Liver injury suppressing compounds from avocado (Persea americana).

To evaluate the protective activity of fruits against liver injury, 22 different fruits were fed to rats with liver damage caused by D-galactosamine, a powerful liver toxin. As measured by changes in the levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), avocado showed extraordinarily potent liver injury suppressing activity. Five active compounds were isolated and their structures determined. These were all fatty acid derivatives, of which three, namely, (2E,5E,12Z,15Z)-1-hydroxyheneicosa-2,5,12,15-tetraen-4-one, (2E,12Z,15Z)-1-hydroxyheneicosa-2,12,15-trien-4-one, and (5E,12Z)-2-hydroxy-4-oxoheneicosa-5,12-dien-1-yl acetate, were novel.

Purification and characterization of a lectin from the mushroom Mycoleptodonoides aitchisonii.

A lectin was isolated from the mushroom Mycoleptodonoides aitchisonii by means of affinity chromatography on bovine submaxillary mucin (BSM)-Toyopearl and gel filtration on Superose 12 HR10/30 using a FPLC system. This lectin is composed of four identical 16 kDa subunits and the molecular mass of the intact lectin was estimated to be 64 kDa by gel filtration. In a hemagglutination inhibition assay, it exhibited strong sugar-binding specificity towards asialo-BSM among the mono- or oligo-saccharides and glycoproteins tested. The binding specificity of the lectin was also examined by surface plasmon resonance analysis.

Purification and characterization of two lectins from a toxic moray, Gymnothrax javanicus.

Two lectins, Gymnothrax javanicus lectin-I (GJL-I) and Gymnothrax javanicus lectin-II (GJL-II) were isolated from the stomach and intestine, and the liver, respectively, of a toxic moray eel, Gymnothrax javanicus. GJL-I is a polymer of two heterogeneous subunits of 67 and 51 kDa. In a hemagglutination inhibition assay, it had sugar-binding specificity toward lactose and lactulose among the mono- or oligo-saccharides and bovine submaxillary mucin (BSM) among the glycoproteins tested. The lectin stimulated nerve growth factor (NGF) synthesis by astroglial cells. GJL-II was a polymer of subunit of 41 kDa. This lectin had N-acetyllactosamine binding specificity.

Suppression of D-galactosamine-induced liver injury by mushrooms in rats.

Six species of edible mushroom were found to suppress D-galactosamine-induced enhancement of plasma alanine and aspartate aminotransferase activities when powdered mushrooms were added to the diet (5%) and fed to rats for 2 wk. Grifola frondosa exhibited the most potent effect in a dose-dependent manner. A significant effect was observed only from the water-soluble low-molecular-weight fraction of G. frondosa. The results indicate that several mushrooms possess a protective effect against liver injury induced by D-galactosamine.

A lectin from an edible mushroom Pleurotus ostreatus as a food intake-suppressing substance.

In an experiment in which rats were allowed free access to food and water, the rats did not eat the diet containing a mushroom Pleurotus ostreatus even if they were emaciated. A P. ostreatus lectin (POL) was isolated from the mushroom as the food intake-suppression principle. In hemagglutination inhibition assays, Me-alphaGalNAc was the most potent inhibitor among the monosaccharides tested. Among all the sugars tested, 2'-fucosyllactose (Fucalpha1-->2Galbeta1-->4Glc) was the strongest inhibitor and its inhibitory potency was five times greater than that of Me-alphaGalNAc. POL exhibited a binding ability to bovine submaxillary mucin (BSM) and asialo-BSM and the other glycoproteins were inert to the binding. The food intake-suppressing activity of POL was dependent on the dose. The diet containing 0.1% POL caused a 50% decrease in the food intake of rats against the control.

Analysis of specific interactions of synthetic glycopolypeptides carrying N-acetyllactosamine and related compounds with lectins.

Analysis of interactions of synthetic glycopolypeptides with lectins was performed with a biosensor based on surface plasmon resonance (SPR). A series of synthetic oligosaccharide-substituted poly(L-glutamic acid)s were immobilized on sensor surfaces via the gamma-carboxyl groups of their peptide moieties by the surface thiol coupling method. Artificial glycopolypeptides: an N-acetyllactosamine-substituted polymer (1), an N-acetylisolactosamine-substituted polymer (2), a (GlcNAc)3-substituted polymer (3), a (GlcNAc)2-substituted polymer (4), and a p-aminophenyl N-acetyl-beta-lactosaminide-substituted polymer (5), were used as the ligands. On analysis by SPR, surface-bound polymers 1 and 5 reacted with Erythrina cristagalli agglutinin (ECA), Lycopersicon esculentum agglutinin (LEA), Ricinus communis agglutinin-120 (RCA120), and wheat germ (Triticum vulgaris) agglutinin (WGA). Polymer 2 reacted with WGA and RCA120, but did not with ECA and LEA. The results indicate that beta-(1-->4)-linked galactosyl residues are needed for binding to ECA and LEA. Polymer 3 reacted strongly with LEA and WGA, but polymer 4 reacted strongly only with WGA. Affinity constants (KA) for surface-bound polymer 5-lectin interactions were also about 4-61 times as strong as those for surface-bound polymer 1-lectin interactions. These artificial glycopolypeptides were shown to be useful as tools and probes of carbohydrate recognition and modeling in the analysis of glycoprotein-lectin interactions.

The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals.

The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein.

Synthesis of artificial N-glycopolypeptides carrying N-acetyllactosamine and related compounds and their specific interactions with lectins.

Artificial N-glycopolypeptides carrying N-acetyllactosamine (LacNAc) or related compounds were synthesized. First, sugars were converted into their corresponding beta-glycosylamines with ammonium hydrogen carbonate. Then, the beta-glycosylamines were condensated with the carboxyl groups of poly(L-glutamic acid). N-Glycopolypeptides with different degrees of substitution of sugars were isolated by passage through a column of Sephadex G-25. These synthetic polymers were used as model compounds in the analysis of oligosaccharide-lectin interactions. Interactions with some lectins were investigated by agar-gel double-diffusion tests and in terms of inhibition of hemagglutination. A glycopolypeptide substituted with LacNAc reacted with Erythrina cristagalli agglutinin (ECA), peanut (Arachis hypogaea) agglutinin (PNA), Ricinus communis agglutinin-120 (RCA120), wheat germ (Triticum vulgaris) agglutinin (WGA) lectins, which recognize either galactosyl or N-acetylglucosamine (GlcNAc) residues. Other synthetic glycopolymers carrying N-acetylisolactosamine, GlcNAc, N,N'-diacetylchitobiose, or N,N', N"-triacetylchitotriose also reacted with WGA, and these last two polymers inhibited hemagglutination most. Of these five glycopolypeptides, only the one substituted with LacNAc reacted with ECA. These sugar-substituted glycopolypeptides interacted specifically with the corresponding lectins, no matter how much shorter the sugar side chains of the glycopolymers were than those of natural glycoproteins.

Dictyophorines A and B, two stimulators of NGF-synthesis from the mushroom Dictyophora indusiata.

Two novel eudesmane-type sesquiterpenes, dictyophorines A and B, and a known compound, teucrenone, were isolated from the mushroom Dictyophora indusiata. Dictyophorines A and B promoted nerve growth factor (NGF)-synthesis by astroglial cells.

Dietary eritadenine modifies plasma phosphatidylcholine molecular species profile in rats fed different types of fat.

The effect of dietary eritadenine on plasma phosphatidylcholine (PC) molecular species composition was investigated in relation to its hypocholesterolemic action in rats fed different types of fats (olive oil, corn oil and linseed oil; 100 g/kg diet). Eritadenine supplementation (50 mg/kg diet) significantly decreased the plasma total cholesterol concentration, irrespective of dietary fat sources, and without change in the order of plasma cholesterol concentration among the fat groups (corn oil > olive oil > linseed oil). Eritadenine significantly decreased the ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in liver microsomes of all the fat groups, while the PC:PE ratio was unaffected by dietary fat type. The fatty acid and molecular species composition of plasma PC was affected either directly or indirectly by the fatty acid composition of dietary fats. The proportion of linoleic acid and linoleic acid-containing molecular species (16:0-18:2 and 18:0-18:2) in plasma PC was the highest in rats fed linseed oil, despite the fact that linoleic acid concentration of linseed oil was only 1/3 that of corn oil. Eritadenine supplementation significantly increased the proportion of linoleic acid and linoleic acid-containing molecular species, especially 16:0-18:2, in plasma PC, irrespective of dietary fat source. Altered plasma PC molecular species composition, as represented by an increase in 16:0-18:2 PC, might contribute to the hypocholesterolemic action of eritadenine.

Effect of capsianoside, a diterpene glycoside, on tight-junctional permeability.

Previous work (Hashimoto et al., (1994) Biosci. Biotech. Biochem. 58, 1345) revealed that a sweet pepper extract enhanced the tight-junctional (TJ) permeability of a human intestinal Caco-2 cell monolayer. In the present study, the substance which modulated the TJ permeability was chromatographically purified from the extract. The active substances were identified as capsianosides A-F, diterpene glycosides. Treatment of the cells with capsianoside F, the most active compound, decreased the cellular G-actin content by 40% and increased the F-actin content by 16%. The effect of capsianoside F was significantly suppressed by disturbing the cytoskeletal structure with cytochalasin D at a low dose (50 ng/ml). These results suggest that capsianosides affected the cytoskeletal function by modulating the reorganization of actin filaments, by which the TJ structure and permeability were changed. The possible involvement of a PKC inhibition in the mechanism of an increase in TJ permeability is also suggested.

A lectin from mycelia of the fungus Ganoderma lucidum.

A lectin (GLL-M) was isolated from mycelia of Ganoderma lucidum using affinity chromatography on BSM-Toyopearl. GLL-M is a monomer in its native form with a M(r) of 18,000. Another lectin was also purified from fruiting bodies of the same fungus. The two lectins were partially compared with each other.

Purification and characterization of a lectin from the toxic mushroom Amanita pantherina.

A lectin (APL) was isolated from the mushroom Amantia pantherina by means of hydrophobic chromatography on Butyl-Toyopearl, affinity chromatography on bovine submaxillary mucin (BSM)-Toyopearl and gel filtration on Superose 12 HR10/30 using a FPLC system. This lectin is composed of two identical subunits of 22 kDa and the molecular mass of the intact lectin was estimated to be 43 kDa by gel filtration. In hemagglutination inhibition assays, it exhibits sugar-binding specificities towards GlcNAc beta 1-->4Man beta-pNP, Gal beta 1-->4GlcNAc beta 1-->4GlcNAc, and Gal beta 1-->4GlcNAc beta 1-->4GlcNAc beta 1-->4GlcNAc among mono- and oligosaccharides tested. Among glycoproteins tested, BSM and asialo-BSM were the strongest inhibitors.

A pyradine-derivative from the mushroom Albatrellus confluens.

A novel pyradine-derivative was isolated from the mushroom Albatrellus confluens. This compound promoted melanin synthesis by B16 melanoma cells.

Two N-acetyl-D-galactosamine-specific lectins from Phaeolepiota aurea.

Two lectins, PAL-I and PAL-II, were isolated from the mushroom Phaeolepiota aurea by affinity chromatography on acid-treated Sepharose CL-4B followed by reverse-phase FPLC on ProRPC. Both of the lectins were tetramers of 16 kDa subunits. The lectins had little agglutination activity against native erythrocytes but Pronase treatment of erythrocytes increased the sensitivity to agglutination by the lectins. Both lectins exhibited slight preferences for type A compared with type B and O erythrocytes. In haemagglutination inhibition assays, N-acetylgalactosamine and both anomers of methyl N-acetylgalactosaminide were the best inhibitors.

Enzymic synthesis of lacto-N-triose II and its positional analogues.

N-acetylhexosaminidase from Nocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (beta-D-GlcNAc-(1-3)-beta-D-Gal-(1-4)-beta-D-Glc-OMe, 3) with its isomers beta-D-GlcNAc-(1-6)-beta-D-Gal-(1-4)-beta-d-Glc-OMe (4) and beta-D-Gal-(1-4)-[beta-D-GlcNAc-(1-6)]-beta-D-Glc-OMe (5) through N-acetylglucosaminyl transfer from N,N'-diacetylchitobiose (GlcNAc2) to methyl beta-lactoside. The enzyme formed the mixture of trisaccharides 3, 4 and 5 in 17% overall yield based on GlcNAc2, in a ratio of 20:21:59. With p-nitrophenyl beta-lactoside as an acceptor, the enzyme also produced p-nitrophenyl beta-lacto-N-trioside II (beta-D-GlcNAc-(1-3)-beta-D-Gal-(1-4)-beta-D-Glc-OC6H4NO2-p, 6) with its isomers beta-D-GlcNAc-(1-6)-beta-D-Gal-(1-4)-beta-D-Glc-OC6H4NO2-p (7) and beta-D-Gal-(1-4)-[beta-D-GlcNAc-(1-6)]-beta-D-Glc-OC6H4NO2-p (8). In this case, when an inclusion complex of p-nitrophenyl lactoside acceptor with beta-cyclodextrin was used the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound 6.

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain of N-glycoprotein.

Transmannosylation from mannotriose (Man beta 1-4Man beta 1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue of N,N'-diacetylchitobiose was regioselectively induced through the use of beta-D-mannanase from Aspergillus niger. The enzyme formed the trisaccharide Man beta 1-4GlcNAc beta 1-4GlcNAc (3.7% of the enzyme-catalysed net decrease of N,N'-diacetylchitobiose) from mannotriose as a donor and N,N'-diacetylchitobiose as an acceptor. Mannobiose (Man beta 1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.

A sialic acid-binding lectin from the mushroom Hericium erinaceum.

A lectin was isolated from the mushroom Hericium erinaceum. This lectin is composed of two different subunits of 15 and 16 kDa and the molecular mass of the intact lectin was estimated to be 54 kDa by gel filtration. It exhibits specificity towards sialic acids, especially N-glycolylneuraminic acid.

Two lectins from the marine sponge Halichondria okadai. An N-acetyl-sugar-specific lectin (HOL-I) and an N-acetyllactosamine-specific lectin (HOL-II).

Two lectins (HOL-I and HOL-II) were isolated from the marine sponge Halichondria okadai by affinity chromatography on a bovine submaxillary mucin (BSM)-Toyopearl and an acid-treated Sepharose 4B columns, respectively. In hemagglutination inhibition assays, GlcNAc, GalNAc, and their methyl glycosides were the most potent inhibitors among the monosaccharides tested against the HOL-I-mediated hemagglutination, suggesting that HOL-I can especially recognize the N-acetyl groups of the sugars. This N-acetyl specificity was supported by 1H NMR analyses; the highest field-shifts of the signal of the N-acetyl group among all the signals in Me beta GlcNAc were observed in 1H NMR spectra of mixtures of HOL-I and the sugar. Among the oligosaccharides tested, GlcNAc beta 1-->4(GlcNAc beta 1-->2)Man alpha 1-O(CH2)2 CH3 was the most potent inhibitor, and the inhibitory potency of the oligosaccharide was 2(4) times greater than those of GlcNAc and GalNAc. On the other hand, N-acetyllactosamine (Gal beta 1-->4GlcNAc) and its analogs were the strongest inhibitors toward HOL-II-induced hemagglutination. The agglutination was completely inert to Gal beta 1-->3GlcNAc, Gal beta 1-->6GlcNAc, Gal beta 1-->3GalNAc, Gal beta 1-->4GalNAc, and Gal beta 1-->6GalNAc. Furthermore, HOL-II exhibited no binding ability to BSM, asialo-BSM, fetuin, asialofetuin, alpha 1-acid glycoprotein, and human transferrin. These results indicate that HOL-II strictly recognizes simple Gal beta 1-->4GlcNAc unit.

Hypocholesterolemic Action of Dietary Grifolin on Rats Fed with a High-cholesterol Diet.

The hypocholesterolemic action of grifolin was investigated in terms of its structure-activity relationship with rats fed on a high-cholesterol diet. The results show that the structure of farnesylorcinol was required for the hypocholesterolemic action, and that the effect of grifolin might be elicited, at least in part, through the augmented excretion of cholesterol into the feces.