Teruhiko Nitoda

Structural Features of N-Glycans Linked to Royal Jelly Glycoproteins: Structures of high-mannose type, hybrid type, and biantennary type glycans

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing β1-2 xylosyl and/or α1-3 fucosyl residue(s) and occurrence of β1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man9~4GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of β-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a β1-4 linkage N-acetylglucosaminyl residue.

Bile acids are new products of a marine bacterium, Myroides sp. strain SM1

Strain SM1 was isolated as a biosurfactant-producing microorganism from seawater and presumptively identified as Myroides sp., based on morphology, biochemical characteristics and 16S rDNA sequence. The strain produced surface-active compounds in marine broth, which were purified, using emulsification activity for n-hexadecane as an indicator. The purified compounds were identified by thin-layer chromatography, 1H- and 13C-NMR spectra and fast atom bombardment mass spectrometry as cholic acid, deoxycholic acid and their glycine conjugates. Type strains of the genus Myroides, M. odoratus JCM7458 and M. odoramitimus JCM7460, also produced these compounds. Myroides sp. strain SM1 possessed a biosynthetic route to cholic acid from cholesterol. Thus, bile acids were found as new products of prokaryotic cells, genus Myroides.

Monoacylglycerols: glycolipid biosurfactants produced by a thermotolerant yeast, Candida ishiwadae

Aims:  To isolate and characterize biosurfactants produced by a thermotolerant yeast isolated in Thailand.

Effects of quercetin on mushroom tyrosinase and B16-F10 melanoma cells.

In searching for tyrosinase inhibitors from plants using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, quercetin was found to be partially oxidized to the corresponding o-quinone under catalysis by mushroom tyrosinase (EC 1.14.18.1). Simultaneously, L-DOPA was also oxidized to dopaquinone and both o-quinones were further oxidized, respectively. The remaining quercetin partially formed adducts with dopaquinone through a Michael type addition. In general, flavonols form adducts with dopaquinone as long as their 3-hydroxyl group is free. Quercetin enhanced melanin production per cell in cultured murine B16-F10 melanoma cells, but this effect may be due in part to melanocytotoxicity. The concentration leading to 50% viable cells lost was established as 20 microM and almost complete lethality was observed at 80 microM.

Pochonicine, a polyhydroxylated pyrrolizidine alkaloid from fungus Pochonia suchlasporia var. suchlasporia TAMA 87 as a potent β-N-acetylglucosaminidase inhibitor

A new polyhydroxylated pyrrolizidine alkaloid designated as pochonicine (1) was isolated from a solid fermentation culture of the fungal strain Pochonia suchlasporia var. suchlasporia TAMA 87. The structure of 1 was determined using NMR and MS techniques as (1R*, 3S*, 5S*, 6S*, 7R*, 7a S*)-5-acetamidomethyl-3-hydroxymethyl-1,6,7-trihydroxypyrrolizidine. Pochonicine (1) showed potent inhibition against beta-N-acetylglucosaminidases (GlcNAcases) of various organisms including insects, fungi, mammals, and a plant but no inhibition against beta-glucosidase of almond, alpha-glucosidase of yeast, or chitinase of Bacillus sp. The GlcNAcase inhibitory activity of pochonicine (1) was comparable to nagstatin, a potent GlcNAcase inhibitor of natural origin.

TMG-chitotriomycin, an enzyme inhibitor specific for insect and fungal β-N-acetylglucosaminidases, produced by actinomycete Streptomyces anulatus NBRC 13369

A novel beta-N-acetylglucosaminidase (GlcNAcase) inhibitor named TMG-chitotriomycin (1) was isolated from the culture filtrate of Streptomyces anulatus NBRC13369. The strain produced 1 only when colloidal chitin was used as the sole carbon source in the production medium. The structure of 1 was determined by spectral and constitutive sugar analyses of the corresponding alditol derivatives to be an equilibrated mixture of alpha-d-N,N,N-triMeGlcNH2-(1,4)-beta-d-GlcNAc-(1,4)-beta-d-GlcNAc-(1,4)-d-GlcNAc and its C-2 epimer of the reducing end residue. TMG-chitotriomycin (1) showed potent and selective inhibition of insect and fungal GlcNAcases with no inhibition of mammalian and plant GlcNAcases. In contrast, the known GlcNAcase inhibitor nagstatin potently inhibited all GlcNAcases. It should be emphasized that synthesized d-N,N,N-triMeGlcNH2, which is the component sugar of 1, showed no inhibition of the insect Spodoptera litura GlcNAcase. These results suggest that the (GlcNAc)3 unit positioned at the reducing end of 1 is essential for its enzyme inhibitory activity. The unique inhibitory spectrum of 1 will be useful to study chitinolytic systems and to develop selective fungicides or pesticides.

Synthesis of Eight Stereoisomers of Pochonicine: Nanomolar Inhibition of β-N-Acetylhexosaminidases

Pochonicine, the first naturally occurring polyhydroxylated pyrrolizidine containing an acetamidomethyl group, which was isolated from Pochonia suchlasporia var. suchlasporia TAMA 87, together with its enantiomer and their C-1 and/or C-3 epimers, have been synthesized from the sugar-derived cyclic nitrones 9D and 9L, respectively. An in-depth NMR study showed that both the (1)H and (13)C NMR spectra of the synthetic pochonicines (1D and 1L) matched very well with those of natural pochonicine in D2O, which unequivocally determined the relative configuration of the natural product as 1D or 1L. In addition, comparison of the optical rotations of the synthetic pochonicines and that of the natural product, but more convincingly their glycosidase inhibition profiles, confirmed the absolute configuration of natural pochonicine as 1R,3S,5R,6R,7S,7aR. Thereby, the structure of natural pochonicine was unequivocally determined as (+)-(1R,3S,5R,6R,7S,7aR)-pochonicine (1D). Glycosidase inhibition experiments showed that natural pochonicine 1D and its epimers 2D, 3D, and 4D all are powerful inhibitors of hexosaminidases (five β-N-acetylglucosaminidases and two β-N-acetylgalactosaminidases) while their enantiomers 1L, 2L, 3L, and 4L are much weaker inhibitors of the same enzymes. (-)-3-epi-Pochonicine (2L) was found to be a potent and selective inhibitor of α-l-rhamnosidase. None of the compounds showed any inhibition of α-GalNAcase.

Tin-Carbon Cleavage of Organotin Compounds by Pyoverdine from Pseudomonas chlororaphis

ABSTRACT The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT. Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized. The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P. chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads. Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I. Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants. F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively. The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production. The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine. On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu2+ and Sn4+, respectively. These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.

Enzymatic Conversion of Cyclic Dipeptides to Dehydro Derivatives That Inhibit Cell Division

The cell-free extract of an albonoursin-producing strain, Streptomyces albulus KO-23, was found to catalyze the conversion of several cyclic dipeptides having Phe and aliphatic side chain-containing amino acid residues to the corresponding dehydro derivatives. 3Z-Benzylidene-6S-methyl-2,5-piperazinedione, 3Z-benzylidene-2,5-piperazinedione, and 3Z, 6Z-dibenzylidene-2,5-piperazinedione were prepared by this conversion system. Among the dehydro cyclic dipeptides prepared, tetradehydro derivatives exhibited inhibitory activity toward the first cleavage of sea urchin embryo, while didehydro derivatives did not. We previously found that cyclo(Leu-Phe) and its didehydro derivatives did not show any inhibitory activity, in contrast to high activity in the case of albonoursin. Taken together, these findings indicate that dehydrogenation at the alpha,beta-positions of both amino acid residues in this type of cyclic dipeptide is required for the inhibitory activity.

Three Antinematodal Diterpenes from Euphorbia kansui

Three compounds, 20-O-acetyl-[3-O-(2′E,4′Z)-decadienoyl]-ingenol (1), 20-O-acetyl-[5-O-(2′E,4′Z)-decadienoyl]-ingenol (2) and 3-O-(2′E,4′Z)-decadienoylingenol (3), were isolated from Euphorbia kansui under the bioassay-guided method. Each compound showed the same antinematodal activity against the nematode, Bursaphelenchus xylophilus, at a minimum effective dose (MED) of 5 μg/cotton ball.