Galbeta1-6Gal, antigenic epitope which accounts for serological cross-reaction in diagnosis of Echinococcus multilocularis infection.

Parasitic helminths express various antigenic carbohydrates, which often account for serological cross-reactions. In serodiagnosis, it is essential to inspect cross-reactivity between the target parasite and other parasites in order to assess diagnostic performance. Our previous study showed that the Galbeta1-6Gal sequence was a common epitope between Echinococcus multilocularis (Em) and E. granulosus (Eg). Furthermore, compounds with this sequence from Fasciola hepatica (Fh) reportedly were recognized by sera with Eg infection. Our aim is to investigate whether this sequence is one of the widely common epitopes in many kinds of parasites. For various parasites, sera with Fh infection cross-reacted at the highest frequency (71.4%) against Em antigen. In patients with other parasitic infections, sera showed cross-reactions against Fh antigen bound to Em antigen with a high frequency (23.7%). Binding inhibition tests with commercial Galbeta1-6Gal disaccharide showed that Galbeta1-6Gal was the common epitope between not only Em, Eg and Fh, but also between various other parasites. Furthermore, the presence of the Galbeta1-6Gal epitope in Em antigen was confirmed by immunoblot testing with the specific antibody for this sequence. This study showed that the Galbeta1-6Gal sequence is one of the antigenic epitopes that accounts for serological cross-reactivity between Em and various other parasites.

Serodiagnostic potential of chemically synthesized glycosphingolipid antigens in an enzyme-linked immunosorbent assay for alveolar echinococcosis.

In the serodiagnosis of alveolar echinococcosis, the detection of specific reactions against not only protein but also carbohydrate antigen is useful and both antigens supplement each other. Though recombinant protein antigens have recently advanced, the preparation of carbohydrate antigen still depends on extraction from crude antigens. In the latter case, it is not conventional to obtain carbohydrate antigen as a single component for examination and research. Therefore, chemically synthesized carbohydrate antigens were prepared for serodiagnosis by the enzyme-linked immunosorbent assay (ELISA). Four antigens with the structure of glycosphingolipids from Echinococcus multilocularis were examined and one antigen, Galbeta1-6(Fucalpha1-3)Galbeta1-6Galbeta1-ceramide, was found to show significant serodiagnostic potential in differentiating alveolar from cystic echinococcosis.

Dissecting Ascaris glycosphingolipids for immunomodulatory moieties--the use of synthetic structural glycosphingolipid analogues.

We have previously shown glycosphingolipids of Ascaris suum to have phosphorylcholine (PC) and non-PC immunomodulatory moieties. In the present study we further investigated the nature of the immunomodulatory moieties by employing three synthetic glycosphingolipids each possessing features of the original molecule to examine effects on macrophage and dendritic cell (DC) cytokine production and surface co-stimulatory molecule expression. Compound 2, which lacked PC but contained ceramide, had no effect on either macrophages or DCs. Surprisingly however, Compound 1, which contained PC and hence arguably most resembled the native material, had, with the exception of a small increase in surface antigen expression, no immunomodulatory properties. Conversely, Compound 3, which contained PC but was otherwise least like the native molecule, demonstrated a number of effects on both macrophages and DCs, including induction of Th-1/pro-inflammatory cytokines, inhibition of such cytokines induced by IFN-gamma/LPS and increased expression of co-stimulatory molecules. Taken together these results indicate: (i) that although PC is an immunomodulatory component of the native molecule other structural feature are necessary to allow it to act; (ii) that carbohydrate rather than ceramide is likely to represent a non-PC immunomodulatory moiety; and (iii) that synthetic PC-containing molecules have the potential to act as immunomodulatory drugs.

Synthetic studies on glycosphingolipids from Protostomia phyla: synthesis of amphoteric glycolipid analogues containing a phosphocholine residue from the earthworm Pheretima hilgendorfi.

Two kinds of amphoteric glycosphingolipid analogues from the earthworm Pheretima hilgendorfi were synthesized as follows: The key reaction is a coupling of a phosphocholine group at the position C-6 of 1 and 6 which was attempted using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by reaction of the resulting cyclic phosphate intermediate with anhydrous trimethylamine to give 2 and 7. Subsequent debenzylation afforded target compounds (3, 8). Their ability to inhibit the histamine release in vitro was examined.

Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers.

Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains. However, norharman shows mutagenicity to S. typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines. We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman). In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix. When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC. The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis. The latter mutagen was identified to be the hydroxyamino derivative. Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix. Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix. These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative. After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S. typhimurium TA98 and YG1024. When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido[3,4-b]indole (amino-2'-methylphenylnorharman) was identified as a mutagen. Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.

Syntheses of model compounds related to an antigenic epitope in pectic polysaccharides from Bupleurum falcatum L.

Stereocontrolled syntheses of model compounds related to a category of the major antigenic epitope against anti-bupleurum 2IIc/PG-1-IgG from an anti-ulcer pectic polysaccharide are described. Glycosylation of the glucuronic acid donors methyl(2,3-di-O-benzoyl-4-O-methyl-alpha-D-glucopyranosyl trichloroacetimidate)uronate and methyl (2,3-di-O-benzoyl-4-O-methyl-beta-D-glucopyranosyl)uronate-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with the common acceptor 2-(trimethylsilyl)ethyl 2,3,4-tri-O-benzyl-beta-D-galactopyranoside in the presence of trimethylsilyl triflate (TMSOTf) gave the desired di- and trisaccharide derivatives. Furthermore the products were transformed into the oligo-valent clustering saccharides, N,N',N"-tri-(5-[4-O-methyl-beta-D-glucopyranosyluronic acid-(1-->6)-beta-D-galactopyranosyloxy]pentylcarbonylaminoethyl)-1,3,5-benzenetriamide and N,N',N"-tri-(5-[4-O-methyl-beta-D-glucopyranosyluronic acid (1-->6)-beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranosyloxy]pentylcarbonylaminoethyl)-1,3,5-benzenetriamide.

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of a glycosphingolipid analogue from the parasite Spirometra erinacei.

Novel neutral glycosphingolipids isolated from the plerocercoids of a tapeworm, Spirometra erinacei, may be expected to be involved in host-parasite interactions. We have synthesized this glycosphingolipid analogue containing 2-branched fatty alkyl residue in place of ceramide. Glycosylation of nonreducing-end trisaccharide derivative 15 with the reducing-end disaccharide derivative 17 in the presence of trimethylsilyl triflate (TMSOTf) gave the desired oligosaccharide derivative in good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycoside 19 was converted to glycosylimidate 20, which was condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated to give the target glycosphingolipid analogue 22.

Synthesis of a model compound related to an anti-ulcer pectic polysaccharide.

A stereocontrolled synthesis of the model compound for an anti-ulcer active polysaccharide (Bupleuran 2IIc) is described. Glycosidation of the disaccharide acceptor, 2-O-acetyl-3-O-benzyl-4-O-(p-methoxybenzyl)-alpha-L-rhamnopyranosyl-(1-- >4)-2,3,6-tri-O-benzyl-alpha-D-galactopyranosyl trichloroacetimidate, with the disaccharide receptor, allyl 3,4-di-O-benzyl-alpha-L-rhamnopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta- D-galactopyranoside, using silver triflate (AgOTf) as a promoter gave the desired tetrasaccharide derivative, which was transformed into the acidic tetrasaccharide, corresponding to a segment of the rhamnogalacturonan (Bupleuran 2IIc) polysaccharide, propyl alpha-L-Rha-(1-->4)-alpha-D-GalA-(1-->2)-alpha-L-Rha-(1-->4)-beta-D-GalA , via removal of the corresponding ether and ester protecting groups, followed by oxidation.

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of neogala-series glycolipid analogues containing a mannose residue from the earthworm Pheretima hilgendorfi.

Two kinds of glycosphingolipid analogues from the earthworm Pheretima hilgendorfi were synthesized as follows: the trisaccharide 2-(tetradecyl)hexadecyl alpha-D-mannopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->6)-beta-D- galactopyranoside (13) and the tetrasaccharide 2-(tetradecyl) hexadecyl alpha-D-galactopyranosyl-(1-->6)-[alpha-D-mannopyranosyl-(1-->4)]-beta-D - galactopyranosyl-(1-->6)-beta-D-galactopyranoside (20) were synthesized by stepwise condensation of suitably protected monosaccharide units. A 2-(trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside derivative (5) was used as the glycosyl acceptor and thiophenyl derivatives of D-galactose and D-mannose were used as donors respectively.

Synthetic studies on glycosphingolipids from the parasite Echinococcus multilocularis.

Novel neutral glycosphingolipids isolated from the metacestodes of Echinococcus multilocularis by Persat, may be expected to be involved in host-parasite interactions. We have synthesized these glycosphingolipid analogues containing 2-branched fatty alkyl residues in place of ceramide. The glycosylation of galactosyl donors 4 and 5 with each of the acceptors 2 and 11 in the presence of N-iodosuccinimide (NIS)/TfOH, and the glycosylation of fucosyl donor 13 with acceptors 12 and 20 in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) gave the desired oligosaccharide derivatives at good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycosides 6, 15, 21, and 26 were converted to glycosylimidates 7, 16, 22, and 27, which were condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated give four target glycosphingolipid analogues.

Structural determination of a mutagenic aminophenylnorharman produced by the co-mutagen norharman with aniline.

Norharman (9H-pyrido[3,4-b]indole), widely distributed in our environment, including cigarette smoke and cooked foodstuffs, is not mutagenic to Salmonella strains, but becomes mutagenic to S.typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. To elucidate the mechanisms of co-mutagenicity, we tried to isolate the mutagen(s) produced by a reaction between norharman and aniline with S9 mix. By HPLC purification, two mutagenic compounds (I and II), one (I) showing mutagenicity with and the other (II) without S9 mix, were isolated. The structure of compound I was deduced to be a coupled compound of norharman and aniline, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), by a variety of spectrometry techniques and this was confirmed by its chemical synthesis. The mutagenic activity of this novel heterocyclic amine was tested using the pre-incubation method and was found to induce 187,000 revertants in TA98 and 1,783,000 revertants in YG1024 per microg with S9 mix. Compound II was shown to be hydroxyaminophenylnorharman. Formation of the same DNA adducts was observed in YG1024 when aminophenylnorharman or a mixture of norharman plus aniline was incubated with S9 mix. The hydroxyamino derivative also yielded the same DNA adducts in YG1024. Thus, the appearance of mutagenicity by norharman with aniline in the presence of S9 mix suggests that the coupled mutagenic compound, aminophenylnorharman, is formed from norharman and aniline, then converted to the hydroxyamino derivative and forms DNA adducts to induce mutations in TA98 and YG1024.

6'-Sulfo sialyl Lex but not 6-sulfo sialyl Lex expressed on the cell surface supports L-selectin-mediated adhesion.

In order to determine if a sulfated oligosaccharide on the cell surface can function as an L-selectin ligand, a novel approach for in vitro transfer of oligosaccharides was utilized (Srivastava, G., Kaun, K. J., Hindsgaul, O., and Palcic, M. M. (1992) J. Biol. Chem. 267, 22356-22361). CHO cells were incubated with synthetic 6'-sulfo sialyl Lex, NeuNAcalpha2-->3(sulfate-6)Galbeta1-->4(Fucalpha1-->3) GlcNAc or 6-sulfo sialyl Lex, NeuNAcalpha2-->3Galbeta1-->4[(Fucalpha1-->3)sulfate--> 6GlcNAc] oligosaccharide linked to C-6 of a fucose residue in GDP-fucose and a milk fucosyltransferase. The resultant CHO cells expressing 6'-sulfo sialyl Lex or 6-sulfo sialyl Lex on their cell surface were tested for adhesion to E-selectin and L-selectin chimeric proteins coated on plates. The results indicate that 6'-sulfo sialyl Lex supports L-selectin-mediated adhesion much better than sialyl Lex similarly tagged on the cell surface. In contrast, 6-sulfo sialyl Lex containing a sulfate group on the N-acetylglucosamine residue did not support adhesion with either selectin. These combined results suggest that 6'-sulfo sialyl Lex is a much better ligand than sialyl Lex oligosaccharide for L-selectin.

Synthetic studies on an oligosaccharide of a glycolipid from the spermatozoa of bivalves. IX. Synthesis of lipids I, II, and IV.

Glycosphingolipids isolated from the spermatozoa of the freshwater bivalve, Hyriopsis schlegelii, have a unique structure containing one or two mannosyl residues and novel linkages, including an internal fucopyranosyl residue, as well as terminal xylosyl and 4-O-methyl-D-glucopyranosyluronic acid groups. The octasaccharide of lipid IV was synthesized as follows. Condensation of methyl (2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-methyl-alpha-D-galactopyranosyl )- (1-->3)-[methyl(2,3-di-O-acetyl-4-O-methyl-beta-D-glucopyranosyluronate) - (1-->4)]-2-O-benzyl-1-thio-alpha,beta-L-fucopyranoside (18) with (3-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1-- >2)- (3,4,6-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->3)-[(2,3,4-tri-O-acetyl -beta - D-xylopyranosyl)-(1-->2)]-(4,6-di-O-acetyl-beta-D-mannopyranosyl)-(1-->4 )- 2,3-di-O-acetyl-1,6-anhydro-beta-D-glucopyranose (14), in the presence of dimethyl (methylthio) sulfonium triflate (DMTST), gave the corresponding octasaccharide (19). Removal of the protecting groups gave 2-acetamido-2-deoxy-3-O-methyl-alpha-D-galactopyranosyl-(1-->3)-[4-O- methyl-beta-D-glucopyranosyl uronic acid-(1-->4)]-alpha-L-fucopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D- glucopyranosyl-(1-->2)-alpha-D-mannopyranosyl-(1-->3)- beta-D-xylopyranosyl- (1-->2)]-beta-D-mannopyranosyl-(1-->4)-1,6-anhydro-beta-D-glucopyranose (22). The other two oligosaccharides that constitute the partial structure of lipid IV, called lipid I and II, were also synthesized.

[Inhibitory effect on anti-lipid IV antiserum binding activity of various synthetic oligosaccharides].

The inhibitory effects of various synthetic oligosaccharides (1-8) on anti-lipid IV antiserum binding activity were examined by ELISA (enzyme linked immunosorbent assay). Compound 8, containing an epitope GlcA-4Me beta 1-4Fuc of lipid IV, inhibited but precursors (1-5) of lipid IV did not inhibit the binding activity. In addition to the nonreducing end disaccharide derivative (6) having a methyl group at position 4 of glucuronic acid, its analogous compound (7) having no methyl groups was synthesized and their inhibition activities were compared. Moreover, the inhibitory effects of compounds (1-5) were examined using each antiserum.