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Biomedical subjects

I Ishizuka

Publications and source records attributed to I Ishizuka.

At least 19 recordsLinked to original sources
PubMed1992

Substrate specificity and distribution of UDP-GalNAc:sialylparagloboside N-acetylgalactosaminyltransferase in the human stomach.

The detailed substrate specificity of the UDP-GalNAc:sialylparagloboside N-acetylgalactosaminyltransferase to form the Sd(a+) blood group active carbohydrate determinant GalNAc beta 1-4(NeuAc alpha 2-3)Gal was studied using a membrane fraction prepared from human gastric fundic mucosa. Various sialosylated oligosaccharides and gangliosides were examined as acceptor substrates. Oligosaccharide substrates were fluorescence-labelled with 2-aminopyridine, and the transferase activity was quantified by h.p.l.c. using a reversed-phase column. The structures of the products were determined by glycosidase degradation and proton n.m.r. 3'-Sialyl-lactose (II3NeuAcLac), 3'-sialyl-lactotetraose (IV3NeuAcLc4), and 3'-sialyl-lactoneotetraose (IV3NeuAcnLc4) were good substrates for the beta 1-4GalNAc transferase in gastric fundic mucosa, but 6'-sialyl-lactoneotetraose (IV6NeuAcnLc4) or 6'-sialyl-lactose (II6NeuAcLac) were not. Gangliosides with a terminal NeuAc alpha 2-3Gal residue such as GM3, sialylparagloboside, GM1b and GD1a were also studied. The activity of beta 1-4GalNAc transfer to sialylparagloboside was much higher than that to GM2, GM1b or GD1a in spite of them having the same terminal residue. Measurement of the activity of the beta 1-4GalNAc transferase in biopsy specimens demonstrated that the activity was localized in gastric fundic mucosa and was absent in pyloric mucosa, intestinal metaplasia and gastric cancer tissue. Thus the beta 1-4GalNAc transferase present specifically in fundic mucosa required a NeuAc alpha 2-3Gal residue connected to either type-1-chain or type-2-chain oligosaccharides. In glycolipids, the acceptor specificity was restricted to NeuAc alpha 2-3Gal beta 1-4GlcNAc because the NeuAc alpha 2-3Gal beta 1-3GalNAc structure in ganglio-series glycolipids was not a good acceptor substrate.

Aminopyridines↗
PubMed1992

Isolation and structural characterization of a mono-sulfated isoglobotetraosylceramide, the first sulfoglycosphingolipid of the isoglobo-series, from rat kidney.

A novel sulfoglycosphingolipid based on the isoglobo-series core structure was isolated from rat kidney and purified by column chromatographies with DEAE-Sephadex and silica beads. The structure was characterized by solvolysis, compositional analysis, proton NMR spectroscopy, Fourier-transform infrared spectroscopy, methylation analysis and liquid secondary ion mass spectrometry (LSIMS). The characteristic fragment ions for a sulfate and a sulfated N-acetylhexosamine were observed in LSIMS spectra. The two-dimensional chemical-shift-correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy experiments evidenced the presence of a 3-O-sulfated N-acetylgalactosamine and a Gal alpha 1-3Gal structure in the molecule. The major ceramide consisted of 4-hydroxysphinganine linked to a C24 nonhydroxy fatty acid, deduced from both compositional analysis and LSIMS. From the above results, the following structure was established for this glycolipid: HSO3-3GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4Glc beta 1-1Cer, isoglobotetraosylceramide (iGb4Cer) IV3-sulfate. Rat kidney also contained globotetraosylceramide (Gb4Cer) IV3-sulfate which has a carbohydrate core identical to that from human kidney. The yields of iGb4Cer IV3-sulfate and Gb4Cer IV3-sulfate were 0.27 and 0.07 nmol/g wet tissue, respectively.

Animals↗
PubMed1992

Sperm immobilizing antibodies react to the 3-O-sulfated galactose residue of seminolipid on human sperm.

It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or women's sera containing sperm immobilizing antibodies, but not by normal women's sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in women's sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.

Animals↗
PubMed1992

[Evaluation of the timing principle with small priming doses of vecuronium].

The intubating conditions using the timing principle combined with small priming doses of vecuronium were evaluated in forty patients who underwent elective surgery. They were randomly assigned to one of two groups: 1) timing, 2) timing with priming. In timing group, vecuronium 0.15 mg.kg-1 was administered, and at the onset of clinical muscle weakness, thiopental 4-5 mg.kg-1 was given promptly. Sixty seconds after thiopental, patients were intubated. In the timing with priming group, vecuronium 0.005 mg.kg-1 was administered as priming doses. Four minutes later vecuronium 0.15 mg.kg-1 was given. The administration of thiopental and the intubation were done in the same way as in timing group. The time to onset of clinical weakness after the administration of vecuronium 0.15 mg.kg-1 was significantly shorter in the timing with priming group than that in the timing group (46.1 +/- 4.8 vs. 57.6 +/- 7.8, P < 0.01). There were no significant differences in intubating score, T1, TR, onset time, and duration between the two groups. We conclude that the timing principle combined with small priming doses of vecuronium might be safe and useful for rapid tracheal intubation.

Adult↗
PubMed1991

Accumulation of sulfoglycolipids in hyperosmosis-resistant clones derived from the renal epithelial cell line MDCK (Madin-Darby canine kidney cell).

1. Two clones (osmR-A and osmR-B) resistant to hyperosmotic media of 700 and 800 mosmol/l, respectively, were selected from Madin-Darby canine kidney (MDCK) cells. 2. When cultured in isosmotic medium (300 mosmol/l), the concentration of galactosyl sulfatide and lactosyl sulfatide in these hyperosmosis-resistant clones was 3.4-5.9 times higher than in the wild-type MDCK. The rate of incorporation of [35S]sulfate into sulfolipids of osmR-A and osmR-B was 1.9-6.7 times higher than MDCK. 3. The stimulation of incorporation into sulfolipids by hyperosmotic culture was completely inhibited by cycloheximide. The pulse-chase studies indicated decreased turnover rate of sulfolipids in osmR-A.

Animals↗
PubMed1990

Accumulation of highly acidic sulfated glycosphingolipids in human hepatocellular carcinoma defined by a series of monoclonal antibodies.

An accumulation of sulfated and very complex, highly acidic glycolipids was observed in cultured human hepatocellular carcinoma cells. Among the cells tested, PLC/PRF/5 cells contained a significant amount of very complex sulfated acidic glycolipids, and HepG2 cells were characterized as having a large amount of relatively simple sulfated glycolipids. Several monoclonal antibodies (all IgM) directed to these sulfated and highly acidic glycolipids were established. Among them, 49-D6 and 7-E10 were both directed to SM3 (LacCer-II3-sulfate), a relatively simple sulfated glycolipid, and 34-A4 was directed to SD1a (GgOse4Cer II3,IV3-disulfate) and more complex sulfated glycolipids. The other four antibodies, 26-A10, 34-B9, 79-C8, and 16-E10, reacted with unknown highly acidic glycolipids, which were eluted in 0.9-2.7 M ammonium acetate in DEAE chromatography, indicating that these antigenic glycolipids were far more acidic than the usual glycolipids described until now. Analysis of the glycolipids extracted from the hepatocellular carcinoma tissues and cirrhotic livers of patients and from a normal liver with these monoclonal antibodies revealed that sulfated glycolipids having simple carbohydrate structures such as SM3 accumulated significantly in the cirrhotic liver (2 of 4 cases) as well as hepatocellular carcinoma tissue (15 of 17 cases, 88%), and more complex sulfated glycolipids and highly acidic glycolipids were much more specific to hepatocellular carcinoma tissues (10 of 17 cases, 59%) compared to the cirrhotic liver (0 of 4 cases).

Antibodies, Monoclonal↗
PubMed1990

Adaptive changes in sulfoglycolipids of kidney cell lines by culture in anisosmotic media.

(1) The effects of osmolarity environments on renal glycolipid composition were examined using established renal cell lines. The profile of glycosphingolipids of Madin-Darby canine kidney cells (MDCK) in culture with anisosmotic media showed that a hyposomotic medium reduced the concentration of GalCer I3-sulfate and LacCer II3-sulfate. (2) The concentrations of sulfoglycolipids were increased by maintaining the culture in a hyperosmotic media prepared by the addition of various sodium salts to the control isosmotic medium, while the contents of most of the neutral glycolipids were reduced. The hyperosomotic medium supplemented with nonelectrolytes, mannitol, sucrose or urea, also increased the concentration of sulfoglycolipids. (3) Both sulfoglycolipids were increased linearly with gradual increases of sodium chloride in the medium. Hyperosmolarity produced by the addition of a nonelectrolyte, mannitol, also increased the levels of sulfoglycolipids. In both series of media, the most prominent accumulation was observed in LacCer II3-sulfate. (4) The incorporation of radioactive sulfate into sulfoglycolipids was elevated in cells adapted to high NaCl or mannitol. The increase of the label was observed not only in MDCK but also in three other established cell lines of renal tubular origin, JTC-12, LLC-PK1 and MDBK. (5) It was established, using the culture system of homogeneous cell lines, that the mechanism of increasing the amount of sulfoglycolipids is independent of the integral regulatory mechanism of animals and resides in the renal epithelial cell itself. These results suggest that by culture in hyperosmotic media, the elevated level of intracellular cations stimulated the activity of GalCer and LacCer sulfotransferase, inducing the increased expression of sulfoglycolipids.

Animals↗
PubMed1990

Isolation and characterization of major urinary amino acid O-glycosides and a dipeptide O-glycoside from a new lysosomal storage disorder (Kanzaki disease). Excessive excretion of serine- and threonine-linked glycan in the patient urine.

Four major sialo compounds, termed GP-M1, GP-D1, GP-D2, and GP-D3 have been isolated from the urine of a novel glycoprotein storage disorder patient with angiokeratoma corporis diffusum which was discovered by Kanzaki et al. (Kanzaki, T., Yokota, M., Mizuno, N., Matsumoto, Y., and Hirabayashi, Y. (1989) Lancet April 22, 875-877). Based on the results of fast atom bombardment mass spectrometry, methylation analysis, and proton nuclear magnetic resonance spectroscopy, their chemical structures were concluded to be: (formula; see text) The yields of GP-M1, GP-D1, GP-D2, and GP-D3 were approximately 15, 6, 50, and 5 mg/liter of urine, respectively. The most major compound GP-D2, was further purified into single molecular species, threonine and serine type, by reversed phase high performance liquid chromatography. NMR analysis of the two purified compounds with single molecular species showed that the chemical shifts of anomeric protons of GalNAc were significantly different between threonine- and serine-linked GalNAc. Neither mannose-containing glycopeptides nor glycosphingolipids were excreted in the patient urine. From these results, this disease is thought to be caused by the deficiency of a lysosomal enzyme(s) acting on O-linked glycan chains.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1990

Surface-active novel glycolipid and linked 3-hydroxy fatty acids produced by Serratia rubidaea.

A Serratia rubidaea isolate with wetting activity when grown at 30 but not 37 degrees C was examined for the production of specific lipids. Two novel lipids (rubiwettins R1 and RG1) were isolated and shown to be able to lower the surface tension of saline to 26 mN/m. These lipids were located in extracellular vesicles found in a 30 degrees C culture of S. rubidaea. Chemical structures of these biosurfactants were determined by degradation product analyses, infrared spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Rubiwettin R1 was proposed to be a mixture of 3-(3'-hydroxytetradecanoyloxy)decanoate, 3-(3'-hydroxyhexadecenoyloxy)decanoate, and minor molecular isomers. The structure of rubiwettin RG1 was proposed to be beta-D-glucopyranosyl 3-(3'-hydroxytetradecanoyloxy)decanoate. The importance of such surface-active exolipids in bacterial occupancy on surfaces was suggested.

Chromatography, Thin Layer↗
PubMed1989

Mono-sulfated globopentaosylceramide from human kidney.

A novel sulfated glycosphingolipid that belongs to the "globo-series" was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform/methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative thin layer chromatography. The structure and the properties were studied by infrared spectroscopy, two-dimensional proton magnetic resonance spectroscopy, negative secondary ion mass spectrometry, solvolysis, compositional and methylation analyses, monoclonal antibodies, and sulfatide-binding proteins. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO3-3Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide. The proton resonance at 3.93 ppm of the H-3 of the sulfated nonreducing terminal galactose of this lipid was downfield-shifted (delta 0.48 ppm), as compared with H-3 of the internal beta-galactose because of the electronegativity of the sulfate ester. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (MC-631) (Kannagi, R., Cochran, N. A., Ishigami, F., Hakomori, S., Andrews, P. W., Knowles, B. B., and Solter, D. (1983) EMBO J. 2, 2355-2361), whose epitope is R-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-R', on thin layer chromatograms and solid-phase radioimmunoassay. This lipid also bound to the 125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 0.19 nmol/g of tissue, which was about 0.09 and 0.5 mol % of galactosyl and lactosyl sulfatides in human kidney.

Chromatography, Ion Exchange↗
PubMed1989

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.

Animals↗
PubMed1989

Mono-sulfated globotetraosylceramide from human kidney.

A novel sulfated glycosphingolipid that belongs to "globo-series" was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform-methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative TLC. The structure and the properties were studied by IR spectroscopy, proton NMR spectroscopy, negative secondary ion-mass spectrometry, solvolysis, periodate oxidation, compositional and methylation analyses, monoclonal antibodies, and a sulfatide-binding protein. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO3-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (stage-specific embryonic antigen-3) (MC-631) (Kannagi, R., Cochran, N.A., Ishigami, F., Hakomori, S., Andrews, P.W., Knowles, B.B., & Solter, D. (1983) EMBO J. 2, 2355-2361), whose epitope is R-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-R', on TLC and solid-phase radioimmunoassay. This lipid also bound to the 125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 34 pmol/g of tissue, which was about 0.028, 0.16, and 18 mol% of galactosyl- and lactosylceramide sulfates, and globopentosylceramide sulfate (Nagai, K.-i., Roberts, D.D., Toida, T., Matsumoto, H., Kushi, Y., Handa, S., & Ishizuka, I. (1989) J. Biol. Chem. 264, in press), respectively, in human kidney.

Animals↗
PubMed1988

Monoclonal antibodies directed to a disulfated glycosphingolipid, SB1a (GgOse4Cer-II3IV3-bis-sulfate), associated with human hepatocellular carcinoma.

Two murine monoclonal antibodies, 2H6G5 (IgM) and 4A9E10 (IgG3), were obtained by using either cultured human hepatocellular carcinoma cells (PLC/PRF/5) or the acidic glycolipid mixture prepared from the same cells as immunogens. The antigen in PLC/PRF/5 cell membranes recognized by both antibodies was identified as a disulfated acidic glycolipid, GgOse4Cer-II3IV3-bis-sulfate (SB1a). Both antibodies reacted specifically with SB1a, and no significant reactivity was noted with other sulfated glycolipids or gangliosides except that the antibody 2H6G5 showed a weak cross-reactivity with LacCer-II3-sulfate (SM3), another sulfated glycolipid which partly shares the same carbohydrate structure as SB1a. The SB1a antigen is a relatively minor glycolipid in PLC/PRF/5 cells, but it was strongly expressed at the surface of PLC/PRF/5 cells as ascertained by cytofluorometry using both antibodies. A significant amount of SB1a antigen was present in 3 of the acidic glycolipid fractions isolated from 15 human hepatocellular carcinoma tissues as well as in the acidic glycolipid fraction prepared from PLC/PRF/5 cells, while all the acidic glycolipid fractions prepared from cirrhotic livers and a normal liver were essentially negative for SB1a, as ascertained by both solid phase enzyme immunoassay and the thin-layer chromatography-immunostaining method. These results strongly suggest that the SB1a antigen as defined by these new monoclonal antibodies is associated with human hepatocellular carcinoma.

Animals↗
PubMed1988

Characterization of the binding epitope of a monoclonal antibody to sulphatide.

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3'-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.

Antibodies, Monoclonal↗
PubMed1987

Biosynthesis of monosulfogangliotriaosylceramide and GM2 by N-acetylgalactosaminyltransferase from rat brain.

Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II3-sulfate (SM2) and N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The Km values for UDP-N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 microM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme.

Animals↗
PubMed1985

Preparation and enzymatic degradation of monosulfogangliotriaosylceramide.

Gangliotriaosylceramide 3'-sulfate (GgOse3Cer-II3-sulfate) contains the sugar sequence similar to that of GM2 ganglioside except that the NeuAc in GM2 is replaced by a sulfate group. Due to this structural similarity, we have studied the in vitro synthesis of GgOse3Cer-II3-sulfate using the system for GM2. Our results showed that GgOse3Cer-II3-sulfate could be synthesized from lactosylceramide 3'-sulfate and UDP-GalNAc catalyzed by N-acetylgalactosaminyltransferase prepared from rat brain (Dicesare, J. L., and Dain, J. A. (1971) Biochim. Biophys. Acta 231, 385-393). As in the case of GM2, the GgOse3Cer-II3-sulfate biosynthesized in vitro or isolated from rat kidney could also be cleaved by human beta-hexosaminidase A in the presence of GM2-activator (Li, S.-C., Hirabayashi, Y., and Li, Y.-T. (1981) J. Biol. Chem. 256, 6234-6240). The fact that the GM2-activator could stimulate beta-hexosaminidase A to hydrolyze both GM2 and Gg-Ose3Cer-II3-sulfate indicates that these two glycolipids may be catabolyzed by the same mechanism.

Animals↗
PubMed1985

Visualization method for sulfolipids and its application to the determination of nanomole quantities of lipid sulfur.

A simple and sensitive visualization method for sulfolipids on a thin-layer chromatogram is described. By spraying with an acidic solution of azure A, a complex was formed between an anionic sulfolipid and a blue cationic compound. After the unbound dye was washed out by brief soaking in methanol, sulfolipids were visualized as clear dark-blue bands on a light-blue background. As little as 0.5 nmol could be detected. Sulfolipid-dye complex was estimated by densitometry or colorimetric measurement after extraction with chloroform/methanol. For the quantitative determination of sulfolipids having long sugar chains, it is necessary to treat thin-layer chromatography plates with acetic anhydride before color development. Of the other tissue lipids not containing sulfuric acid ester that were tested none were stained significantly. A linearity of quantitative determination was observed over the range of 1-8 nmol.

Acetylation↗