Cholera toxin (CT) has been reported to bind specifically to ganglioside on cell surface plasma membranes and activates adenylate cyclase. We examined whether or not ganglioside (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide) accumulates in plasma membranes of cultured skin fibroblasts from gangliosidosis patients by measuring cyclic AMP (adenosine 3′, 5′-cyclic monophosphate; cAMP) production elicited by CT. Optimal conditions for the assay were determined to be 1μg/ml of CT and 1h incubation at 37°C. The responses of intracellular cAMP level to a direct activator of adenylate cyclase, forskolin, and to phorbol 12-myristate 13-acetate (PMA) were not different between patients and controls. Insertion of exogenous ganglioside into the cell membrane caused a linear increase in the cAMP production triggered by CT in control cells. Under the optimal conditions, intracellular cAMP production in response to CT was estimated as 544.3±16.3pmol/h per mg protein in the patients' cells and 284.8±46.8pmol/h per mg protein in the control ones. These data are the first indication of the accumulation of ganglioside in cell surface membranes of patients with gangliosidosis.

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症例1は2才女児で6ヵ月頃より精神運動発達の遅滞がみられ, 1才6ヵ月ころには退行性変化をきたし, さらに痙攣が出現した. ガルゴイル様顔貌はなく, 肝・脾腫, cherry red spotはみられない. 椎体の変形, 末梢リンパ球の空胞化を認め, 末梢白血球の4-methylumbelliferyl-β-D-galactosidase (β-galactosidase) 活性は対照の2.1%であった.
症例2は症例1に次いで2回目の妊娠であり, 妊娠第17週に羊水穿刺をおこない第24週に中絶した胎児である. β-wgalactosidaseは羊水上清で対照と差がなく, 培養羊水細胞では対照の2.3%と低値であった.
胎児の肝・脳におけるβ-galactosidaseは各々6%, 10%であり, また, -ganglioside-β-galactosidase (-β-galactosidase) は1.1%, 1.3%と著明な低下をみた.
電顕的に大脳基底核部の神経細胞内にmembranous cytoplasmic bodyあるいはzebra body様構造物を認めたが, 生化学的検索で脳のtotal N-acetyl-neuraminic acid (NANA) は対照に比しわずかに増加していたがその分画で, -gangliosideの占める割合は対照と差がみられなかった.

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Rat erythrocytes contained ganglio-series gangliosides, , fucosyl , and GDla, in a high concentration. The concentrations of , fucosyl , and GDIa in rat erythrocyte ghosts were 889.0 nmol, 470.6 nmol, and 462.0 nmol per g dry weight, respectively, and the molar ratio of lipid-bound sialic acid, cholesterol and lipid-bound phosphorus was 3.1:73.9:100.0. The reactions of fucosyl and on rat erythrocytes with rabbit anti-fucosyl and anti- antisera were measured by means of haemolysis in the presence of complement and a binding assay of antibodies with a FACS after staining erythrocytes by the indirect membrane immunofluorescence technique. When measured by ELISA, antifucosyl antiserum was found to react almost exclusively with fucosyl with a slight cross-reaction with , but anti- antiserum cross-reacted to a significant extent with asialo . Rat erythrocytes were haemolyzed specifically with anti-fucosyl antiserum, but not with antisera to , asialo , asialo GM2, Forssman and globoside, and the haemolysis was proved to be definitely caused by the specific recognition of fucosyl on rat erythrocytes by anti-fucosyl antibody according to the haemolysis inhibition reaction using various glycosphingolipid-containing liposomes as inhibitors. In addition, although the binding of anti-fucosyl antibody on rat erythrocytes was clearly demonstrated with a FACS, anti- antibody did not bind. The observations that the haemolysis of rat erythrocytes and the binding of antibody to rat erythrocytes were found only with anti-fucosyl antiserum, and not with anti- antiserum, but that nevertheless the titer of anti- antiserum was higher than that of anti-fucosyl antiserum and on rat erythrocytes was more abundant in concentration than fucosyl , seem to be a matter of great importance in assessing the specificity of anti-ganglioside antibody and the surface distribution of gangliosides on the cell.

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The surface states of ganglioside () / dipalmitoylphosphatidylcholine (DPPC), /dioleoylphosphatidylcholine (DOPC) and /DPPC/DOPC monolayers were investigated using atomic force microscopy (AFM), and the effect of surface pressure on the surface states of the membranes was examined. Specific changes in morphology were observed in the /DPPC and /DPPC/DOPC monolayers when the surface pressure was varied from 30 to 40 mN m^-1, while no significant morphological change was detected in the /DOPC monolayer. was likely to bring about a change in morphology in the liquid-condensed film but not the liquid-expanded film. Nevertheless, the changes to monolayers with surface pressure support the participation of in signal transduction or specific cell recognition. Furthermore, the surface pressure-responsive change in morphology of was affected by the matrix, suggesting that the localized to each organ has a specific role.

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A patient considered to represent a variant of -gangliosidosis type 2 is described. Enzymic differences between the two clinical types 1 and 2 in -gangliosidosis could be detected by Sephadex gel filtration procedure.

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Ganglioside (NeuAc), labeled at the C-3 position of sphingosine with tritium, was injected into C3H/He, C57BL/10, B10.AQR mice intraperitoneally. The incorporation and the distribution of the radioactivity in various organs were examined. The injected [³H] (NeuAc) was mainly incorporated in the liver and hydrolyzed sequentially. Sialic acid of ganglioside (NeuAc) and metabolites was converted to N-glycolyl type from N-acetyl type. An appreciable amount of the sphingosine moiety in the administered (NeuAc), moreover, was reutilized, being converted to sphingomyelin, and incorpo-rated into alkyl chain of the ether lipid in phosphatidylethanolamine. The distributions of radioactivity in the metabolites of (NeuAc) administered to the three strains of mice were different from each other. In other organs, (NeuAc) was incorporated and metabolized only slightly. The N-methylamide, at the carboxyl group of the sialic acid, of the labeled ganglioside ( (NeuAc)-NMe) was injected into C3H/He mice. Most of the administered [³H] (NeuAc)-NMe was incorporated in the liver, and was metabolized to GM3 (NeuAc)-NMe, via GM2 (NeuAc)-NMe, within 24 h. GM3 (NeuAc)-NMe was the only radioactive compound in the subsequent 10 weeks, but disappeared from the liver gradu-ally. N-Methylamide-modified gangliosides were resistant to hydrolysis by mouse hepatic sialidase, to elongation by glycosyltransferase and to N-glycolylation at N-acetyl-neuraminic acid by monooxygenase.

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The activity of β-galactosidase in the brain and liver of patients with -gangliosidosis was assayed using -ganglioside tritiated in the terminal galactose. In the cases of -gangliosidosis Types 1 and 2A the activity was less than 0.5% of the control. In the liver of -gangliosidosis Type 2B the activity was observed to be much higher than that of Types 1 and 2A. On Sephadex G-150 gel filtration, three active fractions (I, II and III) for 4-methylumbelliferyl β-galactopyrancside (4MU) and two active fractions (I and II) for , -ganglioside were obtained in the control liver. There was no active fraction for -ganglioside in spite of the preserved fraction I for 4MU in the liver of -gangliosidosis Type 1 or Type 2A. In any of the three cases fraction II for both 4MU and -ganglioside was not detected.

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遺伝子型検査によって- ガングリオシドーシスと確定診断された3症例の柴犬において，臨床診断の過程で頭部MRIが撮像された． これらの症例のMRI検査を受けた月齢は異なっていたが（症例1 ：3カ月齢と7カ月齢，症例2 ：6カ月齢，症例3 ：8カ月齢），すべてに共通して，T2強調画像で大脳白質が左右対称性に高信号を呈し，灰白質と白質のコントラストが不明瞭であった． また，Fluid-attenuated inversion recovery画像でも同様の所見が認められた． このようなMRI所見が本疾患の特徴であると示唆されたため，頭部MRI検査は柴犬の- ガングリオシドーシスの補助的診断に有用であると考えられた． また，これら3症例の所在は近畿・中国地方であり，すべてに血統上の関連性が認められた．

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Five IgM monoclonal antibodies (MAbs), MW-1, MW-2, MW-3, MW-4, and MW-5, against a glycolipid asialo were prepared from hybridoma clones obtained by the fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with asialo adsorbed to naked Salmonella. All the MAbs reacted only with asialo when their reactivities were examined by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC)-immunostaining using structurally related glycolipids. The MAbs showed a complement-dependent lysis of mouse natural killer (NK) cells, but the lytic activities were weaker than that of a rabbit polyclonal anti-asialo antibody. When they were mixed, the anti-NK activity was increased to a level almost comparable to that of the polyclonal antibody. These results suggest that all the MAbs obtained are specific for asialo and that they may be different in fine specificity for the glycolipid. Significance of the MAbs in immunological and neurochemical studies is discussed.

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In my previous research, the fuzzy grey regression model is derived for solving limited time series, and applied to forecast the LCD TV's demand. In this study, we follow the previous research of fuzzy grey regression model, and find good membership functions for the fuzzy-input data of LCD TV demand such that a smaller estimated error between collected fuzzy-input data and forecasting fuzzy-output value is obtained.

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Three kinds of anti- monoclonal antibodies, AGM-1, -2, and -3, of the IgM class were produced by the immunization of BALB/c mice with ganglioside inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides and fusion of the spleen cells with a mouse myeloma cell line. The specificities of the monoclonal antibodies obtained were elucidated through complement-dependent liposome immune lysis assay and enzyme immunostaining on thin-layer chromatograms. All of the monoclonal antibodies reacted only with ganglioside , and structurally related glycosphingolipids, such as fucosyl-, asialo-, GM2, and GD1b, and the other gangliosides (GM3 and GD1a) tested showed no reactivity to the 3 monoclonal antibodies. These findings suggest that the monoclonal antibodies obtained may be specific for ganglioside . These anti- monoclonal antibodies were used to define the expression of ganglioside on small cell lung carcinoma (SCLC) cell lines and tissues. In flow cytometric analysis and immunostaining studies, we observed that ganglioside was highly expressed on the SCLC cell lines. Results obtained with flow cytometry and immunohistochemistry agreed well with the immunochemical determination of ganglioside in lipid extracts of cell lines. Furthermore, expression of ganglioside in tumor tissues from patients with SCLC was ascertained by the immunohistochemical examination of acetone-fixed paraffin-embedded tissue sections. Ganglioside was detected in 5 of 19 SCLC tissues. These results suggest that ganglioside is expressed in SCLC cells.

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The clinical, pathological, chemical and enzymatic differences between three types of -gangliosidosis and a variant of β-galactosidase deficiency were described.

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gangliosidosis is one of the inherited metabolic lysosomal storage disorders characterized by neurological symptoms caused by β-galactosidase deficiency and consequent accumulation of ganglioside in neuronal cells. Shiba dogs affected with gangliosidosis have been found to suffer from corneal opacity. In our morphological analysis, keratocyte enlargement was induced by abnormal intracellular accumulation of neutral carbohydrates, resulting in the loss of normal arrangement of collagen fibrils in the opaque cornea was found to be associated with the disorder. We therefore conclude that corneal opacity in this Shiba dog with gangliosidosis may be caused by neutral carbohydrate accumulation in lysosomes, swelling and dysfunction of keratocytes, and subsequent irregular arrangement of collagen fibrils in the corneal proper substance.

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Being transcriptionally and translationally inactive, sperm must utilize preassembled pathways into specific compartments in which they function to fertilize ovum. Membrane rafts are specific membrane regions enriched in sterols and glycosphingolipids such as ganglioside () and play an important role in a variety of cellular functions. Recent findings have demonstrated that membrane rafts are present in mammalian sperm and are involved in regulating the induction of acrosome exocytosis. However, no information is available on whether avian sperm possess membrane rafts. Thus, we investigated the organization of membrane rafts in chicken sperm. Our localization experiments for and sterols showed that the plasma membrane overlaying the sperm head possesses specific membrane domains enriched in both aforementioned lipids. Caveolin-1, which localizes into membrane rafts in other systems, was localized only to the sperm tail. Based on the biochemical definition that membrane rafts are insoluble membranes when subjected to a Triton X-100 treatment, we isolated detergent-insoluble membranes from chicken sperm and quantified the content, which showed an enrichment of in the membrane fraction relative to the detergent-soluble fraction. Together with the results of localization and biochemical experiments, we demonstrate for the first time that membrane rafts exist in chicken sperm. Thus, our results provide a foundation for investigating a novel cellular pathway inherent in avian sperm membranes that might be involved in functions necessary to achieve fertilization.

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We investigated the influence of the administration of anti-asialo antibody on aberrant crypt foci (ACF) formation induced by a single injection of 1, 2-dimethylhydrazine (DMH). At four weeks after the injection of DMH (20mg/kg body weight), the number of ACF and aberrant crypts were counted. Most ACF appeared in the distal large bowel, accounting for approximately 60% of the total ACF in both groups. Rats administered anti-asialo had significantly more ACF in the distal colon, the rectum and the total large bowel as compared to control rats. A similar tendency was observed for the number of aberrant crypts. The increased number of ACF resulting from the administration of anti-asialo was not accompanied by the enlargement of ACF size in every part of the colon. This study demonstrated that the administration of anti-asialo at the initiation stage leads to an increase in ACF as well as aberrant crypts in the distal colon, rectum and total large bowel probably via the suppression of natural killer cells.

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Two gangliosides (IV-sialosylgangliotetraosylceramide) containing either N-glycolylneuraminic acid or N-acetylneuraminic acid at the terminal galactose of gangliotetraosylceramide were found in the ICR mouse spleen. Their structures were characterized by the behavior on thin layer chromatograms, sugar composition, sus-ceptibility to sialidase and immunobinding activity toward anti-gangliotetraosylcer-amide antibody. The structure of containing N-glycolylneuraminic acid was further confirmed by methylation analysis. gangliosides containing either N-glycolylneuraminic acid or N-acetylneuraminic acid were also purified and chara-cterized by thin layer chromatography, sugar analysis and sialidase treatment. As a result, the presence of four kinds of monosialoganglioside with a gangliotetraosyl core structure, (NeuAc), (NeuGc), (NeuAc), and (NeuGc), were found to exist in the ICR mouse spleen. These four gangliosides accounted for about 50% of the spleen monosialoganglioside content. Additional four gangliosides in the monosialoganglioside fraction were tentatively characterized as G_M3 (NeuAc), G_M3 (NeuGc), G_M2 (NeuGc), and sialosylneolactotetraosylceramide (NeuGc).

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By means of TLC immunostaining with anti-asialo antiserum and conventional structural analyses including exoglycosidase treatment, permethylation and negative ion FABMS, asialo was found to be widely distributed in rat ascites hepatomas, AH130, AH109A, AH44, AH272, AH41C, AH60C, AH414, AH7974, AH66, AH66αF, AH66F and AH13, and Yoshida sarcoma cells. However, reactivity of asialo , when measured by flow cytometry and complement-mediated cytotoxicity assay with anti-asialo antiserum, was only observed on AH13 and AH66F, and did not necessarily correspond to the concentration of asialo on the cells, indicating a cryptic or unreactive nature of glycosphingolipids with respect to their antibodies. On the other hand, although rats injected with 10⁷ cells of AH66F died within 7 days, treatment of the rats with anti-asialo antiserum was found to be effective for their cure or for prolonging their survival, indicating an in situ cytotoxic effect of antiserum. For the in situ cytotoxicity, the timing and period of injection and the dose of antiserum were found to be important.

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Large unilamellar liposomes (LUV), which have a large entrapped volume and efficient drug capture characteristics, are rapidly and efficiently taken up by the RES cells in liver and spleen. A prolonged residence of the drug-entrapped liposomes in the circulation is important for the sustained release of drug form liposomes. Incorporation of 6 mol% of into LUV composed of DSPC/CH (/DSPC/CH 0.13 : 1 : 1 m/m, 200 nm in size) showed a significant increase of circulation time in the blood circulation. The /DSPC/CH liposomes have been tested for their utility as a sustained release system for adriamycin (ADM) in vivo. ADM was encapsulated into /DSPC/CH liposome with>95% in trapping efficiency by employing the pH gradient method. Mice were given i.p. injection of 2 million L1210 leukemia cells. Twenty-four hrs later, mice were treated with single shot of ADM-/DSPC/CH liposome, ADM-DSPC/CH liposome or free ADM (5 mg/kg). Treatment with ADM-/DSPC/CH liposome significantly increased mean survival times as compared with two controls. Our data indicate that can significantly enhance the DSPC/CH liposome residence time in bloodstream and /DSPC/CH liposomes with prolonged circulation time are important for the sustained relase of drug in circulation.

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The present study deals with the role of cells sensitive to anti-asialo antibody treatment in T cell-mediated tumor cell radication in vivo. Rabbit anti-asialo antiserum was injected into C3H/He mice. This treatment not only resulted in almost complete abrogation of natural killer (NK) cell activity but also produced a potent inhibiting effect on the generation of activated macrophage activity induced by inoculating Propionibacterium acnes (P. acnes). Such an immunodepressed state lasted for 20 days or more after 5 consecutive injections of anti-asialo antiserum. These antiasialo antibody-treated C3H/He mice were used as recipients in Winn assays, in which the neutralizing activity of spleen cells immunized to syngeneic X5563 tumor cells was assessed. The results demonstrated that anti-X5563 immune spleen cells depleted of asialo -positive cells by the in vitro treatment with anti-asialo antibody plus complement exhibited potent anti-X5563 tumor-neutralizing activity in antibodyuntreated normal recipient mice. In contrast, the X5563-immune spleen cells depleted of asialo + cells failed to produce tumor protection in asialo antiseurm-treated recipient mice. When T cell-deprived B cell mice were used as recipients in Winn assays, X5563 immune spleen cells depleted of asialo + cells exhibited or failed to exhibit tumor-neutralizing activity in asialo antiserum-untreated or-treated recipient B cell mice, respectively. These results indicate that the implementation of T cell-mediated in vivo protective immunity requires the participation of anti-asialo antibody-sensitive cells, but not necessarily the host's T cells.

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