Anti-Diabetic Effects of a Kaempferol Glycoside-Rich Fraction from Unripe Soybean (Edamame,Glycine max L. Merrill. ‘Jindai’) Leaves on KK-Ay Mice

The anti-diabetic effects of a kaempferol glycoside-rich fraction (KG) prepared from leaves of unripe Jindai soybean (Edamame) and kaempferol, an aglycone of kaempferol glycoside, were determined in genetically type 2 diabetic KK-A^y mice. The hemoglobin A_1c level was decreased and tended to be decreased by respectively feeding KG and kaempferol (K). The area under the curve (AUC) in the oral glucose tolerance test (OGTT) tended to be decreased by feeding K and KG. The liver triglyceride level and fatty acid synthase activity were both decreased in the mice fed with KG and K when compared to those parameters in the control mice. These results suggest that KG and K would be useful to improve the diabetes condition. The major flavonoids in KG were identified as kaempferol 3-O-β-D-glucopyranosyl(1→2)-O-[α-L-rhamnopyranosyl(1→6)]-β-D-galactopyranoside, kaempferol 3-O-β-D-glucopyranosyl(1→2)-O-[α-L-rhamnopyranosyl(1→6)]-β-D-glucopyranoside, kaempferol 3-O-β-D-(2-O-β-D-glucopyranosyl) galactopyranoside and kaempferol 3-O-β-D-(2,6-di-O-α-L-rhamnopyranosyl) galactopyronoside, suggesting that these compounds or some of them may be concerned with mitigation of diabetes.     

Biotransformation of Cinnamic Acid,p-Coumaric Acid,Caffeic Acid,and Ferulic Acid by Plant Cell Cultures of Eucalyptus perriniana

Biotransformations of phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated with plant-cultured cells of Eucalyptus perriniana. The plant-cultured cells of E. perriniana converted cinnamic acid into cinnamic acid β-D-glucopyranosyl ester, p-coumaric acid, and 4-O-β-D-glucopyranosylcoumaric acid. p-Coumaric acid was converted into 4-O-β-D-glucopyranosylcoumaric acid, p-coumaric acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcoumaric acid β-D-glucopyranosyl ester, a new compound, caffeic acid, and 3-O-β-D-glucopyranosylcaffeic acid. On the other hand, incubation of caffeic acid with cultured E. perriniana cells gave 3-O-β-D-glucopyranosylcaffeic acid, 3-O-(6-O-β-D-glucopyranosyl)-β-D-glucopyranosylcaffeic acid, a new compound, 3-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, 4-O-β-D-glucopyranosylcaffeic acid, 4-O-β-D-glucopyranosylcaffeic acid β-D-glucopyranosyl ester, ferulic acid, and 4-O-β-D-glucopyranosylferulic acid. 4-O-β-D-Glucopyranosylferulic acid, ferulic acid β-D-glucopyranosyl ester, and 4-O-β-D-glucopyranosylferulic acid β-D-glucopyranosyl ester were isolated from E. perriniana cells treated with ferulic acid.     

Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans

Delipidated cell walls from Aureobasidium pullulans were fractionated systematically.

The cell surface heteropolysaccharide contains D-mannose, D-galactose, D-glucose, and D-glucuronic acid (ratio, 8.5:3.9:1.0:1.0). It consists of a backbone of (1→6)-α-linked D-mannose residues, some of which are substituted at O-3 with single or β-(1→6)-linked D-galactofuranosyl side chains, some terminated with a D-glucuronic acid residue, and also with single residues of D-glucopyranose, D-galactopyranose, and D-mannopyranose.

This glucurono-gluco-galactomannan interacted with antiserum against Elsinoe leucospila, which also reacted with its galactomannan, indicating that both polysaccharides contain a common epitope, i.e., at least terminal β-galactofuranosyl groups and also possibly internal β-(1→6)-linked galactofuranose residues.

It was further separated by DEAE-Sephacel column chromatography to gluco-galactomannan and glucurono-gluco-galactomannan.

The alkali-extracted β-D-glucan was purified by DEAE-cellulose chromatography to afford two antitumor-active (1→3)-β-D-glucans. One of the glucans (M_r, 1–2 × 10⁵) was a O-6-branched (1→3)-β-D-glucan with a single β-D-glucosyl residue, d.b., 1/7, and the other (M_r, 3.5–4.5 × 10⁵) had similar branched structure, but having d.b., 1/5. Side chains of both glucans contain small proportions of β-(1→6)-and β-(1→4)-D-glucosidic linkages.     

Efficient Production and Purification of Extracellular 1,2-α-L-Fucosidase of Bacillus sp. K40T

When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-α-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5–7.0 and was stable at pH 6.0–9.0. The enzyme hydrolyzed the α(1 → 2)-L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I 〈O-α-L-fucose-(1 → 2)-O-β-D-galactose-(1 → 3)-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze α(1 → 3)-, α-(1 → 4)- and α-(1 → 6)-L-fucosidic linkages in LNF-III 〈O-β-D-galactose-(1 → 4)[O-α-L-fucose-(1 → 3)-]-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, LNF-II 〈O-β-D-galactose-(1 → 3)[O-α-L-fucose-(1 → 4)-]-N-acetyl-O-β-D-galactose-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉 or 6-O-α-L-fucopyranosyl-N-acetylglucosamine.     

Formation of β-Fructosyl Compounds of Pyridoxine in Growing Culture of Aspergillus niger

Two pyridoxine compounds were found to be formed in a culture filtrate of Aspergillus niger and A. sydowi, when grown in a medium containing sucrose and pyridoxine. Each of the two compounds I and II was obtained as a white powdered preparation by preparative paper chromatography, gel filtration on Toyopearl HW-40S and Sephadex G-10 columns, DEAE-cellulose column chromatography, and lyophilization. Compounds I and II were identified as 5?-O-(β-D-fructofuranosyl)-pyridoxine and 5?-O-(β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl]-pyridoxine, on the basis of the various experimental results, viz., elementary analyses, UV, ¹H-, and ¹³C-NMR spectra, products by hydrolysis with acid and yeast β-D-fructofuranosidase, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid. Levansucrase from Microbacterium laevaniformans and yeast β-D-fructofuranosidase did not catalyze the β-D-fructofuranosyl transfer from sucrose to pyridoxine to give rise to β-D-fructofuranosyl-pyridoxine.     

Oligonucleotides Containing Disaccharide Nucleosides: Synthesis,Physicochemical, and Substrate Properties

Abstract

The efficient synthesis of oligonucleotides containing 2′-O-β-D-ribofuranosyl (and β-D-ribopyranosyl)nucleosides, 2′-O-α-D-arabinofuranosyl (and α-L-arabinofuranosyl)nucleosides, 2′-O-β-D-erythrofuranosylnucleosides, and 2′-O-(5′-amino-5-deoxy-β-D-ribofuranosyl)nucleosides have been developed.     

Synthesis of 3-O-β-N-Acetylglucosaminyl Cyclic Tetrasaccharide through a Lysozyme-catalyzed Transfer Reaction

Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→} (CTS). Structural analysis showed that the transfer product was3-O-β-N-acetylglucosaminyl CTS, cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[β-GlcNAc-(1→3)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→}. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.     

Acceptor Specificity of Cyclodextrin Glycosyltransferase from Bacillus stearothermophilus and Synthesis of α-D-Glucosyl O-β-D-galactosyl-(1→4)-β-D-glucoside

Bacillus stearothermophilus CGTase had a wider acceptor specificity than Bacillus macerans CGTase did and produced large amounts of transfer products of various acceptors such as D-galactose, D-mannose, D-fructose, D- and L-arabinose, d- and L-fucose, L-rhamnose, D-glucosamine, and lactose, which were inefficient acceptors for B. macerans CGTase. The main component of the smallest transfer products of lactose was assumed to be α-D-glucosyl O-β-D-galactosyl-(l→4)-β-D-glucoside.     

Syntheses of a Neobiosamine C Derivative and Its Analogs

Methyl 2,5-di-O-p-nitrobenzoyl-β-d-ribofuranoside was prepared via methyl 2,3-O-ethoxyethylidene-β-d-ribofuranoside from d-ribose. It was condensed with 3,4,6-tri-O-acetyl-2-deoxy-2-(2′,4′-dinitroanilino)-α-d-glucopyranosyl bromide and 3,4-di-O-acetyl-2,6-dideoxy-2-(2′,4′-dinitroanilino)-6-phthalimido-α-d-glucopyranosyl bromide by a modified Königs-Knorr reaction to give neobiosamine analogs. The condensation reaction gave α-glucosides as the minor product, and the corresponding β-glucoside as the major product.     

Isolation and Constitution of a Xylan from Bamboo

A xylan from bamboo culm was isolated by extraction with aikali of chlorite holocellulose and fractional precipitation as a copper complex. The structure was investigated by means of examination of acid components by controlled hydrolysis, methylation analysis, and periodate oxidation. As a result, 4-O-methyl-α-D-glucuronic acid and 2-O-(4-O-methyl-α-D-glucopyranosyluronic acid) D-xylose were isolated and identified as acid components of the bamboo xylan. Hydrolysis of the fully methylated products afforded 2,3,5-tri-O- methyl-L-arabinose (1.6 moles), 2,3,4-tri-O-methyl-D-xylose (1.2 moles), 2,3,4,6-tetra-O-methyl-D-glucose(0.4 moles), 2,3-di-O-methyl-D-xylose (35.8 moles) and mono-O-methyl-D-xylose (2.6 moles). In addition to the above methylated sugars, 2,3,4-tri-O-methyl-D-glucuronic acid and partially methylated aldobiouronic acid were separated by cellulose column chromatography and identified. These results suggest that the bamboo xylan consists mainly of a linear backbone of 1,4-linked β-D-xylopyranose unit, to which L-arabinofuranose and 4-O-methyl-D-glucuronic acid were attached as a single side chain unit at C₂ or C₃.

Additional evidence for a linear chain structure has been given by periodate oxidation. On oxidation by periodate, the bamboo xylan consumed 1.09 moles of periodate and produced 0.05 mole of formic acid per anhydroxylose unit.     

Isolation of Oligosaccharides Corresponding to the Branching-point of Konjac Mannan

Ultracentrifugically homogeneous glucomannan acetate derived from konjac mannan was subjected to acetolysis. Besides β-1,4-linked oligosaccharides composed of D-mannose and/or D-glucose, three oligosaccharides corresponding to the branching point of the polysaccharide were isolated and identified as (1) 3-O-β-D-mannopyranosyl-D-mannose, (2) O-β-D-mannopyranosyl-(1→4)-O-β-D-mannopyranosyl-(1→3)-D-mannose, and (3) O-β-D- mannopyranosyl-(1→3)-O-β-D-mannopyranosyl-(1→4)-D-glucose. The average chain length (CL) was, moreover, determined to be about 46 by methylation analysis. The structural pattern of the glucomannan, including the branching point, is discussed.     

Syntheses of Some Derivatives of Glycosyl Pantothenic Acids,Analogues of Growth Factor for MLF Bacteria

A growth factor (TJF) for a malo-lactic fermentation bacterium (Leuconostoc sp.) has been found to be 4′-O-(β-D-glucopyranosyl)-D-pantothenic acid with structural and synthetical studies. Now other 4′-O-glycosides (β-D-ribofuranosyl, α-D-glucopyranosyl, β-D-galacto-pyranosyl, β-maltosyl and β-cellobiosyl) and 2′,4′-O-di-β-D-glucopyranoside of DL-pantothenic acid, and 4′-O-β-D-glucopyranoside of DL-pantethine were synthesized to examine their biological activities. The improved syntheses of TJF were also examined.     

Synthesis of α-L-Mannopyranosyl-containing Disaccharides and Phenols as Substrates for the α-L-Mannosidase Activity of Commercial Naringinase

In order to clarify the substrate specificity of the α-L-mannosidase activity of naringinase (Sigma), the following disaccharides and phenol glycosides were freshly prepared: methyl 2-O-(α-L-mannopyranosyl)­β-D-glucoside (1), methyl 3-O-(α-L-mannopyranosyl)-α-D-glucoside (2), methyl 4-O-(α-L-mannopyranosyl)-α-D-glucoside (3), methyl 5-O-(α-L-mannopyranosyl)-β-D-glucoside (4), methyl 6-O-(α-L-mannopyranosyl)-α-D­glucoside (5), 6-O-(α-L-mannpyranosyl)-D-galactose (6), p-nitrophenyl α-L-mannoside (7), and 4-methyl umbelliferone α-L-mannoside (8).These compounds, except for 3 and 5, were hydrolyzed with naringinase.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.     

Structural Analysis of an Extracellular Polysaccharide Bioflocculant of Klebsiella pneumoniae

The glycoside composition and sequence of an extracellular polysaccharide flocculant of Klebsiella pneumoniae H12 was analyzed. GC and HPLC analysis of the acid-hydrolysate identified its constituent monosaccharides as D-Glc, D-Man, D-Gal, and D-GlcA in an approximate molar ratio of 3.9:1.0:2.3:3.6. To analyze the glycoside sequence, the polysaccharide was partially hydrolyzed by acid and enzyme treatment. GC, HPLC, TLC, MALDI-TOF/MS, and 1H- and 13C- NMR spectroscopy characterized the obtained oligosaccharides.

The results clarified the partial structure of H12 polysaccharide as a linear polymer of a unit of pentasaccharide with a side chain of one D-GlcA to D-Glc moiety (see below). Although the existence of other sequences or other constituent glycosides could not be fully excluded, H12 polysaccharide must be a novel types as such a complicated unit for a polymer has not so far been reported. The partial structure of a H12 polysaccharide flocculant is also discussed in this report.

→4)- α-D-Glcp-(1→2)-α-D-Manp-(1→3)-4,6-Pyr-β-D- 3 Galp-(1→4)-β-D-Galp-(1→ ↓

1 β-D-GlcpA     

Variation of Protein among Eleven Varieties of Common Bean (Phaseolus vulgaris)

The acylated, amidated and esterified derivatives of N-acetylglucosaminyl-α(1 → 4)-N-acetylmuramyl tri- and tetrapeptide were synthesized and examined as to their protective effect on pseudomonal infection in the mouse and pyrogenicity in the rabbit. Modifications of the terminal end function of the peptide moieties in their molecules caused enhancement of resistance to pseudomonal infection and reduction of pyrogenicity. Among the compounds tested, sodium N-acetylglucosaminyl-β(1 → 4)-N-acetylmuramyl-l-alanyl-d-isoglutaminyl-(l)-stearoyl-(d)-meso-2,6-diaminopimelic acid-(d)-amide and sodium N-acetylglucosaminyl-β(1 → 4)-N-acetylmuramyl-l-alanyl-d-isoglutaminyl-(l)-stearoyl-(d)-meso-2,6-diaminopimelic acid-(d)-amide-(l)-d-alanine were found to be advantageous and conceivably worthwhile for further investigation as immunobiologically active compounds.     

Synthesis and Biological Activity of the Lipophilic Derivatives of N- Acetyl-1-thiomuramoyl-l-alanyl-d-isoglutamine

A variety of the lipophilic derivatives at C-1 and C-6 in N-[2-O-(2-acetamido-2,3-dideoxy-1-thio-β-d-glucopyranose-B-yl)-d-lactoy]-l-alanyl-(N¹-fatty acyl)-d-isoglutamine methyl esters were synthesized from 2N-acetyl-1-S-acetyl-4,6-O-isopropylidene-1-thiomuramoyl-l-alanyl-d-isogluta-mine methyl ester. Their immunoadjuvant activity in guinea-pigs, and the protective effect in mice infected with Escherichia coli (E-77156) were examined.     

Characterization of a Thermostable Endo-β-1,4-D-galactanase from the Hyperthermophile Thermotoga maritima

A putative endo-β-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, β-1,4-D-galactobiose, β-1,4-D-galactotriose, and β-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 °C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 °C.     

Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.     

Mutant of Escherichia coli Tolerant to the Lysis Induced by Cephalexin

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes α,β-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5′-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the α,β-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D- and L-β-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.