Three groups of polysaccharides from the edible mushroom "Maitake, " the cultured fruiting body of Grifola frondosa, were extracted with hot water, 3% NH₄-oxalate (100°C), and 5% sodium hydroxide solution (30°C) in this order. The three fractions, FI, FII and Fill, were divided into several sub-fractions using various chromatographic techniques.
The fractions with host-mediated antitumor activity were as follows:
Water-soluble β-(1→ 3)-o-glucan: FI₀-a-β₁, [α]_D +9° (water), MW 1, 000, 000, N 0%, degree of branching = 3, average chain-length = 5, ID₅₀ (the dose level, mg/kg in mice, inhibiting tumor growth in 50% of animal controls) = 5.8.
Water-soluble acidic β-D-glucan: FA-la-β₁, [a]_D +5° (water), MW 500, 000, component sugars; Glc 82.4% and GlcUA 8.8%, ID₅₀=12.9.
Water-insoluble acidic xyloglucan: FII-3, [α]_D + 56° (NaOH), MW 50, 000, component sugars; Glc:Xyl=100:82, GlcUA 16.5%, ID₅₀ = 23.8.
Acidic hetero-glycan: Fill-la, [α]_D +6° (NaOH), MW 100, 000-250, 000, protein content 3.8%, component sugars; Glc:Xyl: Man: Fuc= 100: 58:34:14, GlcUA 20.4%, ID₅₀ = 16.1.
Acidic glycoproteins: FIII-2a, FIII-2b and FIII-2c: [a]_D +58°, +43°, -11° (NaOH); MW 1, 000, 000, 70, 000- 100, 000, 20, 000- 50, 000; ID₅₀= 38.5, 13.9, 9.3 respectively; component sugars; major = Glc, minor = Fuc, Xyl, Man, and Gal.
None of the polysaccharides intraperitoneally active against mouse-implanted Sarcoma 180 had any activity orally.

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Fusarium oxysporum highly produced α-L-fucosidase inductively in the culture broth when grown in a medium containing L-fucose, which is the reaction product of the enzyme. Production of the enzyme was also slightly induced by D-arabinose, but not by other sugars including glucose. To efficiently produce the enzyme, the fungus was grown in a glucose medium at first, and then the growing mycelia were transferred to an inducing medium containing only L-fucose.
α-L-Fucosidase induced by L-fucose was purified from the inducing medium by fractionation with ammonium sulfate, DEAE-Sephadex A-50 column chromatography and gel filtration on a Sephadex G-100 column. The purified enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis. The enzyme is a glycoprotein with a molecular weight of 75, 000. The enzyme can hydrolyze p-nitrophenyl α-L-fucoside, a synthetic substrate, unlike the enzymes of other microorganisms. The Km value for the substrate was 0.48 mM. The optimum pH range was 4.5-6.0 and the stable pH range 5.5-10.0. The enzyme hydrolyzed natural substrates such as porcine mucin and fucoidan to produce L-fucose.

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Several kinds of anthracyclines having γ-rhodomycinone as the aglycone were isolated from Streptomyces cosmosus TMF 518, and their derivatives were prepared by chemical modification. We tested their differentiation inducing activity in Friend leukemia cells and clarified their structure activity relationship as follows: 1) The aglycone, γ-rhodomycinone, had no differentiation inducing activity but was cytotoxic; 2) the compounds with two sugar chains at both C7 and CIO had more potent differentiation inducing activity than those with only a sugar chain at C-10; 3) cosmomycin C was the most favorable candidate for an anticancer agent of all anthracyclines tested, because the value of ED₅₀ (cytotoxicity)/ED₅₀ (differentiation) was as high as 3000; and 4) the increase in differentiation inducing activity and cytotoxicity was not always in parallel.

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A β-xylosidase (β-D-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360, 000 and 380, 000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 HIM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 HIM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosaccharides prepared by steam-explosion of cotton seed waste (DP≤10, 53%, total sugars =150 g/liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160g/liter).

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We immobilized many classes of pyrimidine compounds on the carrier Sepharose^® 4B via alkyl spacers by constructing various spacers using cyanogen bromide activation of the carrier. A crude enzyme solution of Escherichia coli with cytosine- and 5-fluorocytosine-deaminating activity was studied by adsorption and desorption chromatography with columns of the sixty-eight kinds of gels we made. Gels made with the following five ligands were effective. 2-Mercaptopyrimidine or 2-thiobarbituric acid, when coupled with 1, 6-diaminohexane and then with the activated carrier, was suitable. So was 2-amino-4, 6-dihydroxypyrimidine or 5-aminouracil, when linked by carbodiimide coupling to a carrier coupled with 6-aminohexanoic acid. Orotic acid, when linked in the same way with a carrier coupled with 1, 4-diaminobutane, was also effective.

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We wanted to obtain a large amount of cytosine deaminase from Escherichia coli. This enzyme deaminates 5-fluorocytosine and is thermostable. It is needed for trial use in a novel chemotherapy for cancer in combination with 5-fluorocytosine. We improved the conditions of its production by examining the culture conditions. A pH-stat culture with the culture pH controlled at around 8.5 using citric acid gave efficient production of cell mass and enzyme activity. The enzyme was purified 1200-fold from the cell-free extract, to homogeneity, using two kinds of affinity adsorbents prepared for this purpose. The enzyme had a molecular weight of 200, 000 (gel permeation), and seemed to be an SH-enzyme. The purified enzyme was thermostable.

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Bacillus thuringiensis kurstaki strain HD-1 produces two morphologically different parasporal inclusions of δ-endotoxin, bipyramidal and cuboidal inclusions. We found that the bipyramidal inclusion was composed of a 130, 000-dalton sub)unit protein and was toxic to the silkworm, Bombyx mori (a lepidopteran insect), but not to the mosquito, Aedes aegypti (a dipteran insect) and that the cuboidal inclusion was composed of a 65, 000-dalton subunit protein and was associated with the mosquitocidal activity. The distribution of the two proteins, the toxic fragment with a molecular weight of about 60, 000 derived from the bipyramidal inclusions and the cuboidal toxin, was studied in various strains of B. thuringiensis by an enzyme-linked immunosorbent assay.

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Recombinant Rhizopus glucoamylase produced by a yeast containing the Rhizopus glucoamylase gene was purified, characterized, and compared with native Rhizopus glucoamylase. The recombinant glucoamylase was similar to Glue 1, one of three forms of Rhizopus native glucoamylase, but it degraded raw starch more efficiently than the native glucoamylase. Recombinant glucoamylase and Glue 1 adsorbed to gelatinized soluble starch as well as raw starch. These findings suggested that Rhizopus glucoamylase consists of two domains; one for causing adsorption to the starch and the other for catalyzing degradation of the starch molecule.

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Two endoproteases, proteinase-I and proteinase-II, and one aminopeptidase were isolated from the culture supernatant of Aeromonas hydrophila by ammonium sulfate precipitation, gradient elution on diethylaminoethyl-Sephadex A-50 and Sephadex G-75 column chromatography. The molecular weights of the proteinases and aminopeptidase ranged between 30, 000 to 48, 000. The proteinases had comparable pH (8.5) and temperature (48-50°C) optima. The aminopeptidase assayed using L-leucine-p-nitroanilide showed maximum activity at pH 8.0 and at a temperature of 70°C. The aminopeptidase was remarkably heat stable, while the proteinases were heat sensitive. Both the proteinases hydrolysed several proteins including fish myofibrillar proteins. Divalent cations like Fe²⁺, Cu²⁺ and Zn²⁺ inhibited the activities of the two proteinases while Ca²⁺ and Mg²⁺ did not affect either the activities of the proteinases or that of the aminopeptidase. The aminopeptidase was inhibited by metal chelating agents as well as thiol reagents.

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To establish an index of the freshness of meats, we examined the changes in amount of ATP-related compounds in beef and rabbit muscle kept in cold storage. We found an accumulation of hypoxanthine (Hx) and xanthine (X) in beef on the one hand and the accumulation of inosine (HxR) in rabbit muscle on the other. We inferred that this difference was caused by the outstandingly high level of activity in beef of nucleoside phosphorylase having HxR as substrate. The speed at which IMP decreased was greater in beef than in rabbit muscle.
From these results we have come to the conclusion that such indexes of the freshness of fish meats as the K value, K₁ value, IMP ratio, HxR ratio, and Hx ratio are not adequate to indicate the freshness of animal meats and that the K_o value, defined below, is the most effective. The distinctive merit of the K_o value is that it is applicable not only to animal meats and fish but also to marine invertebrates and crustaceans.
K₀=(HxR+Hx+xanthine(X))/(ATP+ADP+AMP+IMP +adenosine (AdR)+HxR+Hx+X) × 100

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β-, γ-, δ- and aza-β-lactam, and thiaziadine compounds induced the differentiation of Friend leukemia cells and hemoglobin was produced. Modification of the lactam rings of γ- and δ-lactam compounds with aromatic side chains and bromine atoms enhanced their differentiation-inducing-activities to Friend leukemia cells. On the other hand, 4 thiaziadine compounds (AKT-1, AKT-2, AKT-3 and AKT-4), which commonly contain a -N(CH₃)-N(CH₃)-moiety, also promoted the differentiation of Friend leukemia cells. They had specific differentiation-inducing-abilities without toxic effects to Friend leukemia cells. 5-Aza-β-lactam compounds showed differentiation-inducing-activity toward all of three types of Friend leukemia cells, two of which were dimethylsulfoxide (DMSO)-sensitive and the other of which was DMSO-resistant. But we did not find any correlation between the differentiation-inducing-activity and the chemical group at the 1-imino position. The presence of an amino bond, which commonly exist in differentiation agents, was suggested to be necessary for the induction of the differentiation of Friend leukemia cells, and this ability was enhanced with the addition of aromatic side groups.

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To identify the compounds in machine cutting-fluid emulsion that have an obnoxious odor, we separated volatile components from the emulsion using both steam distillation with a Nickerson-Likens apparatus and vacuum distillation. These components were analyzed by gas chromatography and gas chromatography-mass spectrometry using a fused silica capillary column coated with cross-linked 5% phenylmethyl silicone. 2, 6-Dimethyl-3-methoxypyrazine was detected. The main odorous compounds were dimethyl disulflde, dimethyl trisulfide, and 2-butene-l-thiol, the last compound being tentatively identified by its mass spectrum.

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A recombinant Bacillus subtilis phage, ρ11-AA248, contains the tmrA7-amyR2-amyE⁺-tmrB⁺-aroI⁺ region of the B. subtilis N7 chromosome on a 22.4kb DNA fragment. The amyE⁺-tmrB⁺ gene region in the phage genome of the B. subtilis 207-21 transductants by ρ11-AA248 was amplified to approximately 10 copies after cultivation in the presence of tunicamycin (10 μg/ml) and to two copies without tunicamycin. The amplification of the gene region caused hyper-production of extracellular α-amylase. In contrast, no amplification of the gene region was detected in the transductants of B. subtilis 207-25, a recE-deficient derivative of 207-21 strain.

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The reaction between lipid hydroperoxide and hemoglobin (Hb) under aerobic condition was studied kinetically by a Spectrophotometric and a spin trapping method. The reaction did not follow simple first order or second order kinetics, possibly because Hb was also decomposed during the decomposition of lipid hydroperoxide. A spin trap reagent decreased the rate of the reaction. Some free radicals were trapped by the spin trap reagent and their structures were found from ESR spectra of spin adducts. These results were explained by a free-radical chain reaction mechanism initiated by lipid hydroperoxide and Hb.

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In order to investigate the steric requirement of the C-terminal hydrophobic amino acid of the bitter peptide, Arg-Arg-Pro-Pro-Phe-Phe, for the production of bitterness, we prepared analogs of it containing o-phenylalanine, in place of L-phenylalanine. The analogs with L-phenylalanine at the C-terminal exhibited stronger bitterness than those with D-phenylalanine at the C-terminal. We confirmed that the configuration of the C-terminal hydrophobic amino acid is important for the increase in bitterness.

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The fatty acid compositions of membrane phospholipids in selected freezing-resistant cells of Lavandula vera and unselected original cells subjected to freeze-thaw procedure were studied. The degree of the fatty acid unsaturation of phosphatidylcholine in the freezing-resistant cells increased significantly when the cells were treated with cryoprotectant at 0°C for 2hr and the cells survived subsequent cryostorage in liquid nitrogen. However, the increase in the degree of the fatty acid unsaturation of phosphatidylcholine in the unselected original cells was slight and the cells could not survive subsequent cryostorage. These results suggest that the increase in the unsaturation of membrane phospholipid fatty acids plays an important role in the freezing resistance of cultured green Lavandula vera cells.

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Six new isoflavones each having a prenyl (3, 3-dimethylallyl) or a modified prenyl group at C-6 or C-8 of the isoflavone skeleton, lupiwighteone [5, 7, 4'-trihydroxy-8-(3, 3-dimethylallyl)isoflavone, 1], luteone hydrate [5, 7, 2', 4'-tetrahydroxy-6-(3-hydroxy-3-methylbutyl)isoflavone, 3 (already known as a fungal metabolite of luteone)], lupiwighteone hydrate [5, 7, 4'-trihydroxy-8-(3-hydroxy3-methylbutyl)isoflavone, 4], 2, 3-dehydrokievitone hydrate [5, 7, 2', 4'-tetrahydroxy-8-(3-hydroxy3-methylbutyl)isoflavone, 5], hydroxywighteone [5, 7', 4'-trihydroxy-6-(3-hydroxymethyl-3-methylallyl)isoflavone, 6], hydroxyparvisoflavone A [a 2-hydroxymethyl-2-methylpyrano-isoflavone, 7], and a new coumaronochromone, lupilutin [5, 7, 4'-trihydroxy-8-(3-hydroxy-3-methylbutyl)-coumaronochromone, 8], were isolated from the roots of yellow lupin (Lupinus luteus L., cv. Barpine). Together with these new isoflavonoids, the presence of simple isoflavones (genistein and 2'-hydroxygenistein) and complex isoflavones (wighteone, luteone, 2, 3-dehydrokievitone (2), parvisoflavones A and B, and lupinisoflavones A and B) in the yellow lupin roots has also been confirmed.

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In addition to 5-O-methylgenistein (1), a further investigation of the isoflavonoid constituents in roots of the yellow lupin (Lupinus luteus L. cv. Barpine) has yielded five new 5-O-methylisoflavones named barpisoflavone A (2), 5-O-methyl-lupiwighteone (3), barpisoflavone B (4), 5-O-methylderrone (5) and barpisoflavone C (6). These isoflavones were identified by physicochemical methods involving the use of other lupin isoflavones as reference compounds.

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7-Aza, 7-thia and 6-deoxo analogs of (22S, 23S)- and (22R, 23R)-homobrassinolide were synthesized. They were found to possess weak bioactivity in a lamina-inclination test.

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An efficient synthesis of 3-hydroxyisoxazoles from β-ketoesters and hydroxylamine was investigated. The reaction of the sodium salts of β-ketoesters and hydroxylamine at a low temperature, followed by quenching with an excess of cone. HC1 under heating gave predominantly 3-hydroxyisoxazoles involving 4-sulfenylated 3-hydroxyisoxazoles. Starting from the prepared 4-(2, 4-dichlorobenzyl)-3-hydroxy-5-methylisoxazole, a potential herbicidal compound, 4-(2, 4-dichlorobenzoyl)-3-hydroxy-5-methylisoxazole, was also synthesized in five steps.

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Differential growth of two strains of Ceratocystis fimbriata EII. and Halst. was demonstrated in liquid media containing exudates from the infected sweet potato root tissues; the germ tube growth of the taro strain, incompatible with sweet potato, was specifically inhibited in medium containing the exudate from the taro-strain-infected tissues which had been incubated for 18hr, whereas germ tube growth of the sweet potato strain, compatible with sweet potato, was not inhibited in media containing the same dilution of any exudates. Furanoterpenoid phytoalexins and umbelliferone were shown to be the main cause of differential inhibitory activity of exudates from the infected tissues. The taro strain was more sensitive than the sweet potato strain to inhibition by furanoterpenoid phytoalexins in both germination and germ tube growth. Umbelliferone inhibited germ tube growth of the taro strain only but not the seet potato strain, at the concentrations tested.
The content of umbelliferone increased before that of furanoterpenoid phytoalexins in both tissues infected by the sweet potato and taro strains. Umbelliferone accumulated to the same extent in the early stages in both tissues. On the other hand, the content of furanoterpenoid phytoalexins in the tissues infected by the taro strain started to increase earlier (12 hr) than that in the tissues infected by the sweet potato strains (18hr).

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Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52, 000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27, 000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2 mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by D-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.

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An efficient method for the preparation of Pleurotus ostreatus protoplasts (more than 10⁸/ml/2hr) and for colony regeneration from the protoplasts (more than 10%) was developed by adding the components of sulfite pulp wastes to the media and by using an enzyme mixture of Cellulase Onozuka RS, chitinase, Zymolyase 100, 000 and β-glucuronidase. Several different specimens of sulfite pulp waste derivatives (including commercially available ones) stimulated the growth of mycelia suitable as protoplast sources, and markedly increased the regeneration frequency of the protoplasts. The colonies of the regenerated protoplasts could develop fruit bodies effectively within 3 weeks cultivation on media supplemented with the sulfite pulp waste components. The effective components in sulfite pulp wastes were discussed with reference to the studies of K. Inaba et al. and N. Kawamura et al.

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A new convenient assay method for nematode attractants was developed. Using this method, the attracting activity of volatile components in a pine, Pinus densiflora, was examined for the pine wood nematode, Bursaphelenchus xylophilus, which causes serious damage to pine trees in Japan. Among the volatile components, β-myrcene showed the strongest attracting potency. This compound was assumed to play an important role in the transmigration of the nematode from the sawyer to the pine tree and for the movement of the nematode inside the pine wood.

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An efficient synthesis of the less stable cis-isomer, methyl dihydroepijasmonate 8, is described.

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N-[2-O-(2-Acetamido-1, 5-anhydro-2, 3-dideoxy-D-glucitol-3-yl)-D-lactoyl]-L-alanyl-Disoglutamine (1-deoxy-MDP) and its lipophilic analogs were synthesized from 2-acetamido-3, 4, 6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride. The immunoadjuvant activity was examined in order to clarify the structural requirements of the carbohydrate moiety and the effects of the lipophilic character for this activity in N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP).

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In order to produce 5'-guanylic acid (GMP) from 5'-xanthylic acid (XMP), we attempted to increase the activity of the conversion enzyme, XMP aminase (EC 6.3.4.1), in Escherichia coli. We subcloned the guaA gene coding for XMP aminase from plasmid pLC34-10 into pBR322. By connecting the tryptophan (trp) promoter at a suitable position upstream of the guaA gene, we obtained plasmid pXAR33. E. coli K294/pXAR33 showed an increase in activity of approximately 80 times when compared with K294, and the amount of the enzyme protein represented approx. 10% of the total cellular protein. The production of GMP with the plasmid carrying strain was also investigated.

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