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Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the non reducing end oligosaccharides of glycosphingolipids from Ascaris suum have been accomplished. Galα1→3GalNAcβ1→OR (1), Galβ1→3Galα1→3GalNAcβ1→OR (2), Galβ1→6Galα1→3GalNAcβ1→OR (3), Galβ1→6(Galβ1→3)Galα1→3GalNAcβ1→OR (4) and GlcNAcβ1→6Galβ1→6(Galβ1→3)Galα1→3GalNAcβ1→OR (5) (R = biotinylated probe) were synthesized by stepwise condensation (1-4) and block synthesis (5) using 5-(methoxycarbonylpentyl) 2-O-benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (12) as a common precursor. Compound 12 was converted into two kinds of glycosyl acceptors and was condensed with suitable galactosyl donors, respectively.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the non reducing end oligosaccharides of glycosphingolipids from Ascaris suum have been accomplished. Galα1→3GalNAcβ1→OR (1), Galβ1→3Galα1→3GalNAcβ1→OR (2), Galβ1→6Galα1→3GalNAcβ1→OR (3), Galβ1→6(Galβ1→3)Galα1→3GalNAcβ1→OR (4) and GlcNAcβ1→6Galβ1→6(Galβ1→3)Galα1→3GalNAcβ1→OR (5) (R = biotinylated probe) were synthesized by stepwise condensation (1-4) and block synthesis (5) using 5-(methoxycarbonylpentyl) 2-O-benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-β-D-galactopyranoside (12) as a common precursor. Compound 12 was converted into two kinds of glycosyl acceptors and was condensed with suitable galactosyl donors, respectively.

A novel asymmetric direct Mannich-type reaction of α-iminophenylacetate esters with thionolactones, bearing a substituent at the α-position, as a less acidic pronucleophile was developed. Using bis(guanidino)iminophosphorane as the chiral organosuperbase catalyst, the reaction afforded densely functionalized amino-acid derivatives having vicinal quaternary stereogenic centers, one of which is an all-carbon quaternary stereogenic center, in good yield with high diastereo- and enantioselectivities.

A chiral bis(guanidino)iminophosphorane catalyzes enantioselective addition reactions of a 1,3-dithiane derivative as a pronucleophile. The chiral uncharged organosuperbase facilitates the addition of benzyloxycarbonyl-1,3-dithiane to aromatic N-Boc-protected imines to provide optically active α-amino-1,3-dithiane derivatives, which are valuable versatile building blocks in organic synthesis.

Helically chiral spirocyclic P3 phosphazenes were designed as a novel family of chiral organosuperbases. The newly designed chiral P3 phosphazenes were synthesized from commercially available sources in several steps and characterized by X-ray crystallographic analysis of their onium salts. The optically pure P3 phosphazenium salt was obtained by using preparative chiral stationary phase HPLC and the absolute configuration of the helical chirality was determined.

Propolis, a complex resinous material collected by honeybees from buds and exudates of certain plant sources neighboring its hive, is considered to possess broad spectrum of biological activities and has historical utilization in folk medicine. Thus, it is extensively being used in health food, pharmaceutical preparations etc. The chemical consistency of propolis is highly dependent on the flora of the region from where it is collected. In this study, we investigated propolis from different geographical locations in Nepal and isolated several known compounds. As a result, we clarified the origins of propolis in Nepal.

A novel asymmetric direct Mannich-type reaction of α-iminophenylacetate esters with thionolactones, bearing a substituent at the α-position, as a less acidic pronucleophile was developed. Using bis(guanidino)iminophosphorane as the chiral organosuperbase catalyst, the reaction afforded densely functionalized amino-acid derivatives having vicinal quaternary stereogenic centers, one of which is an all-carbon quaternary stereogenic center, in good yield with high diastereo- and enantioselectivities.

A chiral bis(guanidino)iminophosphorane catalyzes enantioselective addition reactions of a 1,3-dithiane derivative as a pronucleophile. The chiral uncharged organosuperbase facilitates the addition of benzyloxycarbonyl-1,3-dithiane to aromatic N-Boc-protected imines to provide optically active α-amino-1,3-dithiane derivatives, which are valuable versatile building blocks in organic synthesis.

Fractionation of acetone and methanol extracts of Viola yedoensis, under the guidance of inhibition against Clostridium histolyticum collagenase (ChC), resulted in the isolation of esculetin (1) (IC(50) 12 μM) and scopoletin (2) (IC(50) 1.8 μM) as the active constituents, together with trans-p-coumaric acid (3), cis-p-coumaric acid (4), 3-O-β-D-glucosyl-7-O-α-L-rhamnosylkaempferol (5), rutin (6), isovitexin (7), isoorientin (8), vicenin-2 (9), isoscoparin (10), vanillic acid (11) and adenosine (12). Modification of phenolic hydroxy groups of 1 showed that small O-alkyl groups largely increased the activity, whereas larger O-alkyl groups decreased the activity, and 6,7-dimethoxycoumarin (scoparone 13) potently inhibited ChC (IC(50) 24 nM).

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions with a Galβ1-3GalNAc core of the Em2 glycoprotein antigen obtained from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Galβ1-3(GlcNAcβ1-6)GalNAcα1-R (G), tetrasaccharide Galβ1-3(Galβ1-4GlcNAcβ1-6)GalNAcα1-R (J) and pentasaccharide Galβ1-3(Galα1-4Galβ1-4GlcNAcβ1-6)GalNAcα1-R (K) (R = biotinylated probe) were synthesized by block synthesis by the use of 5-(methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-β- d -galactopyranosyl-(1→3)-2-azido-4-O-benzyl-2-deoxy-α- d -galactopyranoside as a common glycosyl acceptor. Moreover, linear trisaccharide Galα1-4Galβ1-3GalNAcα1-R (H) and branched tetrasaccharide Galα1-4Galβ1-3(GlcNAcβ1-6)GalNAcα1-R (I) were synthesized by stepwise condensation. We examined the antigenicity of these five oligosaccharides by an enzyme linked immunosorbent assay (ELISA). Our results demonstrate that biotinylated oligosaccharides H, I and K show good serodiagnostic potential to detect infections caused by the parasite E. multilocularis. Among them the linear sequence Galα1-4Galβ1-3GalNAcα1-R in oligosaccharide (H) appears to show the highest sensitivity (95%). Moreover, our study clarified the dominant carbohydrate epitope of Em2 antigen.

Synthetic access to three neutral glycosphingolipids from the parasite Schistosoma mansoni adult worm has been achieved. These structures differ significantly from those of other parasites and exhibit a unique structural motif termed "schisto-core" consisting of GalNAcbeta1--4Glcbeta1--sequence. We have synthesized glycosphingolipids, beta-D-GalNAcp-(1--4)-beta-D-Glcp-(1--1)Cer (1), beta-D-GlcNAcp-(1--3)-beta-D-GalNAcp-(1--4)-beta-D-Glcp-(1--1)Cer (2) and beta-D-Galp-(1--4)-beta-D-GlcNAcp-(1--3)-beta-D-GalNAcp-(1--4)-beta-D-Glcp-(1--1)Cer (3).

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.

This review describes the carbohydrate study and the natural product related to the glycoside chemistry. What shall the people in the field of pharmacognosy and natural products chemistry search in scene in future? Forty years before while isolating dimeric compound having naphthoquinonepyrone skeleton from the coloring material produced by the pathogen that hosted in wheat and caused rotten root disease, silica gel has to be treated with oxalic acid to reduce the absorbency before separation. However now a days, availability of reversed phase adsorbents for liquid chromatography has made the separation and isolation of complex compounds possible, easy and rapid. With the advancement of mechanical/physicochemical analytic methods, it has even been possible to isolate traces of compounds present in complex. This advancement has made it possible to determine structure of saponins and complex polysaccharides without decomposition and carry out in vitro bioassay at the same time using various cells on-line. Further, this review describes the oligosaccharide syntheses and biological activities of glycosphingolipids, focusing especially on those found in invertebrates.

Lysichiton camtschatcense is a well-known plant in Japan where it has been used as a traditional medicine by the "Ainu" people for the treatment of acute nephritis. It is presumed that L. camtschatcense has an inhibitory effect against nephritis caused by reactive oxygen species (ROS) owing to its antioxidant activities. Consequently, the antioxidant effect of L. camtschatcense extracts was assessed against Fe(2+)/ascorbic acid (AsA)-induced lipid peroxidation in rat kidney and brain homogenates. The antioxidant effect of the chloroform extract (CE) was more potent than that of the methanol extract (ME) for both homogenates. The antioxidant effect of both extracts was similar to those of alpha-tocopherol, a lipid-soluble antioxidant, and glutathione (GSH), a water-soluble antioxidant, which were used as reference compounds. Although CE showed a low radical-scavenging effect for superoxide anion radicals (O(2)(*-)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, assessed by using an electron spin resonance (ESR) method, hydroxyl radicals (*OH) were markedly scavenged by more than 80%. On the other hand, ME showed more significant scavenging effect for DPPH radicals and O(2)(*-) than CE. These results suggest that the inhibitory effects of the L. camtschatcense extract on lipid peroxidation in rat kidney and brain are based on its high radical-scavenging effect against *OH, O(2)(*-) , and lipid-derived radicals generated from the cell membrane.

Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Gal beta 1--3GalNAc core in the glycan portion of the glycoprotein antigen from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Gal beta 1--3(GlcNAc beta 1--6)GalNAc alpha 1-OR (A), tetrasaccharide Gal beta 1--3(Gal beta 1--4GlcNAc beta 1--6)GalNAc alpha 1-OR (D), and pentasaccharides Gal beta 1--3(Gal beta 1--4Gal beta 1--4GlcNAc beta 1--6)GalNAc alpha 1-OR (E) and Gal beta1--3(Gal alpha 1--4Gal beta 1--4GlcNAc beta 1--6)GalNAc alpha 1-OR (F) (R = 2-(trimethylsilyl)ethyl) were synthesized by block synthesis. The disaccharide 2-(trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1--3)-2-azido-4-O-benzyl-2-deoxy-alpha-d-galactopyranoside served as a common glycosyl acceptor in the synthesis of the branched oligosaccharides. Moreover, linear trisaccharide Gal beta 1--4Gal beta 1--3GalNAc alpha 1-OR (B) and branched tetrasaccharide Gal beta 1--4Gal beta 1--3(GlcNAc beta 1--6)GalNAc alpha 1-OR (C) were synthesized by stepwise condensation.

Several fruit juices have been reported to cause food-drug interactions, mainly affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on conjugation reactions. Among several fruit juices tested (apple, peach, orange, pineapple, grapefruit, and pomegranate), pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in Caco-2 cells. This inhibition was both dose- and culture time-dependent, with a 50% inhibitory concentration (IC(50)) value calculated at 2.7% (vol/vol). In contrast, no obvious inhibition of glucuronidation of 1-naphthol in Caco-2 cells was observed by any of the juices examined. Punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in Caco-2 cells with an IC(50) of 45 microM, which is consistent with that of pomegranate juice. These data suggest that punicalagin is mainly responsible for the inhibition of sulfoconjugation by pomegranate juice. We additionally demonstrated that pomegranate juice and punicalagin both inhibit phenol sulfotransferase activity in Caco-2 cells in vitro, at concentrations that are almost equivalent to those used in the Caco-2 cells. Pomegranate juice, however, shows no effects on the expression of the sulfotransferase SULT1A family of genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity by punicalagin in Caco-2 cells is responsible for the reductions seen in 1-naphthyl sulfate accumulation. Our data also suggest that constituents of pomegranate juice, most probably punicalagin, impair the enteric functions of sulfoconjugation and that this might have effects upon the bioavailability of drugs and other compounds present in food and in the environment. These effects might be related to the anticarcinogenic properties of pomegranate juice.

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.

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 fromEchinococcus multiloculariswere examined and one antigen, Galβ1-6(Fucα1-3)Galβ1-6Galβ1-ceramide, was found to show significant serodiagnostic potential in differentiating alveolar from cystic echinococcosis.

Two new dalbergiones (1–2) and a new flavanone (3) were isolated along with nine other known flavonoids (4–12) from the methanolic extract of propolis collected from Chitwan, Nepal. The structures were determined on the basis of spectral analysis.

Apyrases (E.C.3.6.1.5; NTP-NDPases) are distributed in the cytosol, nuclei, cytoskeleton, and on the surface of plant cells. Some may play an important role in signal transduction from exogenous stimuli. We previously found a protein of ca. 55-kDa (CWP-55) in an ATPase-rich fraction from the pea cell wall bound to the elicitor and supprescins (suppressors of defense) from pea pathogen Mycosphaerella pinodes. We cloned the cDNA of CWP-55 that coincided with PsAPY1, one of two NTPase clones in a pea cDNA library. An analysis with a green fluorescent protein fusion protein indicated that PsAPY1 was distributed in the cell wall, nucleus, and cytoplasm. The recombinant PsAPY1 expressed in Escherichia coli had ATP-hydrolyzing activity responsive not only to the elicitor and supprescins from the pea pathogen but also to other elicitors such as a bacterial harpin, a yeast extract, and a synthetic glycopeptide. Biotinylated fungal signal molecules were bound to the recombinant PsAPY1 specifically. Resonant mirror detection confirmed such binding characteristics of PsAPY1. Based on these results, we discuss the role of cell-wall-bound NTPases in recognizing and responding to microorganisms on the cell wall surface.

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules.

6-N-[2-(Tetradecyl)hexadecanamido]hexyl beta-D-glucopyranosyluronic acid-(1--6)-beta-D-galactopyranosyl-(1--6)-beta-D-galactopyranoside (1) and its clustering compound (2) carrying a tetravalent sugar unit, which are new model compounds related to a major antigenic epitope from antiulcer pectic polysaccharide of Bupleurum falcatum L., were synthesized and the distributions of 1 and 2 in mixed ganglioside (GM1, GD1a or GT1b)/phospholipid (DPPC) monolayers were observed using atomic force microscopy (AFM). AFM images showed that 1 was distributed in the GM1, GD1a and GT1b region of the mixed monolayers, in which 1 was miscible with GD1a. Specific distribution of 1 was observed in the mixed GM1/DPPC monolayer. Compound 2 was miscible with GM1, while 2 formed associations with GD1a and GT1b in the mixed monolayers. The distribution mode of 1 and 2 was different among the mixed ganglioside/DPPC monolayers.

Three known monodesmosidic saponins: 3-O-β-d-glucopyranosyl-2β,3β,16α,23,24-pentahydroxyolean-12-ene-28-oic acid, 3-O-β-d-glucopyranosyl polygalacic acid, and 3-O-β-d-glucopyranosyl-(1→3)-β-d-glucopyranosyl polygalacic acid; and two known nonsaponin compounds: a mixed compound of n-tetracosanoic acid (lignoceric acid), n-hexacosanoic acid (cerotic acid), and n-octacosanoic acid, and α-monopalmitin; were isolated for the first time from the root of Platycodon grandiflorum A. DC. together with another seven known compounds: platycoside G1 (3-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl-2β,3β,16α,23,24-pentahydroxyolean-12-ene-28-oic acid 28-O-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside), deapio-platycodin D, Polygalacin D, deapio-platycodin D3, platycoside A, α-spinasterol, and α-spinasteryl-3-O-β-d-glucopyranoside. Alkaline hydrolysis of platycoside G1 afforded a new monodesmosidic prosaponin: 3-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl-2β,3β,16α,23,24-pentahydroxyolean-12-ene-28-oic acid. Their chemical structures were elucidated on the basis of their spectral data and chemical evidence.

Four new diterpenoids, salvifulgenolide (1), trans-1,2-dihydrosalvifaricin (2), ent-19- O-acetoxy-15,16-epoxy-3,13(16),14-clerodatrien-6,18-diol (3) and ent-19-acetoxy-15,16-epoxy-6-hydroxy-3,13(16),14-clerodatrien-18-al (4) along with two known diterpenoids, 10β-hydroxybacchotricuneatin A (5) and dehydrokerlin (6) were isolated from the aerial parts of Salvia fulgens Cav. (Labiatae). The structures were established by spectroscopic methods, including the X-ray analysis of salvifulgenolide and trans-1,2-dihydrosalvifaricin.

A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo , exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (III B ) infection in MT-4 cells with EC 50 values of 0.5 μg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC 50 > 1000 μg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC 50 of 11.56 μg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2–400 μg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100 °C in 1N HCl or 1N NaOH, and after treatment with 100 mM NaIO 4 . The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000–50,000.

We have previously found that oral or intravenous (i.v.) administration of the polysaccharide fraction PB-2, extracted from the lichen Flavoparmelia baltimorensis, facilitated the induction of long-term potentiation (LTP) in the dentate gyrus (DG) in vivo. In this study, the mechanism underlying the effect of PB-2 on the induction of LTP was investigated in the DG of anesthetized rat focusing on the contribution of the interleukin-1 (IL-1) receptor and the adrenaline beta-receptor. An i.v. injection of IL-1ra (10(-9) g/kg), an antagonist of the IL-1 receptor, had no effect on the basal response in the DG; however, this treatment augmented the enhancement of LTP induced by a single i.v. injection of PB-2 (10(-3) g/kg). This potentiating effect was also observed following intracerebroventricular (i.c.v.) injection of IL-1ra (10(-15)-10(-11) g). An i.v. injection of IL-1beta (3.5 x 10(-15)-3.5 x 10(-9) g/kg) inhibited the induction of LTP, which was diminished by the previous application of IL-1ra. These results suggest that the activation of the IL-1 receptor induces the suppression of LTP in PB-2-treated rats, and that endogenous IL-1beta contributes to the IL-1 receptor activation. An i.c.v. infusion of metoprolol (7.5 x 10(-6) g), an antagonist of the adrenaline beta(1)-receptor, attenuated the enhancement of LTP induced by an i.v. injection of PB-2. These results suggest that PB-2 has two different effects on the LTP, an enhancing effect and an inhibiting one, and that it exhibited the significant enhancing effect on the LTP as a total balance of these effects.

Efficient and systematic syntheses of four neutral glycosphingolipids that have been isolated from the metacestodes of Echinococcus multilocularis have been achieved. A key step is the direct glycosylation of galactosyl donors using thioglycosides with benzoyl ceramide in the presence of N-iodosuccinimide (NIS)/TfOH, which gave the desired oligosaccharide derivatives. The fully protected glycosides 13, 20, 22 and 25 were deprotected to give four target glycosphingolipids (1-4).

Effects of a Kampo (Japanese herbal) medicine "Sho-seiryu-to (SST, Xiao-Qing-Long-Tang in Chinese)", which has been used for the treatment of allergic bronchial asthma clinically, were examined on ovalbumin (OVA)-sensitized allergic airway inflammation model (i.e., bronchial asthma) in a mouse. When SST was orally administered at 0.5 g/kg/day from day 1 to 6 days after OVA inhalation, SST reduced the OVA-specific IgE antibody titer in bronchoalveolar lavage (BAL) fluids at 7 days after the OVA inhalation. CD4(+) T cells obtained from the mouse lung produced more interleukin (IL)-4 and IL-5 but less interferon (IFN)-gamma than T cells from nonsensitized control animals. However, oral administration of SST reduced the production of IL-4 and IL-5 and the production of IFN-gamma returned to the control level. In addition, the IL-4 level was increased in the BAL fluid of the OVA-sensitized animals compared to the nonsensitized control, while the IFN-gamma levels decreased. SST reduced the IL-4 levels in the BAL fluids and returned the IFN-gamma level to control levels. Nerve growth factor (NGF) was increased in the BAL fluids of the OVA-sensitized mice over that of nonsensitized mice, but oral administration of SST augmented the NGF levels to approximately 2 times higher than in the sensitized mice. Although lung cells obtained from sensitized mice produced higher levels of NGF than nonsensitized mice, oral administration of SST augmented the production of NGF by the lung cells even higher ( approximately 2 times more than cells from sensitized mice). Administration of anti-NGF antibody to the airway blocked the effects of SST. These results suggest that SST modulates Th1/Th2 balance in the lungs and augmentation of NGF in the lungs may be related to the effects of SST. Pinellic acid (9S, 12S, 13S-trihydroxy-10E-octadecenoic acid), one component of the herbs of SST [Int. Immunopharmacol. 2 (2002) 1183], was purified from the tuber of Pinellia ternata Breitenbach. Oral administration of pinellic acid (50 microg/kg/day) also reduced the OVA-specific IgE antibody titer in BAL fluids from the sensitized mouse. This result suggests that pinellic acid is one of active ingredient(s) in SST.

We found for the first time that Zygomycetes species showed resistance to Aureobasidin A, an antifungal agent. A novel family of neutral glycosphingolipids (GSLs) was found in these fungi and isolated from Mucor hiemalis, which is a typical Zygomycetes species. Their structures were completely determined by compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry, and (1)H NMR spectroscopy. They were as follows: Gal beta 1-6Gal beta 1-1Cer (CDS), Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTS), Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTeS), and Gal alpha 1-6Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CPS). The ceramide moieties of these GSLs consist of 24:0, 25:0, and 26:0 2-hydroxy acids as major fatty acids and 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid. However, the glycosylinositolphosphoceramide families that are the major GSLs components in fungi were not detected in Zygomycetes at all. This seems to be the reason that Aureobasidin A is not effective for Zygomycetes as an antifungal agent. Our results indicate that the biosynthetic pathway for GSLs in Zygomycetes is significantly different from those in other fungi and suggest that any inhibitor of this pathway may be effective for mucormycosis, which is a serious pathogenic disease for humans.

We studied the intraocular pharmacokinetics of dorzolamide hydrochloride eye drops and the effect of dorzolamide on carbonic anhydrase activity and localization in ocular tissues. Carbonic anhydrase activity was detected in normal ocular tissues. The activity was inhibited in corneal endothelial cells, the ciliary body, lens epithelial cells, or the retina 1 to 8 hours after instillation of dorzolamide eye drops. In lens epithelial cells and the retina, the enzyme activity had not recovered even 10 hours after instillation of the drug. Immunostaining did not reveal any differences between the group administered dorzolamide eye drops and the control group administered a physiologically balanced solution. Time-related changes in dorzolamide concentrations in ocular tissues were measured by high-performance liquid chromatography (HPLC). In the cornea, anterior aqueous, iris, ciliary body and retina, drug concentrations increased 15 minutes after the instillation and peaked within 1 hour. These results suggest that dorzolamide immediately suppresses carbonic anhydrase activity in ocular tissues, and is rapidly distributed among the tissues of the eye when administered as eye drops.

Two new phenylethanoid glycosides integrifoliosides A (2) and B (3), along with a known phenylethanoid glycoside alyssonoside (1) and a flavone glucoside chrysoeriol-7-O-β-ᴅ-glucopyranoside (4) were isolated from the aerial parts of Phlomis integrifolia. The structures of the new compounds were identified as 3,4-dihydroxy-β-phenylethoxy-O-β-ᴅ-apiofuranosyl- (1 →4)-α-ʟ-rhamnopyranosyl-(1 →3)-4-O-feruloyl-β-ᴅ-glucopyranoside (2) and 3-hydroxy-4-methoxy-β-phenylethoxy-O-β-ᴅ-apiofuranosyl-(154)-α-ʟ-rhamnopyranosyl-(1→3)-4-O-feruloyl-β-ᴅ-glucopyranoside (3), on the basis of spectroscopic (UV, IR, 1D- and 2D-NMR, and HR-FABMS) methods.

Ganglioside GM3 (GM3) liposomes were prepared by the Bangham method and a reverse-phase evaporation method (RPE method), and the particle size, dispersibility, trapping efficiency, adsorption amount of bovine serum albumin (BSA) on the liposomes and leakage of aqueous-space markers from the liposomes in the presence of BSA were examined. The particle size of the liposomes prepared by the Bangham method was smaller and the trapping efficiency was low, while reasonable liposomes being approximately 150 nm in diameter and having about 3% trapping efficiency were able to be prepared by the RPE method. The entrapped efficiency of glucose in the liposomes was higher than that of calcein because of the electrostatic repulsion between negatively charged GM3 liposomes and calcein. No significant adsorption of BSA on the GM3 liposomes and thus no significant leakage of aqueous-space markers from the liposomes in the presence of BSA were observed. The GM3 liposomes were relatively stable in the presence of BSA. The dispersibility and uniformity of the GM3 liposomes were also superior. In addition, the leakage of aqueous-space markers from the GM3 liposomes by freeze-drying was extremely low.

Liposomes composed of ganglioside GM3 (GM3), Gal β 1(3←2 α NANA)→4Glc β1→1Cer, and l -α-dipalmitoylphosphatidylglycerol (DPPG) were prepared by varying the amount of GM3, and the effects of GM3 on the membrane properties of liposomes in the presence of bovine serum albumin (BSA) were examined at pH 7.4 and 37 °C in terms of adsorption amount of BSA and permeability of the liposomal bilayer membranes. GM3 incorporated into DPPG liposomes inhibited the adsorption of BSA on the liposomes, and the leakage of calcein as an aqueous-space marker from liposomes through adsorption of BSA decreased. However, a large amount of GM3 incorporated into liposomes brought about a probable phase separation in the liposomal bilayer membranes, thereby increasing their permeability. The leakage of calcein from the liposomes in the presence of BSA showed a minimum value at 0.10 mole fraction of GM3.

A novel amphoteric glycosphingolipid, cholinephosphoryl-(-->6)-beta-D-GlcpNAc-(1-->3)-beta-D-Manp-(1-->4)-beta-D-Glcp-(1-->)-Cer, isolated from the porcine parasitic nematode, Ascaris suum, may be expected to be involved in host-parasite interactions. This glycosphingolipid analogue containing octyl residue in place of ceramide was synthesized as follows: The key reaction of this synthetic procedure is the formation of a intramolecular aglycon delivery (IAD) approach for beta-selective mannosylation. Then, a coupling of phosphocholine group at the position C-6'' of 16 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 17. Subsequent debenzylation and debenzylidenation afforded target compound (2).

Two novel phosphonoglycolipids, named PnGC1 and PnGC2 were isolated from the marine crab, Erimacrus isenbeckii by successive column chromatography on ion exchange Sephadex (DEAE- and QAE-Sephadex) and silicic acid (Iatrobeads). Their chemical structures were characterized as phosphonocerebrosides, 4’-O-(2-aminoethylphosphonyl) Glcpβ1-1 ceramide for PnGC1 and 4’-O-(N-methyl-2-aminoethylphosphonyl) Glcpβ1-1 ceramide for PnGC2 by IR, MALDI-TOF MS, 1H-NMR, GC and GC-MS analyses of the water-soluble products after acid hydrolysis, and methylation of the product of hydrogen fluoride degradation. In both phosphonocerebrosides, the ceramide moieties were composed of tetradeca-4-sphingenine as the sole sphingoid, and stearic, arachidic, behenic and docosamonoenoic acids as the major fatty acids.

Newly developed bis(guanidino)iminophosphoranes possess the highest basicity among chiral organocatalysts reported to date.

The neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides (GL-1, GL-2, GL-3, GL-4a and GL-4b), were identified from whole tissues of the marine crab Erimacrus isenbeckii by successive column chromatography with ion exchange Sephadex (QAE-Sephadex), magnesium silicate (Florisil) and silicic acid (Iatrobeads) resins. Through component analysis, sugar analysis, methylation studies, exoglycosidase cleavage, and various chromatographic and spectrometric techniques, their structures were proposed to be as follows: GL-1, Glcβ1-1Cer; GL-2, Manβ1-4Glcβ1-1Cer; GL-3, Galβ1-3Manβ1-4Glcβ1-1Cer; and GL-4a and GL-4b, Gal3Meα1-4Galβ1-3Manβ1-4Glcβ1-1Cer. The main molecular species of the aliphatic moiety in each purified glycolipid were 18:0, 22:0, 22:1-d14:1 (fatty acid-sphingoid) and 18:0-d16:1 for GL-1; 18:0-d16:1 and 22:1-d14:1, d16:1 for GL-2; 22:1, 24:1-d16:1 for GL-3; 22:1, 24:1-d16:1 for GL-4a; and h22:1, h24:1-d16:1 for GL-4b, respectively. By immunological detection, an arthro-series glycosphingolipid (At3Cer; GlcNAcβ1-3Manβ1-4Glcβ1-1Cer) was also detected as a minor component. The characteristic arthro-series glycosphingolipid has been observed in most animals belonging to the phylum Arthropoda.

Micellar inhibition effect of gangliosides on a degradation of drug was investigated, where ganglioside G(M1) (GM1), G(D1a) (GD1a) and G(T1b) (GTlb) whose sialic acid residue is one, two and three, respectively, were used. The base-catalyzed isomerization of prostaglandin A(2) (PGA(2)) to prostaglandin B(2) (PGB(2)) was chosen as a model experiment. The rate for the isomerization of PGA(2) was determined by measuring the concentration of PGA(2) (and PGB(2)) with a high-performance liquid chromatography. Gangliosides micelles inhibited the isomerization of PGA(2). The inhibition effect of GT1b micelles was larger than that of GD1a micelles. This result would be due to the larger absolute value of surface potential of GT1b micelles, which brings about a larger electrostatic repulsion between micellar surface and OH(-). The terminal sialic acid residue of ganglioside was effective to inhibit the isomerization of PGA(2). GM1 micelles without terminal sialic acid residue but with large aggregation number exhibited a superior steric shielding effect rather than an electrostatically repulsive effect. The inhibition effect of GM1 micelles was enhanced by the mixed micellization with the other ganglioside with a terminal sialic acid residue. GM1-GD1a or GM1-GT1b mixed micelles remarkably inhibited the isomerization of PGA(2). The physiological activity of PGs in the biological membranes containing gangliosides was also discussed.

Six new minor saponins, together with known ginsenosides, were isolated from the leaves of Panax ginseng. The new saponins were named as ginsenoside-Rh5, -Rh6, -Rh7 -Rh8, -Rh9 and -Rg7, and their structures were elucidated on the basis of chemical and physicochemical evidence to be as follows: ginsenoside-Rh5: 3beta,6alpha,12beta,24zeta-tetrahydroxy-dammar-20(22),25-diene 6-O-beta-D-glucopyranoside (1), -Rh6: 3beta,6alpha12beta,20(S)-tetrahydroxy-25-hydroperoxy-dammar-23-ene 20-O-beta-D-glucopyranoside (2), -Rh7: 3beta,7beta,12beta,20(S)-tetrahydroxy-dammar-5,24-diene 20-O-beta-D-glucopyranoside (3), -Rh8: 3beta,6alpha,20(S)-trihydroxy-dammar-24-ene-12-one 20-O-beta-D-glucopyranoside (4), -Rh9: 3beta,6alpha,20(S)-trihydroxy-12beta,23-epoxy-dammar-24-ene 20-O-beta-D-glucopyranoside (5) and -Rg7: 3-O-beta-D-glucopyranosyl 3beta,12beta,20(S),24(R)-tetrahydroxy-dammar-25-ene 20-O-beta-D-glucopyranoside (6).

Examination was made of the effects of 2-(trimethylsilyl)ethyl 4-Ο-methyl-β-D-glucopyranosyluronic acid-(1→6)-β-D-galactopyranoside (A), 2-(trimethylsilyl)ethyl 4-Ο-methyl-β-D-glucopyranosyluronic acid-(1→6)-β-D-galactopyranosyl-(1→6)-β-D-galactopyranoside (B) and N, N',N''-tri-{5-[4-Ο-methyl-β-D-glucopyranosyluronic acid-(1→6)-β-D-galactopyranosyloxy]pentylcarbonylaminoethyl}-1,3,5-benzenetriamide (C), each possessing the glucuronic acid residue, on drug degradation. Oligosaccharide mixed micelles containing the nonionic surfactant, heptaethyleneglycol dodecylether (HED), were studied so as to assess oligosaccharides A∼C for drug stabilization potential. The nonionic surfactant was required since oligosaccharides A∼C do not form micelles in single systems. Base-catalyzed dehydration and then isomerization of prostaglandin E2 (PGE2), PGE2→PGA2→PGB2, were conducted as model experiments. The rate of the degradation of PGE2 with base was determined based on concentrations of PGA2 and PGB2 using high-performance liquid chromatography. Mixed oligosaccharide-HED micelles inhibited the dehydration and isomerization of PGE2, possibly owing to suppression of the approach of OH- as catalysis toward PGE2 in mixed oligosaccharide-HED micelles by electrostatic repulsion between negatively charged micellar surfaces and OH-. The clusterized molecular structure of oligosaccharide C was the reason for the inhibition of both these processes. Oligosaccharide C may possibly be situated on the micellar surface and this would lead to greater steric shielding and the above electrostatic repulsion compared to A or B.

Gangliosides participate in cellular interactions and signal transductions. An initial step in those processes is glycolipid interaction. We observed the conformational change of ganglioside GM1 (GM1) at the membrane surface by means of atomic force microscopy (AFM), and revealed the following changes of GM1 with increasing surface pressure : a uniform pattern at 29 mN/m, a swelling pattern at 33 mN/m, and a return to a uniform pattern at 40 mN/m. This behavior is thought to be related to specific cell recognition or signal transduction, and may prove useful for elucidating the functions of glycolipid microdomains in biological membranes.

A novel series of fucolipids, provisionally named CPS and CHS in the previous study, was obtained from whole tissues of the marine annelid, Pseudopotamilla occelata. The structures of these fucolipids were determined by compositional analysis, methylation analysis, gasliquid chromatography, gas chromatograph-mass spectrometry, proton nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The structures were shown to be Xylβ1-4Fucα1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer (CPS1) and Gal2Meα1-3Fucα1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer (CPS2) for CPS, and Xylβ1-4 (Gal2Meα1-3) Fucα1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer for CHS, respectively. They were structurally related to the previously described ceramide trisaccharide (GlcNAcβ1-3Galβ1-4Glcβ1-1Cer, Amino-CTH) except that a fucose-containing di- and/or trisaccharides were linked to the N-acetylglucosamine residue of the latter lipid. The predominant fatty acids were monoenoic acids, C20:1- and C22:1 acids amounting to about 80% of the total acids, and octadeca-4-sphingenine was the sole sphingoid base. The fatty acid and sphingoid base compositions showed nearly the same distribution in these fucolipids.

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.

A novel glycosphingolipid, β- d -Manp-(1→4)-[(α- l -Fucp-(1→3)]-β- d -Glcp-(1→1)-Cer, from the millipede, Parafontaria laminata armigera, was synthesized. A key reaction of this synthetic procedure is the formation of a spiro-orthoester and its reduction for β-selective mannosylation.

Four new triterpenoid saponins, ardisimamilloside C (1), 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]-3beta,16al pha,28,30-tetrahydroxy-olean-12-en, ardisimamilloside D (2), 3-O-?alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]-3beta,15al pha,28,30-tetrahydroxy-olean-12-en, ardisimamilloside E (3), 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosl]-13beta,2 8-epoxy-3beta,16alpha,29-oleananetriol, and ardisimamilloside F (4), 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)-[beta -D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl]-3beta,16al pha-dihydroxy-13beta,28-epoxy-oleanan-30-oic acid were isolated from the roots of Ardisia mamillata Hance. Structure assignments were established on the basis of highresolution (HR)-FAB-MS, 1H-, 13C-, and two-dimensional (2D)-NMR spectra, and on the chemical evidence.

New C 2 -symmetric chiral 1,2-diols, 1,2-bis(1-adamantyl)-1,2-ethanediol and 3,3,6,6-tetramethyl-1,2-cyclohexanediol, were synthesized by the use of a new resolution method.

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.

Micellar surface potentials (Δψ) of icosapolyenoic acids (IPA) having 25 double bonds were determined at 25°C from fluorescence intensity of ammonium 8-anilino-1-naphthalenesulfonate as a fluorescent probe. The surface potential, |-Δψ|, of IPA micelles decreased with increase in the number of double bond in the IPA molecule. Decrease in |-Δψ| by the introduction of a double bond in the IPA molecule was approximately 13.12mV at ionic strength 0.02mol dm-3. The relationship was represented well by the following equation : Δψ/mV=-108.0+13.12n, where n is the number of C=C double bonds in an IPA molecule. Surface charge density |-σ| of IPA micelles based on the Gouy-Chapman theory was found to diminish as n increased. The relationship can be expressed well by the following equation : σ/c cm-2=-3.386×10-6+2.463×10-6·(√n-√2), where n≥2.