KAMIO, K., JIN, T., GASA, S., OHHIRA, M., HONKE, K., KASAI, N. and MAKITA, A. Inconsistent Expression of Glycolipid Sulfotransferase Activity between Hepatoma and Serum. Tohoku J. Exp. Med., 1992, 168 (1), 29-35 Glycolipid sulfotransferase activity in human and rat hepatocellular carcinoma tissues was studied, since an elevated level of the enzyme activity in serum had been demonstrated in cancer patients. The level of the enzyme activity in carcinoma tissues, however, could not be distinguished from that of normal controls. Similar observations were made for rat hepatoma. A higher level of enzyme activity was observed in the female than in the male. Inconsistent expression between hepatoma tissue and serum suggests that humoral factor(s) derived from hepatoma tissue stimulates production of the sulfotransferase, which is released into the blood-stream in the host. glycolipid sulfotransferase ; hepatocellular carcinoma; LEC rat

Introduction. We synthesized sulfo-glycolipid, P-SQAG9 (designate rectangle beta-SQAG9 liposome, because it efficiently forms a liposome structure) that possessed immunosuppressive effects such as inhibition of T-cell responses in human allogeneic MLR and skin allograft survival in rats, and bound to CD62L (L-selectin) in vitro. In this study, we further investigated the immunosuppressive mechanism in vivo by P-SQAG9 liposome in a skin-allografted rat model.Methods. ACI rats (RT1(a)) were grafted skin of LEW rats (RT1(1)) treated with PBS or beta-SQAG9 liposome IV once a day for 7 days. Subsequently, we investigated the population of T cells and CD62L(+) T-cell subset in the spleen, axillary lymph nodes (ALNs), and peripheral blood of skin-allografted rats by two-color flow cytometry.Results. Five of 11 (45.5%) rats that were treated with 50 mg/kg beta-SQAG9 liposome showed graft survival and another showed moderate rejection in graft. The CD62L(+) T-cell subset population in ALNs of beta-SQAG9 liposome-treated rats decreased in a dosedependent manner. No significant difference in the T-cell population was observed between the beta-SQAG9 and control groups. These data suggest that beta-SQAG9 could bind to the CD62L(+) T-cell subset in vivo as well as in vitro and affect T-cell migration, which might lead to T-cell tolerance in vivo.

The aim of this study was to measure objectively the adherence of burned tissue to bipolar forceps to evaluate the coagulation performance of forceps made of different types of metals. Coagulation performance of bipolar forceps made of gold, titanium, and stainless steel was determined by comparing the amount of protein in the adhered coagulum on the tips. The amount of adhered coagulum was significantly less on the gold-plated bipolar forceps than on those made of the other two materials. The ease with which coagulum could be removed was compared using the cleaning cycle of an ultrasonic rinsing device. This ease of removal was also significant with the gold-plated forceps. Electron microscopy observations of the surface of the forceps tips revealed a significant difference in roughness among these materials, and there were also significant differences in wetting tensions. Measuring adherence based on three different types of roughness and wetting tensions of forceps made from the same metal (titanium) also demonstrated a significant difference in the cleaning cycle. Histological examination of an artery coagulated with the gold-plated bipolar forceps showed that the structure had been completely collapsed without destruction of the layers, whereas arteries coagulated with the other materials revealed severely damaged structures. Adherence to bipolar forceps was dependent on both the material in the tips and the roughness of this material. The gold-plated bipolar forceps demonstrated the best performance.

We previously reported that synthetic sulfo-glycolipid, 3-O-(6-deoxy-6-sulfono-β-d-glucopyranosyl)-1,2-di-O-acylglycerol (β-SQDG(18:0)) which was deduced from sulfonoquinovosyl-diacylglycerols of sea urchin possessed immunosuppressive effects, such as human mixed lymphocyte reaction (MLR) and skin allograft in rat, and that these effects were caused by contact inhibition between T-cells and antigen presenting cells (APCs). Here, we investigated the mechanism of these immunosuppressive effects on human MLR by β-SQAG9 which had been newly synthesized from β-SQDG(18:0) to improve structural stability in water solution. CD62L+ T-cells in peripheral blood predominantly respond to APCs, and β-SQAG9 inhibited the response of CD62L+ T-cell subset in human allogeneic MLR. Surprisingly, it was demonstrated that β-SQAG9 bound to L- and P-selectin (CD62L and P) molecule in vitro. Meanwhile, β-SQAG9 efficiently formed liposome structure and bound to L-selectin on the cell surface of CD62L+ T-cell subset but might not be incorporated into the cells. Because the immunosuppressive effects of β-SQAG9 disappeared when β-SQAG9 liposome was changed to soluble form by detergent, the liposome structure of β-SQAG9 was presumed to be essential for these effects. These findings suggested β-SQAG9 to be a novel drug with a unique immunosuppressive action.

Structures and phase behavior of multilamellar vesicles of 1,2-dipalmitoyl-L-phosphatidylcholine (DPPC) containing various amount of ganglioside GM3 with a C18:1 sphingoid base and a 24:0 acyl chain (GM3(18,24)) were investigated by small-angle X-ray diffraction. Below 3.5 mol% GM3 content, the phase behavior was similar to that of pure DPPC except for a slight increase of lamellar repeat distance in the L(beta'), the P(beta') and the L(alpha) phases and a decrease of the pretransition temperature. In the range of 4-12 mol% GM3 content, another phase which has larger repeat distances coexisted with the phase observed below 3.5 mol% GM3 content. This has been interpreted that the phase separation into GM3-poor phase (denoted as A-phase) and GM3-rich phase (denoted as B-phase) took place. Above 13 mol% GM3 content, the B-phase became dominant. This phase separation may be related to the formation of GM3-enriched microdomains that had been observed on the cell surfaces which express large amounts of GM3, such as murine B16 melanoma (J. Biol. Chem. 260 (1985) 13328).

BACKGROUND:It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. METHODS:We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG) that contains a saturated C18 fatty acid, which is designated as beta-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of beta-SQDG(18:0). RESULTS:beta-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. beta-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, beta-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by beta-SQDG(18:0) treatment. CONCLUSIONS:beta-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by beta-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge–charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

Glycolipids were extracted from primary bladder tumors of 14 patients and 2 normal counterparts. Their expression pattern was assessed by thin-layer chromatography (TLC). The most remarkable change was massive accumulation of GM3 in superficial bladder tumors compared with invasive tumors. This change was also confirmed by immunohistochemistry using anti-GM3 monoclonal antibody. The activities of glycosyltransferases responsible for GM3 synthesis (GM3 synthase, Gb3 synthase and GD3 synthase) were consistent with upregulated expression of GM3 in superficial humors. It was suggested that the marked GM3 accumulation in superficial tumors was caused not only by upregulated GM3 synthase but also by downregulated activities of Gb3 and GD3 synthase. Histopathologic examination revealed an inverse correlation of the amount of GM3 expressed with invasive potential. Exogenously supplemented GM3 suppressed invasion potential in human bladder tumor cell lines (T-24, KK-47). These results indicate that the amount of GM3 expressed may serve as an indicator of the invasion potential of bladder tumor. Furthermore, new antiinvasion therapeutics may be possible by administration of GM3. (C) 2001 Wiley-Liss, Inc.

A novel galactosylalkylglycerol modified with a long-chain cyclic acetal at the sugar moiety, 3-O-(4′6′-plasmalogalactosyl) 1-O-alkylglycerol, was isolated from equine brain. The presence of cyclic acetal linkage, its linked position, and the length of the acetal chain of the natural plasmalo lipid were determined by proton NMR spectroscopy and fast-atom bombardment–mass spectrometry, as well as gas chromatography–mass spectrometry and gas–liquid chromatography. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from 3-O-galactosyl 2-O-acyl 1-O-alkyl glyceride through acetalization after deacylation. As a result, the direction and position of the acetal chain of the natural plasmalo lipid were characterized as an “endo”-type 4′,6′-O-acetal derivative linked to galactoside by comparison with the NMR data of the synthesized product. The chain lengths of alkyl and acetal groups were C14 for the former and C16 and C18 for the latter, and those for the latter group were mostly similar to those of plasmalogalactosyl ceramide, which was previously isolated from equine brain.—Yachida, Y., M. Kashiwagi, T. Mikami, K. Tsuchihashi, T. Daino, T. Akino, and S. Gasa. Novel plasmalogalactosylalkylglycerol from equine brain.

BACKGROUND:A spontaneously hypercholesterolemic Imai rat has recently been reported as a model of focal glomerulosclerosis that causes nephrotic syndrome followed by renal failure. This study was designed to determine if an oral adsorbent, AST-120, ameliorates renal lesions and TGF-beta 1 expression in the rats.METHODS:AST-120 was given orally to the Imai rats for 32 weeks, and renal function and pathology were compared between the AST-120-administered and control Imai rats.RESULTS:AST-120-administered rats showed significantly lower level of blood urea nitrogen, serum creatinine, urinary protein, serum total-cholesterol, serum triglyceride, and serum and urinary indoxyl sulfate, and significantly higher levels of serum albumin and creatinine clearance than control rats. AST-120 reduced the glomerular sclerosis index, interstitial fibrosis area, and the extent of glomerular lipid deposition. Immunohistochemistry demonstrated that AST-120 reduced the expression of transforming growth factor (TGF)-beta 1 and tissue inhibitor of metalloproteinase (TIMP)-1 as well as interstitial infiltration of macrophages in the renal cortex of the Imai rats.CONCLUSIONS:AST-120 prevented the progression of nephrotic syndrome and renal failure in the Imai rats by ameliorating glomerular sclerosis and interstitial fibrosis, accompanied with reduced expression of TGF-beta 1 and TIMP-1, and reduced infiltration of macrophages in the kidneys.

To determine O-esterified positions, a rapid and complete acetalization to prepare an intermediate was established using ethyl vinyl ether as a new reagent. The new method was applied to O-esterified glycolipids followed by GC–MS analysis of the monosaccharide derivatives after methylation and methanolysis, revealing the derivatives with correctly substituted positions. This method was superior in terms of its shorter reaction time and complete acetalization, particularly of the N-glycolyl hydroxyl residue, to previously reported methods using methyl vinyl ether.

A sulfatide, O-fatty-acylated 3-sulfogalactosylceramide at C6-O on galactoside. was isolated from equine brain and the chemical structure was characterized by proton NMR and MS. The O-acylation site of the acylated sulfatide was determined by the down-field shift of protons attached to a carbon having an O-acyl group in the NMR spectrum and by analysis of a partially methylated derivative before and after acetalization of the intact sulfatide using GC-MS. The O-acyl chain length was determined by GLC, revealing that it exclusively had palmitoyl and stearoyl residues as the major fatty acids. The enzymatic conversion to the O-acyl sulfatide was further examined using equine brain microsomes as an enzyme source and different lipid substrates, resulting in O-acylation of 3-sulfogalactosylceramide from stearoyl CoA, while 6-O-acyl galactosylceramide was not O-sulfated from phosphoadenosine phosphosulfate. The results were supported by the comparably different N-linked fatty acid components between two lipid substrates, in which the component of 6-O-acyl sulfatide was mostly similar to that of sulfatide, but not to 6-O-acyl galactosylceramide.

Dihydroceramide desaturase, which catalyzes the introduction of a double bond at the 4,5-position of the sphingosine base in a dihydroceramide, was assayed in vitro using radiolabeled D-erythro-C18-dihydroceramide (N-stearoyl sphinganine) and homogenates of fetal rat skin, and some enzymatic properties, including substrate specificity, were determined. The ceramide structure, as the enzymatic product, was confirmed by (i) oxidation of the product with 2, 3-dicyano-5,6-dichlorobenzoquinone, which revealed the conversion to 3-ketoceramide (3,3'-didehydroceramide), indicating that a double bond was introduced at the adjacent to the C-3 hydroxyl residue of sphinganine, and (ii) mass spectrometry of a long chain base released from the enzymatic product, which revealed a spectrum identical to that of authentic sphingenine. A short chain dihydroceramide, which was radiolabeled at sphinganine through a newly established method, having a C2- or C6-fatty acid was not desaturated by the skin enzyme, whereas that having a C10-, C14-, or C18-acid was desaturated, maximal reactivity being observed for the C14-dihydroceramide. Other enzymatic properties were confirmed: NAD(H) or NADP(H) and a detergent were required for elevation of the activity; the optimum pH was approximately 6.7; and metal cations were not essential, but Zn2+, Cu2+, and Fe2+ were rather inhibitory. These properties of rat skin desaturase were partly similar to those of rat liver microsomes, as reported recently, however, their substrate specificities were different.

Three sulfolipid compounds, 1, 2 and 3, have been isolated from a higher plant, a pteridophyte, Athyrium niponicum, as potent inhibitors of the activities of calf DNA polymerase α and rat DNA polymerase β. The inhibition by the sulfolipids was concentration dependent, and almost complete inhibition of DNA polymerase α and DNA polymerase β was achieved at 6 and 8 μg/mL, respectively. The compounds did not influence the activities of calf thymus terminal deoxynucleotidyl transferase, prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase and Taq polymerase, the DNA metabolic enzyme DNase I, and even a DNA polymerase from a higher plant, cauliflower. Similarly, the compounds did not inhibit the activity of the human immunodeficiency virus type 1 reverse transcriptase. The kinetic studies of the compounds showed that DNA polymerase α was inhibited non-competitively with respect to the DNA template and substrate, whereas DNA polymerase β was inhibited competitively with both the DNA template and substrate. The binding to DNA polymerase β could be stopped with non-ionic detergent, but the binding to DNA polymerase α could not.

Fourteen patients with untreated hypercholesterolemia were given simvastatin 5 mg/d for 12 weeks. Cholesteryl ester transfer protein (CETP) activity and lecithin-cholesterol acyltransferase (LCAT) activity were determined every 4 weeks during treatment. Total cholesterol (TC) decreased from 279.1 ± 55.1 mg/dL before treatment to 224.6 ± 30.7 mg/dL after 12 weeks, and high-density lipoprotein cholesterol (HDL-C) rose from 56.2 ± 13 mg/dL to 61.2 ± 15.2 mg/dL. These changes were statistically significant. CETP activity decreased significantly from 26.8 ± 6.4% before treatment to 23.6 ± 6.3% after 12 weeks, and LCAT activity at 37 °C increased significantly from 43.3 ± 33.5 nmol/mL per hour to 68.4 ± 32.6 nmol/mL per hour. The changes in TC correlated with those in CETP activity at each time point (r = .674), but not with those in LCAT activity. The HDL-C level was negatively correlated with CETP activity at each time point (r = .655) but not with LCAT activity. These findings show that CETP activity may play a more important role than LCAT activity in simvastatin-induced HDL-C elevation and suggest that simvastatin increases HDL-C by reducing CETP activity secondary to low-density lipoprotein cholesterol reduction.

Equine brain gangliosides were isolated and their structures were characterized, to examine whether equine brain has N-glycolyl neuraminic acid in gangliosides, since other mammals predominantly possess N-acetyl neuraminic acid in brain gangliosides, and equine erythrocytes and organs except the brain have gangliosides exclusively containing N-glycolyl neuraminic acid. The gangliosides purified from the brain were identified by proton NMR spectroscopy and mass spectrometry, as well as GLC, resulting in their identification as GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. Of these gangliosides, GM3 possessed N-glycolyl neuraminic acid as a minor component (18% of the total GM3), whereas other gangliosides exclusively contained N-acetyl neuraminic acid. The N-glycolyl neuraminic acid residue of the GM3 was confirmed by TLC immunostaining. The possibility of contamination of the GM3 by erythrocytes was eliminated based on the finding that the lipid compositions were characteristic of brain gangliosides. The presence, even as a minor component, of the N-glycolyl neuraminic acid in equine brain gangliosides is exceptional among the sialic acid species in mammalian central nervous system.

Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment-mass spectrometry, as well as gas chromatography-mass spectrometry and gas-liquid chromatography. The orientation of the acetal chain linked to galactoside was characterized by connectivity between the cyclic acetal proton and ring proton(s) on the sugar moiety using the homonuclear Overhauser effect. This revealed that, of the two positional isomers of the acetal linkage with 4,6-O-acetal and 3,4-O-acetal derivatives obtained from the acetalization reaction, the former positional isomer, separated into two spots, was identified to 'endo'- and 'exo'-type acetal chains. In comparison to the NMR data of the synthesized derivative, equine brain acetalized lipid was found to be an 'endo'-type 4,6-O-acetal derivative.