Matsutake [Tricholoma matsutake (S. ITO et IMAI) SING. ] was stored under conditions of low O₂ and elevated CO₂ concentrations. The storage conditions were as follows : with an O₂ concentration of 2.5±0.5%, the CO₂ concentrations were 5%, 10%, 15%, and 20%, and relative humidity (RH) was about 100% ; with an O₂ concentration of 2.0±0.5%, the CO₂ concentrations were 0%, 5%, 10%, and 15%, and RH was about 100% ; the storage temperature was 1.0±0.1°C. The fruit was also stored in air and under 100% N₂ as controls. Quality factors such as 'neto' (slimy microbial flora which develop on the moist surface of the fruiting body), weight loss, whiteness, firmness, and off-odor were measured. The development of neto and browning (loss of whiteness) of the inner stipe were suppressed for more than 14 days, except with storage under 100% N₂. Storage in air and under 0% or a high concentration (>10%) of CO₂ caused an early development of off-odor, compared to storage under 5% and 10% CO₂. In air, the development of mold was observed after 14 days. Under a low O₂ concentration and 5% to 10% CO₂, the quality factors of matsutake were most retained, and the fruit was still acceptable after 14 days of storage. A weight decrease of the fruit was recognized as the CO₂ concentration was increased.

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A novel immunomodulating fraction, SP-MAF1, was isolated from soybeans as a polysaccharide-protein complex by hot water extraction followed by acid treatment, ethanol treatment, and chromatography on a DEAE-Sepharose CL-6B column, and was characterized in some biological activities. SP-MAF1 increased glycolysis and IL-1 production by macrophages, but did not affect TNF production. SP-MAF1 also increased splenocyte proliferation that was induced by LPS and ConA, and ConA-induced production of IL-2 by splenocytes. In an in vivo test, the mean survival times of SP-MAF1-treated mice bearing FM3A were significantly longer than those of non-treated group. Since SP-MAF1 affected macrophages and splenocytes but not tumor cell growth, this effect may be mediated by the host immune system. Furthermore SP-MAF1 had a capacity to suppress the TNBS-induced DTH reaction. These results suggested that SP-MAF1 could either enhance or suppress immune functions.

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Candida guilliermondii H-404, isolated from soil, produced thermostable α-galactosidase, but small amounts of other glycosidases (such as β-galactosidase, α-glucosidase, and β-glucosidase). The enzyme was separated into two fractions by DEAE-Toyopearl 650M chromatography, and the two enzymes were designated galactosidase I and II. These two enzymes had the same molecular weight (270, 000 by gel filtration, 64, 000 by SDS-PAGE). The isoelectric points of α-galactosidase I and II were 6.16 and 6.21, respectively. These two enzymes were different from each other in pH stability, temperature stability, and effects of Fe²⁺ and Cu²⁺ ion on α-galactosidase activity. The enzyme had stronger transfer activity and wider acceptor specificity than α-galactosidases which have been reported.

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A biosensor based on a glass electrode was developed to determine free sulfur dioxide in wine. The biosensor consists of a microbial membrane of Thiobacillus thiooxidans JCM7814 and a flat glass electrode. A porous gas-permeable membrane was incorporated in the biosensor system to separate free sulfur dioxide from bound sulfur dioxide and to avoid the buffer action of the sample solution. This biosensor gave a linear relationship between the pH decrease and concentration of sulfur dioxide up to 50mg/liter with a response time of 20min. The limit of detection for sulfur dioxide was 5.0mg/liter, and the life-time of the microbial membrane was ca. 30 days at 4°C. The biosensor could specifically determine free sulfur dioxide in wine, and was not influenced by temperature or oxygen concentration. The relative standard deviations were 7.41% for red wine and 5.00% for white wine.

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Myosin dissolved in 0.5_M KCl at pH 6.0 was exposed to hydrostatic pressure up to 210MPa for 30min. Turbidity of myosin solutions did not change after application of pressure at 70MPa, while it gradually increased with extending duration of pressurization at 140MPa, and it markedly increased within 5min at 210MPa. Sedimentation velocity measurements indicated that myosin molecules associated to form aggregates upon pressurization. Electron microscopic observation confirmed that increasing hydrostatic pressure induced aggregation of myosin molecules. After exposure to and release of 70MPa, most myosin molecules were in the monomeric state like the unpressurized control ; however, some monomeric myosin had a one-headed shape instead of the two heads of an intact molecule. With increasing pressure, myosin heads were tightly packed, forming a clump and the tails of myosin molecules extended radially from the clump. Neither tail to head nor tail to tail interaction occurred. The shape of pressure-induced oligomer looked like a daisy-wheel and its morphology was similar to that of heat-induced oligomer reported previously (Yamamoto et al., 1990). Myosin solution did not form a gel by pressurization alone, though pressure-treated myosin formed a gel upon following heating. Pressurization up to 210MPa did not affect the gel strength or the microstructure of heat-induced gel.

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A fungus, FO401B, was isolated as a microorganism capable of assimilating sardine oil as a sole carbon source and producing a large amount of extracellular lipase ; it was identified as Geotrichum sp. The fungus produced a lipase mixture that was more specific towards the 2 position, as judged by the positional specificity index, when the initial pH of a rice bran medium was raised. The lipases in the broth were purified by ion exchange and hydrophobic interaction chromatographies and termed Lipases A, B, and C. A and C gave a single band each by SDS-PAGE, but fraction B was presumed to be a mixture of Lipases A and C. A was incompletely 1, 3-specific towards triolein (able to hydrolyze the 2-positioned ester at a slower rate) and, in contrast with A, C was incompletely 2-specific. The positional specificity of B was completely non-specific. The optimum pHs of A and C were 7.0 and 8.5 and their molecular weights were 62, 000 and 58, 000, respectively.

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Shimizu et al. and ourselves have reported some enzymatic properties of an α-amylase from Thermoactinomyces vulgaris R-47 that hydrolyzed pullulan to produce panose [M. Shimizu et al., Agric. Biol. Chem., 42, 1681-1688 (1978) ; Y. Sakano et al., Agric. Biol. Chem., 46, 1121-1129 (1982)]. In this study, we cloned a gene for an α-amylase, which was different from the one mentioned above but also hydrolyzed pullulan to produce panose, from T. vulgaris R-47, and analyzed the entire primary structure of the gene. We designated the previously reported enzyme as T. vulgaris α-amylase I (TVA I), and this novel enzyme as T. vulgaris α-amylase II (TVA II). The nucleotide sequence had an open reading frame of 1755 base pairs corresponding to a protein of 585 amino acid residues. Although this novel α-amylase, TVA II, hydrolyzed both pullulan and starch, the ratio of pullulan-hydrolyzing activity to starch-hydrolyzing activity of the enzyme was higher than that of TVA I, and the primary structure of the enzyme resembled neopullulanase, which scarcely hydrolyzed starch, rather than that of TVA I.

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Microdialysis was used for continuous sampling of biogenic amines from the haemolymph of Spodoptera litula (tobacco cutworm). Six different biogenic amines, including norepinephrine, epinephrine, dopamine, octopamine, tyramine, and serotonin in the microdialysates were simultaneously analyzed by HPLC with a coulometric electrochemical detector. In these samples, dopamine and tyramine existed in relatively high concentrations and octopamine could also be detected. We conclude that the "Insect Microdialysis System" can provide a continuous sampling method from the insect haemolymph without physical stress and consequently increase the sensitivity for detection of changes of the haemolymph biogenic amine level.

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Induction of phytoalexin formation in suspension-cultured rice cells by a series of N-acetylchitooligosaccharides and chitooligosaccharides was studied. N-acetylchitooligosaccharides larger than hexaose induced the formation of momilactones A and B as well as oryzalexins A, B, and D at very low concentrations like 10^-9-10^-6_M (N-acetylchitoheptaose). GlcNAc oligomers smaller than trimers had almost no activity and a series of deacetylated chitooligosaccharides were also inactive. Strict requirement for the size and structure of GlcNAc oligomers as well as the sensitivity to them strongly indicates the presence of recognition systems specific for these compounds in rice cells. The level of momilactone A produced reached 100-500μg/g of cultured cells, which appeared to be enough to prevent the growth of pathogenic fungi such as Pyricularia oryzae, thus indicating the importance of this phenomenon in the defense systems of rice plants. Suspension-cultured cells obtained only from a suitable period of cultivation, mainly those from lag phase, could respond to the elicitor and produce phytoalexins.

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We prepared serum-free medium supplemented with growth factors, hormones, nutrients, and egg yolk low density lipoprotein (LDL), and examined the growth-promoting activities of growth factors and culture conditions on human hepatoma Hep G2 cells. Collagen type I was appropriate for precoating tissue culture vessels in which Hep G2 cells were cultured in serum-free medium. William's medium E and a mixture of Dulbecco and Vogt's modification of Eagle's medium and Ham's F-12 (1:1) were used as basal medium. LDL was the most effective growth promoter of Hep G2 cells among the supplemented factors. High density lipoprotein (HDL) prepared from human serum showed 10 times more growth promoting activity than LDL, but HDL is more expensive. Therefore, the serum-free medium supplemented with growth factors, hormones, nutrients, and egg yolk LDL was suitable for Hep G2 cell culture.

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A sandwich enzyme immunoassay for rat transthyretin using a recombinant antigen approach is described. Rat transthyretin cDNA was cloned by PCR. Rat transthyretin fused with the IgG-binding ZZ domain of protein A was expressed in E. coli using the fusion vector pEZZ18. Antibody against the fusion protein was specific to rat transthyretin in plasma as shown by immunoblotting. Affinity-purified anti-rat transthyretin-ZZ was biotinylated and used for the sandwich enzyme immunoassay. In this assay, the measurable range was 1.2-50ng/ml and the coefficients of variation within and between the assay series (assay range : 2.5-20ng/ml) were 1.31±0.08% and 3.50±1.46%, respectively. Cross-reactivity was examined using bovine, human, and mouse serum. There was a cross-reaction only with mouse transthyretin. In an in vitro experiment, transthyretin secreted by rat hepatocytes could be measured by the sandwich enzyme immunoassay.

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2, 4-Dimethyl-5-hexanolide (1), 2, 4-dimethyl-5-heptanolide (2), and 2, 4-dimethyl-5-octanolide (3) are the volatile substances identified in the heads of the male black garden ant, Lasius niger. (2R, 4R, 5S)-Isomers of these lactones were synthesized by employing the iodolactonization reaction (5→4) as the key step.

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Gas chromatographic separation of the enantiomers of 5-alkanolides (alkano-δ-lactones) without conversion was attempted in a capillary column coated with a liquid phase containing heptakis (2, 3, 6-tri-O-methyl)-β-cyclodextrin (CD column) ; however, separation was difficult, especially in the case of lactones with a high carbon number. Therefore, conversion by a simple process using popular chemicals was tried. It was found that 2-methoxy-6-alkyl-tetrahydropyrans (alkano-δ-lactol methyl ethers) were good derivatives for enantiomeric separation of the lactones in the CD column. The δ-lactones were converted to δ-lactol methyl ethers in one step by using sodium borohydride and methanol in the presence of aluminum chloride and water. This conversion method is applicable for enantiomeric separation of the lactones from hexano- to octadecano-δ-lactone.

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The enantiomeric composition of 5-alkanolides (alkano-δ-lactones) from octano(C-8)- to do-decano-δ-lactone contained in coconut and dairy products was determined by the method described in the preceding paper. The enantiomeric contents of the (R) series of δ-lactones were C-8, 91-96% ; C-10, 68-84% ; and C-12, 36-81% in all the analyzed fresh coconut and coconut products ; and C-8, 12-17% ; C-10, 77-86% ; C-12, 90-94% ; and C-14, 91-96% in all the analyzed dairy products. These data demonstrate that the enantiomeric content of each lactone in the two series of products, except for the C-12 lactone of coconut, was almost constant independent of the variety of the product. The (R) content in the series of coconut δ-lactones decreased with increasing carbon number of the lactones, this trend being the opposite in the series of dairy products. These facts are considered to reflect on the enzymatic process of lactone formation, or on the characteristics of biological species.

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Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI', FII', and FIII', respectively. Enzyme fractions FI', FII', and FIII', respectively. Enzyme fractions FI', FII', and FIII' showed specific activities of 15.2×10^-4, 226.7×10^-4, and 33.8×10^-4μmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII'. The optimum pH for chlorophyllase activity was 8.0 for FI' and 8.5 for both FII' and FIII'. Apparent K_m values for enzyme fractions FI', FII', and FIII' were 2.1n_M, 2.3n_M, and 2.0n_M, respectively. Enzyme fractions FII' and FIII' showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI' had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29m_M, 2.14m_M, and 0.71m_M for FI'. FII', and FIII', respectively.

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A plasmid, pSA1.1, of Streptomyces azureus elicited conjugative pocks and inhibited the sporulation of its host mycelia. pSA1.1 was studied with a kanamycin resistance gene derived from S. kanamyceticus as a selective marker. The deletion analysis and sequencing of the derivative plasmid found an open reading frame (ORF909b) that was involved in the sporulation-inhibitory function of pSA1.1 and in pock formation. The nucleotide sequences of ORF909b were compared with those of the genes registered in GenBank, but no similarity was found. However, the predicted amino acid sequence of ORF909b showed a significantly high similarity with that of the spoIIIE gene of Bacillus subtilis. This detected gene might be a new sporulation-regulatory gene in streptomycetes.

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A chitosanolytic enzyme was purified from Enterobacter sp. G-1 by fractionation of 30% saturation with ammonium sulfate, isoelectric focusing, and Sephadex G-100 gel chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the molecular mass was estimated to be 50kDa. The enzyme degraded N-acetyl-chitooligosaccharides, glycol chitin, colloidal chitin, and colloidal chitosan (about 80% deacetylated), but did not degrade chitooligosaccharides, colloidal chitosan (100% deacetylated), or Micrococcus lysodeikticus cell walls. It hydrolyzed GlcNAc_4-6 and colloidal chitin to GlcNAc₂, finally. The main cleavage site with GlcNAc_3-6 was the second linkage from the non-reducing end, based on the pattern of pNp-GlcNAc_2-5. Colloidal chitosan was hydrolyzed to GlcNAc₂ and to similar partially N-acetylated chitooligosaccharides.

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Productivity and some properties of anti-Human Rotavirus (HRV) hen egg yolk antibody (IgY) were compared with those of anti-HRV rabbit serum antibody (IgG). The hens immunized with HRV (Wa strain, serotype 1 and Mo strain, serotype 3) were found to continuously to lay eggs without any change in the egg laying rate and the yolk of the eggs laid over a year showed a high level of neutralization titer against HRV. The production of anti-HRV IgY by a hen (one year) was at least 15 times (anti-Wa) and 120 times (anti-Mo) more effective than those by an immunized rabbit in the neutralization titer of the antibodies. The stability of anti-HRV IgY at temperature above 70°C and low pH 2-3 was less than that of anti-HRV rabbit IgG. The temperature corresponding to the maximum of denaturation endotherm (T_max) of IgY was 73.9°C while that of rabbit IgG was 77.0°C in the analysis by differential scanning calorimetry. This discrepancy in heat and acidic pH stability found between the two antibodies as discussed with regard to their protein structures.

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Two bacterial strains, CA06 and CA10, that assimilate carbazole (CAR) as the sole source of carbon and nitrogen were isolated from 202 farm soil samples and 4 activated sludge samples, and identified as Pseudomonas spp. Growth conditions for strains CA06 and CA10 on CAR were examined. Anthranilic acid (AN) and catechol (CAT) were identified as the main metabolites of CAR by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). When strains CA06 and CA10 were cultivated in a medium containing 17mM CAR, 1.4mM AN, and 0.1mM CAT were accumulated in the culture broth, but AN disappeared after 140h of incubation. An initial oxidation product, 2'-aminobiphenyl-2, 3-diol, and a meta-cleavage product, 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2, 4-dienoic acid, were tentatively identified in the culture broth of CAR by GC-MS. When AN was used for a substrate in culture by these strains, CAT and a small amount of cis, cis-muconate was detected by HPLC. This conversion suggested the existence of an ortho-cleavage. The activities of the meta-cleavage enzymes for bipheny-2, 3-diol (the initial oxidation intermediate analog of 2'-aminobiphenyl-2, 3-diol), 3-methylcatechol, and CAT were measured using the crude cell extracts of CAR- and AN-grown cells. The meta-cleavage enzymes of two strains for biphenyl-2, 3-diol was induced during the growth on CAR, but not induced by AN. Based on these results, a CAR degradation pathway is proposed.

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Several enzymes involved in the L-threonine and L-lysine biosynthesis in gram-negative obligate methylotrophs, Methylobacillus glycogenes ATCC 21276 and ATCC 21371, were characterized. The activities of aspartokinases were inhibited by L-threonine or L-lysine. The homoserine dehydrogenases were inhibited by L-threonine or L-phenylalanine. The dihydrodipicolinate synthase activities were inhibited by L-lysine. No activity of meso-α, ε-dihydrodipicolinate dehydrogenase, the by-path enzyme of L-lysine biosynthesis, was detected. The biosynthetic enzymes in L-threonine-producing and L-lysine-producing mutants of M. glycogenes were also characterized. The aspartokinases of all the mutants were insensitive to inhibition by L-threonine, and some were also insensitive to inhibition by L-lysine. The homoserine dehydrogenases of the mutants had the wild type-profiles. The dihydrodipicolinate synthase of the L-lysine-producing mutant was partially desensitized to inhibition by L-lysine.

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Two chitinases were purified from the culture filtrate of Streptomyces sp. AJ 9463, which is a producer of a chitinase inhibitor, allosamidin. They had similar molecular masses (44.5kDa and 43.7kDa, estimated by SDS-PAGE) and both of them had slightly acidic optimum pH ranges, but showed quite different sensitivities to allosamidin. One chitinase, termed chitinase S, was strongly inhibited by allosamidin with the IC₅₀ value of 0.5μg/ml. The other one, termed chitinase IS, was not inhibited by the drug even at the concentration of 2mg/ml.

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A nitrogen-sparing action of sulfur-containing amino acids and threonine in rats fed a non-protein diet has been reported from our laboratory. The nutritional significance of dietary sulfur-containing amino acids and threonine on the expression of the albumin gene was investigated. An elevated level of serum albumin and its mRNA in liver was observed in rats fed a non-protein diet supplemented with sulfur-containing amino acids and threonine, but the level of albumin mRNA of the group supplemented with cystine and threonine was much higher than that of the methionine and threonine supplemented group. Nuclear run-on assay showed an enhanced rate of albumin gene transcription in liver nuclei of rats fed the cystine and threonine supplemented diet. We showed the evidence that cystine is a more efficient amino acid than methionine to maintain the expression of the albumin gene at a high level in rats fed a non-protein diet.

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