-LM1, approximately one third of the total ganglioside sialic acid. Otherwise, there were marked differences between the two cell sources. The cells grown in culture had a more simple ganglioside pattern than those grown in xenografts. In the latter instance, more complex gangliosides of the lactoseries, including 3'8'-LD1, sialyllactonorhexaosylceramide and a branched structure with two terminal NeuAc alpha 2-3Gal beta 1- 4GlcNAc chains, and the gangliotetraose series were found. Another marked difference involved GM2, which in the cultured cells was a major fraction, indicating that the synthesis of the gangliotetraose series gangliosides in the former stopped at the level of GM2. These results show that the ganglioside composition of a glioma cell line is strongly influenced by environmental factors.     

Lipid accumulation in liver, spleen, lungs and kidneys of miniature-pigs after chloroquine treatment.

Chronic chloroquine treatment of type-Göttingen miniature-pigs induced lipid accumulation in the liver, spleen, lungs and kidneys. The lipid analyses showed marked quantitative and qualitative differences between the organs. In the liver the lipids affected most were cholesteryl esters and glucosylceramides, which were increased at the most 20 times. Cholesterol and ganglioside concentrations were also increased, though less markedly. The concentration of acidic phospholipids was slightly increased but that of the neutral phospholipids was unaffected. There was a considerable inter-individual variation in the lipid changes. Spleen and lung showed significant increases of all the major lipids. Glucosylceramide was increased more than the other lipids, namely 6-fold in the spleen and 10-fold in the lung. The concentration of acidic phospholipids as well as that of gangliosides was increased by 50% in the spleen and by 100% in the lung. The organ affected least was the kidney, in which only the glycolipids, both acidic and neutral, were significantly increased. Common to all the organs of the chloroquine-treated pigs was the large increase of glucosylceramide, ganglioside CM2 and bis(monoacylglyceryl)phosphate. The ganglioside increase affected all the individual gangliosides and, except for the increased proportion of ganglioside GM2, there were not remarkable changes in the ganglioside pattern in any of the organs.     

Cobalt mapping of the nervous system: evidence that cobalt can cross a neuronal membrane

Exposing intact nerves of Aplysia to exogenous CoCl2 was sufficient to fill neurons by axonal iontophoresis. There were no significant differences between filling intact nerves and nerves whose ends had been cut. Nonneuronal elements were not filled appreciably. These results show that Co2+ can cross intact axonal membranes and suggest that in tracing neuronal pathways Co2+ may not remain confined to neurons whose interiors are directly exposed to it.     

Expression of the Gm1-Species, [NeuN]-GM1, in a Case of Human Glioma

Altered glycosylation is a common feature in tumors of various kind and particular interest has been focused on the expression of tumor-associated gangliosides. We have previously identified some human glioma-associated gangliosides and in this study yet another, not previously described, ganglioside has been isolated. The ganglioside was prepared from human glioma tissue taken at autopsy. The new ganglioside bound cholera-toxin B-subunit and its structure was confirmed by fast atom bombardment—mass spectrometry to be NeuN-GM1 (II³NeuNH₂-GgOse₄Cer). In the dissected tumor specimen, the concentration of NeuN-GM1 was 0.1 mol/g wet weight and accounted for approximately 20% of the monosialoganglioside fraction. Normal human brain tissue specimens (n = 10) did not contain detectable (>0.5 nmol/g wet weight of tissue) amounts of NeuN-GM1, indicating that this ganglioside might be associated with human glioma. However, none of the 17 other tumour specimens reveal any detectable amounts of this ganglioside. In conclusion, NeuN GM1 is a glioma-associated ganglioside but its exceptional expression limits its relevance as a molecule involved in general tumor biology.