A simple determination of cyclopiazonic acid by HPLC is elucidated. The zinc cation in the mobile phase and indomethacin as an internal standard made quantitation higher and peak identification of cyclopiazonic acid easier. In the combination with a TLC analysis, production of cyclopiazonic acid by some species of aspergilli could be analyzed both in a liquid culture and in a solid culture on wheat bran without a prior clean-up operation.

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The interaction between bovine lactoferrin (bLf) and ascorbate (Asc) was investigated through malondialdehyde (MAD) formation in a solution containing DNA, bleomycin (BLM), and Fe²⁺ or Asc. The inhibition by bLf on MDA formation in the presence of Asc was not changed even by adding carbonate or oxalate ions to the solution. The percentage inhibition by the hydrolysates of bLf treated with pepsin, trypsin, and both enzymes on MDA formation was almost the same as that by the untreated bLf in the presence of Asc. The inhibition of MDA formation also occurred with the filtrate obtained from a solution containing bLf and Asc, but not with that from a solution of bovine serum albumin and Asc. The interaction of bLf and Asc was observed by gel filtration in a Sephadex G75 column. The binding amount of Asc was estimated to be 87 mol per mole of bLf.

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Streptolysin O (SLO) is a hemolytic, extracellular protein produced by Streptococcus Ryogenes. A hybrid gene consisting of the promoter and signal sequence fused to the region encoding the mature sequence of the slo gene was constructed to secrete SLO in Bacillus subtilis. To increase secretion of SLO into the culture supernatant, several SLO expression vectors containing various combinations of promoters and pre-pro sequences were constructed. B. subtilis harboring pPA consisting of the P-43 promoter and the coding sequence of the pre region of the alkaline protease gene that was fused to the pro-mature region of the slo gene secreted SLO into media. The degree of hemolytic activity was found to be about 40-fold higher in the geneticaly engineered B. subtilis strain than that of S. Ryogenes. Recombinant SLO was reacted with patients' sera infected by group A streptococci.

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An extracellular proteinase secreted by Candida pulcherrima KSY 188-5 was purified about 60-fold to electrophoretical homogeneity from its culture supernatant, by ammonium sulfate fractionation, anion-exchange chromatography, and gel filtration. The proteinase had a molecular weight of approximately 36, 500 and an isoelectric point of pH 4.7. The enzyme had an optimum pH of around 2.5-3.5 for activity and 3.0-5.0 for stability. The optimum temperature was around 45°C at pH 3.0. The enzyme showed a broad substrate specificity for a variety of proteins to hydrolyze casein, BSA, hemoglobin keratin, and collagen. Among several proteinase inhibitors, pepstatin A completely abolished the enzyme activity;indicating that the extracellular proteinase from C. pulcherrima KSY 188-5 was classified in the group of carboxyl proteinases.

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The acidogenicity of commercially available isomaltooligosaccharides mixture (IMO) and its hydrogenated derivative (IMO-H) were evaluated by the in vivo pH response under dental plaque of slx subjects (aged 25-28) using intraoral apparatus. The apparatus, an indwelling pH sensor of a hydrogen ion-sensitive field effect transducer (ISFET), was placed on the buccal site of the mandibular first molar, and the plaque was accumulated for four days. The test sugars were applied with three method-dropping the solution directly on the plaque, rinsing the oral cavity with the solution, and sucking a candy made of the test sugar (weight3-4g). IM0 could not be evaluated as a type of sugar with low acidogenicity, especially by the candy method, but IMO-H could be evaluated as a type of sugar with. very low acidogenicity in a similar manner as maltitol or sorbitol in all type of applications. The acidogenlc response of dental plaque should be assumed to be closely related to salivary parameters such as the secretion rate or to mutans streptococci level of subjects and types of application.

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An acidic polysaccharide was isolated from the water-soluble mucilage extracted from dried leaves of Corchorus olitorius, known as Moroheiya in Japan (3. 0g per 100g). This polysaccharide showed a single peak in a Sepharose CL-6B column, and the specitic rotation in H₂O at 25°C was +250°. The polysaccharide was rich in uronic acid (65%), and consisted of rhamnose, glucose, galacturonic acid, and glucuronic acid in a molar ratio of 1. 0 : 0. 2 : 0. 2 : 0. 9 : 1. 7, in addition to 3. 7% of the acetyl group. A methylation analysis, Smith degradation study and fragmentation analysis suggested that this polysaccharide mainly consisted of O-4 substituted galacturonic acid and glucuronic acid, and O-2 substituted rhamnose residues, and that most of the (1→4)-linked uronic acid residues were substituted at the O-3 position with glucuronic acid residues. This polysaccharide showed proliferative activity toward the murine splenocyte.

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As the first step for production of human apolipoprotein E (hApoE) in Saccharomyces cerevisiae, the hApoE cDNA was cloned in Escherichia coli, on the basis of the nucleotide sequence reported previously. When the hApoE cDNA including its pre-sequence-encoding region was expressed under the control of the GAL7 promoter, no protein immunoreactive with anti-hApoE antibody was detected either in the culture medium or inside the cells. For efficient production and secretion of hApoE in S. cerevisiae, the mature hApoE-encoding region was fused to the prepro-sequence region of Rhizomucor rennin (MPR) and to the whole MPR gene including its prepro- and mature-MPR regions. When the fusion gene consisting of the prepro-sequence-encoding region and hApoE regions was expressed in S. cerevisiae, no protein reactive with the anti-hApoE antibody was detected in any fraction of the yeast cells, probably due to rapid degradation of the hApoE protein by yeast proteases. On the othe hand, when hApoE was expressed as a fusion to the whole MPR protein, a considerable amount of the fused protein was secreted into the medium. The prepro-sequence of MPR was correctly processed from the fused protein in the medium by autocatalytic activity of MPR and by a protease(s) of the host cell. Integration of the fusion gene into the chromosome at a copy number of eight led to secretion of the fused protein in a larger amount than the case when the fusion gene was carried on a 2-μm plasmid with its copy number of a few hundreds, because the 2-μm derived plasmid containing the fusion gene was very unstable in the yeast cells. The secretion level was also improved by changing the culture conditions. A maximum yield of hApoE part in the secreted fused protein was estimated to be 23. 7mg per liter and the amount of the fused protein was calculated to be 53. 0mg per liter.

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Three kinds of peptides (H, S, and R) with 16 amino acid residues were synthesized, and their secondary structure and emulsifying properties (emulsifying activity and oil-binding property) were investigated to clarify the effects of conformational amphiphilicity. The amino acid compositions of the three peptides were the same (8 Leu and 8 Glu residues) but their sequences were different. Circular dichroism analysis showed that peptide H contained an α-helix at pH 5. 5 but took a random structure at pH 7. 0. Peptide S also cotnained an α-helix at pH 5. 5, while a β-sheet structure was predominant at pH 7. 0. Peptide R took mainly a random structure at both pH 5. 5 and pH 7. 0. The helical properties of peptide H and peptide S were different ; the α-helix of peptide H was amphiphilic, while that of peptide S was not. Under conditions where peptide H formed an amphiphilic α-helix, both the emulsifying activity and oil-binding property of the peptide signiticantly increased. The emulsifying properties of peptide S, which formed an amphiphilic β-sheet at pH 7. 0, were also good at that pH. We conclude that a peptide with an amphiphilic α-helix or β-sheet structure has better emulsifying properties, hence a good peptide emulsifier could be designed by incorporating such a structure.

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The pH of a lysine aqueous solution was quickly lowered by about one unit when it was irradiated with a ceramics radiator with cooling. The maximum point of difference in pH of the irradiated lysine from that of the non-irradiated one occurred near the first equivalent point of the α-carboxyl group. The molar extinction of lysine also decreased with the irradiation time. Dissociation constants of α-carboxyl, and ε- and α-amino groups signiticantly decreased. The entropy dependency of dissociation of the α-carboxyl group was changed to an enthalpy one by the irradiation, while the enthalpy dependency of the ε- and α·amino groups tended to be changed to an entropy one by the irradiation. The mechanism of promotion of the dissociation by far-infrared waves irradiated from the ceramics has been estimated by a thermodynamic analysis as a hydration effect of the waves on the dissociable groups.

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Trehalase inhibitors are promising as a specific insect growth regulator. Microbial culture filtrates are a good source of bioactive compounds, but some of them retain a significant amount of glucose, a major component of the culture medium, which makes it difficult to detect trehalase inhibitors because of high blank values for the glucose determination. To find a variety of trehalase inhibitors in glucose-rich microbial culture filtrates, an effective method for removing glucose by enzymatic conversion prior to a routine trehalase-inhibitory assay was devised. Conversion with glucose oxidase and catalase could completely remove 1Omg/ml of glucose, and a wide variety of microbial culture filtrates with high glucose content could then be subjected to the routine bioassay. This method can also be applied to screen inhibitors for other enzymes whose activity is assayed by the amount of glucose formed.

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2-(2-Hydroxyalkylidene)cyclopentanones (1) and 5-(2-hydroxyoctylidene)-2-cyclopentenone (2) were synthesized by the aldol reaction of cyclopentanone-enolate (3)/cyclopentenone-enolate (19) with 2-bromoalkanals (4)/2-bromooctanal (4c) and subsequent treatment of the products with sodium acetate. The stereochemistry of the condensation, substitution and elimination was elucidated. The bio-antimutagenic activity of 1 increased as the 2-hydroxyalkylidene group increased in length up to =CHCH(OH)C₈H₁₇.

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When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB ; 0. 01M, pH 8. 0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-D-gluconate and ammonia : α, β-elimination reaction, B). The ratio of the activities (A : B) was about 1 : 4. However, in potassium phosphate buffer (KPB ; 0. 04M, pH 8. 0), the aldolase reaction was inhibited to 3-4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0. 1M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn^{2 +} + pyridoxal 5'-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn²⁺ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn²⁺, PLP, and the phosphate group of glycero1-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn²⁺ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.

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This investigation concerns the effects of the ingestion during pregnancy of a sucrose diet compared with a dextrin diet on the lipid and glycogen metabolism in the liver of pregnant rats and their fetuses at days 15 and 19 of gestation. At the two time points, the pregnant rats fed with the sucrose diet had higher serum glucose and triglyceride concentrations. On day 15 of pregnancy, the hepatic triglyceride, total, and esterified cholesterol concentrations were higher in the sucrose-fed rats than in the dextrin-fed rat, but by day 19, the triglyceride and esterified cholesterol concentrations only increased in the sucrose-fed rats. In the liver of 15-day fetuses from dams fed with the sucrose diet, the concentrations of triglyceride, total, free and esterified cholesterol increased, whereas in the liver of 19-day fetuses the concentration of all the lipid fractions decreased. The hepatic fatty acid synthase activity and the ³H₂O incorporation into hepatic lipids and glycogen increased in the sucrose-fed rats at days 15 and 19 of pregnancy and in the liver of 19-day fetuses. These results suggest that sucrose feeding to pregnant rats causes an alteration of the hepatic lipid metabolism in them and in their fetus, associated with the changes in carbohydrate metabolism.

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The effects of human milk fractions on clolera toxin B subunit binding to monosialoganglioside 1 (G_M1) were investigated. Human milk, human defatted milk, whey, and a low-molecular-weight fraction of human milk inhibited the binding, but casein did not inhibit it. The inhibitory activity of whey from bovine-milk-based infant formula was less than that of whey from human milk. Differences in composition between human and bovine whey seemed to influence the extent of the inhibitory activity. Sialylated oligosaccharides were considered to be the possible components that inhibited cholera toxin. The effects of sialyllactose, a predominant sialylated component of human milk, on cholera toxin-induced diarrhea were investigated by the rabbit intestinal loop method. Sialyllactose inhibited the cholera toxin inducing fluid accumulation, although neither sialic acid nor lactose had an effect on it. The results suggest that sialyllactose is responsible for the inhibitory activity of milk on cholera toxin.

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We constructed an assay system to measure the annealing activity that is one of the functional features of nuclear proteins, using ssDNAs derived from M13 phage recombinants which contained a complementary 406-bp portion each. Histone H1 variants were puritied from porcine thymus by separation of chromatin, extraction with 5% perchloric acid, and reversed-phase HPLC. Three types of histone H1 variants were found by analysis of amino acid composition and on SDS-PAGE. All of these could promote the annealing. According to Hill's analysis all had similar numbers of binding sites to DNA strands but dissociation constants and annealing activity were different. The number of binding sites, dissociation constants, and annealing activity were changed by dephosphorylation of histone H1 variants. This result suggests that histone H1 variants have different affinities for DNA molecules and ssDNA-annealing activity, which is regulated by phosphorylation.

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Indonesian dried-salted fish (DSF) was produced from skipjack tuna by soaking the flesh in 15% NaCl (DSF I) or 25% NaCl (DSF II). The DSFs were then hydrolyzed by trypsin, chymotrypsin, Pronase E, and pepsin. Angiotensin I-converting enzyme (ACE) inhibitory activity was measured. The pepsin digest showed the highest inhibitory activity (IC_5o ; 0. 63mg protein/ml). DSF II hydrolysate had higher inhibitory activity than that in DSF I. A three-month storage period of DSF gave higher ACE-inhibitory activity than that of 6 months. An oral administration of pepsin hydrolysate significantly decreased the blood pressure of rats. From the purification steps, at least 4 inhibitor peptides were found. The amino acid sequences of the peptides were Val-Ala-Trp-Lys-Leu, Trp-Ser-Lys-Val-Val-Leu, Ser-Lys-Val-Pro-Pro, and Cys-Trp-Leu-Pro-Val-Tyr, with an lC^5o value of 31. 97, 156. 28, 74. 22, and 22. 20μM, respectively.

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Three inherent acidic chitinases were puritied from yam aerial tuber by the application of quaternary ammonium ion detergent. For removal of the mucilage in the crude extract of aerial tuber, the effects of various quaternary ammonium ion detergent were compared. Cetylpyridinium chloride effectively removed the mucilage and this method proved to provide high recovery of chitinase activity. Three acidic chitinase isoforms were purified by anion-exchange chromatography, gel-filtration chromatography, and anion-exchange HPLC from the supernatant of cetylpyridinium chloride treatment. The three purified acidic chitinases had the molecular masses of 23, 25, and 31kDa and proved to be class III, class III, and class I chitinases, respectively, by the comparison of tryptic peptide maps and amino acid compositions.

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An iron-oxidizing bacterium Thiobacillus ferrooxidans resistant to bisulfite ion (strain OK1-50) was isolated from stream water from Okayama City, Japan and characterized. When grown statically on FeSO₄. 7H₂O (3%)-salts medium without sodium sulfite, OK1-50 and T. ferrooxidans strain AP19-3 had the same level of cell growth (200 × 10⁶ cells/ml). However, when cultured in FeSO₄. 7H₂O (3%)-salts medium with 5mM sodium sulfite, OK1-50 and AP19-3 gave maximal cell yield of 105 × 10⁶ and 35 × 10⁶ cells/ml, respectively, suggesting that OK1-50 was much more resistant to bisulfite ion than AP19-3 is. The inhibition site of bisultite ion on iron oxidase system is cytochrome c oxidase, which is one of the essential constituents of the iron oxidase system. Iron-oxidizing activity of AP19-3 was completely inhibited by 0. 2mM bisulfite ion. In contrast, the activity of OK1-50 was not inhibited by 2mM sodium sulfite. Cytochrome c oxidase activity of AP19-3 was completely inhibited by 2mM bisulfite ion. However, 5mM bisulfite ion did not inhibit the activity of OK1-50. A similar level of sulfite : ferric ion oxidoreductase activity was observed in both strains. Hydrogen sulfide : ferric ion oxidoreductase activity of OK1-50 was one half of the activity of AP19-3. NADH-dependent sulfite reductase activity of OK1-50 was three times higher than that of AP19-3. These results indicate that having an iron oxidase system resistant to bisulfite ion makes strain OK1-50 much more resistant to bisulfite ion than AP19-3 is.

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Twelve strains of methanol-assimilating yeast species were examined for their partial base sequences of 18S and 26S rRNAs. In the partial base sequencings of 18S rRNA (positions 1451-1618, 168 bases), P. kodamae had the same partial base sequence as Ogataea minuta, and P. finlandica, P. pini, P. trehalophila, C. maris, and C. methanolovescens were the same in the partial base sequences as O. glucozyma. Candida boidinii and C. methylica were similar to O. glucozyma, but somewhat different from O. minuta (the base differences, one and four, respectively). Pichia naganishii had seven, four, six, and nine base differences with O. minuta, O. glucozyma, P. anomala, and P. membranaefaciens, respectively, and C. methanosorbosa did four, three, five, and twelve base differences. Pichia pastoris was quite different (the base differences among the methanol-assimilating yeasts, 32-31), and no bases were found in the fingerprint segment. In the partial base sequencings of 26S rRNA (positions 1611-1835, 225 bases ; positions 493-622, 130 bases), P. finlandica, P. kodamae, P. pini, P. trehalophila, C. maris, C. methanolovescens, and C. methanosorbosa were similar to O. minuta and O. glucozyma. Pichia naganishii, C. boidinii, and C. methylica had somewhat different partial base sequences. The base differences and the percent similarities of P. pastoris were quite high (101-89) and quite low (40-47). Based on the sequence data obtained, the methanol-assimilating yeast species are discussed taxonomically and phylogenetically. A new genus, Komagataella was proposed for P. pastoris with a new combination, Komagataella pastoris.

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Sixteen strains of the sixteen species, including the type species, Y. philogaea (CBS 6696, type strain), of the genus Yamadazyma were examined for their partial base sequences of 18S and 26S rRNAs. The genus Yamadazyma BILLON-GRAND was found to have a heterogeneous nature Phylogenetically. In the partial base sequences in positions 1451-1618 (168 bases) of 18S rRNA, the number of base differences was 4-0 within the genus except for Y. spartinae, Y. inositovora, Y. ohmeri, and Y. besseyi. The base differences numbered 6-1, 11-8, and 8-4 with D. hansenii, P. membranaefaciens, and S. cerevisiae, respectively. In the partial base sequences in positions 1611-1835 (225 bases) of 26S rRNA, the number of base differences was 14-0 within the genus. The base differences numbered 19-0, 31-24, and 25-17 with D. hansenii, P. membranaefaciens, and S. cerevisiae, respectively. In the partial base sequences in positions 493-622 (130 bases) of 26S rRNA, the percent similarities were 73-93. The percent similarities were 77-90, 64-71, and 68-79 with D. hansenii, P. membranaefaciens, and S. cerevisiae, respectively. Yamadazyma inositovora, Y. spartinae, and Y. ohmeri were not closely related phylogenetically. Yamadazyma besseyi (Q-7) was separate phylogenetically from the species mentioned above of the genera Yamadazyma, Debaryomyces, Pichia, and Saccharomyces (base differences, 13-7 and 62-17 ; percent similarities, 48-63). The discussion was made phylogenetically and taxonomically, especially on transferring Y. besseyi to a separate taxon.

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A fructose 1, 6-bisphosphate [Fru(1, 6)P₂] and divalent cation-regluated allosteric L-lactate de-hydrogenase (L-LDH) (EC 1. 1. 1. 27) of Lactobacillus casei was highly produced in Escherichia coli cells, together with its mutant enzyme, in which His-188 was replaced by Asp. Under acidic conditions, the mutant enzyme showed positive allosteric regulations by the substrate pyruvate and its analogues, like the wild-type enzyme, but not by Fru(1, 6)P₂, which even inhibited the stimulative effects of the alternative activation factors. In addition, Mn²⁺ ions also showed greatly reduced inhibitory effects on the mutant enzyme. Under neutralconditions, on the other hand, the reaction of the mutant enzyme was slightly enhanced by Fru(1, 6)P₂, but not further stimulated by additional Mn²⁺ ions, unlike the case of the wild-type enzyme. These results indicate that His-188 is, though not essential for the regulation by the alternative factors, essential for the cooperative regulation by Fru(1, 6)P₂ and divalent cations in L. casei L-LDH.

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The modification of amino acid residues in sugar beet α-glucosidase with conduritol B epoxide (CBE), an affinity labeling reagent, inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The enzyme was protected from inactivation by a competitive inhibitor, Tris, and the partially inactivated enzymes showed only the decrease of V values and no change in Km value. An ³H-CBE labeled peptide isolated from the digest of the inactivated enzyme with Lys-C protease was sequenced. The -COO - group of Asp was found to be specifically labeled, implicating that it is a catalytic group of the enzyme. The sequence around the essential Asp was determined to be -DGIWIDMNE-, which showed a high homology with those of other α-glucosidases.

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The precise chemical structures of two carotenoid glucoside esters, keto-myxocoxanthin glucoside ester isolated from Myxococcus sp. MY-18 and dehydrorhodopin glucoside ester from cells cultured in a nicotine-containing medium, were determined as 1'-[(6-O-acyl-β-D-glucopyranosyl)oxy]-3', 4'-didehydro-1', 2'-dihydro-β, ψ-caroten-4-one and 1-[(6-O-acyl-β-D-glucopyranosyl)oxy]-3, 4-didehydro-1, 2-dihydro-ψ, ψ-carotene, respectively. They had β-D-glucoside, whose C-6 hydroxyl group was esterified. The major fatty acid was 11-methyldodecanoate with minor components of n-dodecanoate, n-tetradecanoate, and 13-methyltetradecanoate. This composition is different from that of cellular lipids, and the unusual accumulation of phytoene (more than 70% of total carotenoids) is characteristic of this strain. The possible biosynthetic pathway for the carotenoid glucoside ester starts with lycopene being cyclized to γ-carotene, from which myxocoxanthin is produced by hydration and desaturation. β-D-Glucose was attached to the carotenoid, and the fatty acid ester was next. The pathway for the introduction of a keto group to the carotenoid moiety was not clear, however. The final product was keto-myxocoxanthin glucoside ester. Cyclization was completely inhibited by 6. 0mM nicotine in the medium, in which case, the final product was dehydrorhodopin glucoside ester.

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Two novel trypsin inhibitors, MCTI-II' and BGIT, were isolated from the seeds of bitter gourd (Momordica charantia) and their amino acids sequenced. MCTI-II' was a squash type trypsin inhibitor and lacked the N-terminal arginine residue of Momordica charantia trypsin inhibitor (MCTI)-II. It consists of 27 amino acid residues and forms a dimeric structure. BGIT had 88% homology with bitter gourd inhibitor against an acidic amino acid-specific endopeptidase (BGIA) consisting of 68 amino acid residues and inhibited not only trypsin but also subtilisin Carlsberg. The amino acid replacements occurred in 8 positions of which that of Gln2 by Arg or of Ala44 by Lys is suggested to be responsible for the trypsin-inhibitory action of BGIT. Inhibitory activity of BGIT for trypsin was greatly decreased by acetylation, while that for subtilisin was slightly increased. From these results and the sequence comparison with eglin-c superfamily inhibitos, the reactive site of BGIT is assumed to be Lys44

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Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was puritied to an electrophoretically homogeneous state and an activity of more than about 20, 000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.

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Small intestinal epithelial cells interact with a rather high concentration of fatty acids derived from the diet. These fatty acids directly or indirectly regulate the functions of the small intestine. We report here that linoleic acid and oleic acid markedly increased the influx of ⁴⁵Ca²⁺ into the small intestinalepithelial cell line (IEC 6). By contrast, octanoic acid, methyllinoleate, and linolyl alcohol had no effect on the influx. Acidic amino acids, methyl linoleate, and linolyl alcohol, inhibited the linoleic acid-induced influx of ⁴⁵Ca²⁺, indicating that activation of the influx by linoleic acid depended on the chain length and was affected by the presence of a carboxyl group. Of the gastrointestinal hormones, somatostatin specifically inhibited the linoleic acid-induced influx of ⁴⁵Ca²⁺

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Ergosterol is a major sterol component of fungal plasma membranes. The effects of disrupting the Saccharomyces cerevisiae SYR1/ERG3 gene, which encodes sterol C-5 desaturase, an enzyme of ergosterol biosynthesis pathway, were markedly different for different S. cerevisiae strains and growth temperatures. The null mutation of SYR1 (Δsyr1) in strain RAY-3A had only a slight effect on the growth rate at 28°C. However, at this temperature, the same mutation caused poor growth in strain KA-311A and no growth in strain W303-1A. The Δsyrl disruptant of these strains were able to grow at 37°C, as well as their parental strains. Moreover, the growth of the Δsyrl disruptant of W303-1A and KA-311A strains were severely inhibited at 16°C. These results indicated that ergosterol is essential for growth at low temperatures, and the effects of the gene disruption are variable by the genetic background. The growth defect at low temperatures appeared to be due to the defect of tryptophan uptake in the Δsyrl mutants. The Δsyrl mutants were sensitive to a wide variety of drugs, chemicals, and ions, suggesting that yeast ergosterol is important as permeability barrier against various chemical stresses.

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Fructosyl lysine oxidase (FLOD) was examined for its use in the enzymatic measurement of the level of glycated albumin in blood serum. To isolate micoorganisms having such an enzyme activity, we used N^ε-fructosyl N^α-Z-lysine (ε-FL) as a sole nitrogen source in the enrichment culture medium. The isolated fungus, strain S-1F4, showed a high FLOD activity in the cell-free extract and was identified as Fusarium oxysporum. FLOD was purified to an apparent homogeneity on SDS-PAGE. The molecular mass of the subunit was 50 kDa on SDS-PAGE and seemed to exist in a monomeric form. The enzyme had an absorption spectrum characteristic of a flavoprotein and the flavin was found to be covalently bound to the enzyme. The enzyme acted against N^ε-fructosyl N^α-Z-lysine and N^α-fructosyl N^ε-Z-lysine and showed specificity for fructosyl lysine residues.

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To improve the stability of IgY antibody in oral administration, encapsulation of IgY in a W/O/W emulsion was attempted. A stable W/O/W emulsion containing 1% IgY was prepared by using polyglyceryl condensed ricinolate (PGCR) and dextran-casein conjugate as the primary and secondary emulsifier, respectively. However, the activity of IgY antibody was reduced to less than 20% by encapsulation, suggesting that denaturation/inactivation of IgY had occurred at the oil/water interface. Adsorption of IgY to the inner water droplet surface was observed by electron microscopy. Rabbit IgG, α-amylase, and lysozyme also lost their activity after being encapsulated, although the rate of inactivation was lower than that of IgY. Molecular characterization of these proteins suggested that the rate of inactivation after encapsulation is likely to be dependent on the surface hydrophobicity and molecular stability of each protein.

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All four stereoisomers of coronamic acid were synthesized from both enantiomers of 1, 2-butanediol. Cyclopropanation of cyclic sulfate with the dibenzyl malonate anion gave cyclopropane dibenzyl ester in a quantitative yield. Transformation into a protected amino acid was achieved by stereoselective hydrolysis and Curtius rearrangement as the key steps. Saponification and subsequent acidic hydrolysis in one pot and ion exchange gave a free amino acid in a high yield.

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1-O-[5-(Carboxy) pentanoyl]-2-deoxy-2-(2, 2-difluorotetradecanamido)-3-O-[(R)-3-(tetradecanoyl-oxy)tetradecanoyl]-4-O-phosphono-α-D-glucopyranose (13) and its analogues (16 and 19) were synthesized. Compound 13 showed strong LPS-agonistic activity.

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The structures of N-linked sugar chains from 350 kDa royal jelly glycoprotein (RJG), which stimulates the proliferation of human monocytes, have been analyzed. The structural analyses of the Nlinked sugar chains released by hydrazinolysis were done by twodimensional sugar mapping of the corresponding pyridylamino derivatives and 500MHz ¹H-NMR. All four structures were found to fall into the category of oligomannose-type sugar chains as follows ; Manα1→6(Manα1→3)Manα1→6(Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc, Manα1→2Manα1→6(Manα1→3) Manα1→6(Manα1→2Manα1→3) Manβ1 →4GlcNAcβ1 →4GlcNAc, Manα1→2Manα1→6(Manα1→3)Manα1→6(Manα1 →2 Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc, and Manα1→2Manα1→6(Manα1→2Manα1→3)Manα1→6(Manα1→2Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc.

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Casein phosphopeptides (CPPs), which inhibit the precipitation of calcium phosphate in the intestines, were prepared as CPP-calcium phosphate complexes from casein micelles by the ultrafiltration method. The prepared CPPs hardly contained any other peptides, so there was no need to treat with active carbon to remove the bitter peptides. The sum of α_s1-CN-5P(f59-79) and β-CN-4P(fl-25) comprised 60% of the CPP composition by an analysis of the elution pattern from a Q-Sepharose FF column. The content of α_s1-CN-5P(f59-79), which has high retention ability for calcium phosphate, was the highest in the CPPs.

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Guanidinobutyrase (EC 3.5.3.7) involved in the arginine oxygenase pathway of Brevibacterium helvolum IFO 12073 was found to catalyze also the hydrolysis of 2-ketoarginine (2-keto-5-guanidinovalerate) to 2-ketoornithine (2-keto-5-aminovalerate) and urea, the second step of the arginine aminotransferase pathway. No other enzyme that degraded 2-ketoarginine was found in cells grown on L-arginine. The enzyme hydrolyzed 2-ketoarginine with a relative rate of about 0.7% of that toward 4-guanidinobutyrate. The K_m for 2-ketoarginine was 33mM.

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A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40. The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments. The enzyme was identified as a 31kDa protein which is probably an N-acetylmuramyl-L-alanine amidase. The aminoterminal 15 residues of the enzyme were : VLPEPVVPEYIVVHN.

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The brominative cyclization of 5-bromo-2-geranyl-1, 4-benzenediol bismethoxymethyl ether (2) with 2, 4, 4, 6-tetrabromocyclohexadienone was reexamined. This cyclization of 2 resulted in the formation of methoxymethyl ether 10 of 2, 4, 6-tribromophenol, together with previously described tricyclic compound 7, thus supporting the intervention of oxonium ion 12 in this reaction. An analogous reaction of bismethoxyethoxymethyl ether 4 led to 8. Finally, 7 was converted to (±)-4-isocymobarbatol(6).

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We analyzed 18S and 26S rRNA partial base sequences [positions 1451-1618 (168 bases) of 18S rRNA and positions 1611-1835 (225bases) and 493-622 (130 bases) of 26S rRNA] of a total of three strains of Pichia jadinii and Candida utilis. The three strains had identical base sequences with the type strain of P. jadinii (IFO 0987)in the 18S rRNA partial base sequencings. In the 26S rRNA partial base sequencings, there were partial base sequences similar to each other (1-0 base difference and 87-95 percent similarities). The sequence data obtained are discussed taxonomically and phyloge-netically, especially in connection with Williopsis saturnus, the type species of the genus Williopsis ZENDER.

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Specific detection of β-xylanase activity on isoelectric focusing gels was done by using 2, 3, 5-triphenyltetrazolium chloride (TTC). Reducing sugars released from water-soluble xylan with β-xylanase reacted with TTC, and then xylanase activities appeared as pink-red bands on the gel. The sensitivity of detection for β-xylanase by this procedure was higher than that of protein detection with Coomassie Brilliant Blue R-250.

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DL-Homocysteine thiolactone hydrochloride showed growth inhibition toward the roots of Brassica campestris L. subsp. rapa HooK. fil. et ANDERS, Lactuca sativa L. var. longifolia LAM, and Echinochloa utilis 0HWI et YABUNO even at the low concentration of 5.0 × 10^-5 M. This inhibitory activity is higher than that of dihydro-2(3H)-thiophenone reported previously. On the other hand, the N-acetyl and N-benzoyl derivatives of DL-homocysteine thiolactone exhibited much less activity. These facts suggest that the free amino group at C-3 in DL-homocysteine thiolactone is essential to enhanced inhibitory activity. The amounts of chlorophyll in the cotyledons of B. campestris treated with DL-homocysteine thiolactone were decreased when compared with the control.

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Three glucosides (1-3), together with known phenylpropanoids and polyacetylenes were isolated from Oenanthe javanica Blume (DC.). Glucoside 1, named oenanthoside A, is a new phenylpropanoid glucoside and was determined to be 2, 3-methylenedioxy-5-allylphenylβ-D-glucopyranoside, mainly by NMR techniques. Glucosides 2 and 3 were identified as eugenylβ-D-glucopyranoside and pinoresinolβ-D-glucopyranoside, respectively. These were found for the first time in this plant.

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Both chitinase A1 and D from Bacillus circulans WL-12 specifically hydrolyzed the N-acetyl-β-D-glucosaminidic bonds in 50% N-acetylated chitosan molecules to produce hetero-oligo-saccharides with GlcNAc at the reducing end residues, together with GlcNAc and (GlcNAc)₂. GlcN-GlcNAc and GlcN-GlcNAc-GlcNAc were produced as major hydrolysis products with chitinase A1 and D, respectively, but GlcN-GlcNAc was not detected in the digest of 50% N-acetylated chitosan with chitinase D.

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For secretion of human apolipoprotein E (hApoE) by Saccharomyces cerevisiae, the hApoE gene was fused to truncated human serum albumin (HSA)-encoding sequences and expressed under the control of the GAL7 promoter. When the mature region of the hApoE gene was fused to the HSA-encoding sequence without its pro-region and expressed in galactose-containing medium, the HSA-hApoE fusion protein was efficiently secreted into the medium at a maximum yield of 6.3 mg per liter.

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When higher alcoholic solvents were added to the reaction medium, and the enhancement of the enzyme activity, followed by its reduction then inactivation, were observed in thermolysin-catalyzed peptide hydrolysis and synthesis. The organic solvent content used was less than the saturating concentration in the buffer (i.e., water-organic one-phase system). The kinetic parameters, K_m and k_cat, at the alcoholic concentration giving maximal enzyme activity in these reactions changed linearly with increasing log P values of the alcohols and consequently k_cat/K_m as well. When the branched isomers of alcohols with structurally minimal variation of which log P was equivalent theoretically, were used as annexments, the kinetic parameters were also changed. The results, especially the changes of K_m for each organic solvent, suggested that each alcohol should act at the active site of the enzyme in its own effective mode.

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Three distinct extracellular xylanases (FIa-, FIb-, and FIII-xylanases) from a culture filtrate of Aspergillus aculeatus No. F-50 were purified to homogeneity by SDS polyacrylamide gel electrophoresis. The molecular weights of FIa-, FIb-, and FIII-xylanases were estimated to be 18, 000, 26, 000, and 52, 000, and their pI values were pH 5.6, 9.0, and 3.8, respectively. The pH optima of xylanase activities were from 4.5 to 5.0. The optimum temperatures for enzyme activities were from 50°C to 70°C. All three xylanases were highly specific for xylan hydrolysis, and they did not cleave xylobiose.

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Restriction patterns of plasmids isolated from ten Japanese wild strains of Agrobacterium rhizogenes were analyzed and the homology of the plasmid digestion patterns within these strains is described. From the size of homologous fragment against the EcoRI digested 7. 5kb T-DNA of pRi1724, these strains could be divided into two groups which was consistent with opine types.

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Galactosylkojic acid, in which the hydroxymethyl group of the kojic acid moiety is β-galactosylated, was synthesized by using a transgalactosidation reaction with an immobilized β-galactosidase from Bacillus circulans. With 5% kojic acid and 20% lactose as the substrates, the yield with respect to the initial amount of kojic acid was around 24% as a maximum. The inhibitory effect of galactosylkojic acid towards the oxidation reaction of L-β-(3, 4-dihydroxyphenyl)alanine (L-DOPA) with tyrosinase was similar to that of kojic acid.

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This work was done to characterize the structure of a photosynthetic polysaccharide and its metabolizing enzymes in cyanobacteria, which represent a link between bacteria and green plants in evolutionary terms. Filamentous cyanobacteria, occurring in an alkaline hot spring (45-50°C, pH 8.5-9.0) in Kagoshima Prefecture, were morphologically classified in the genera Lyngbya-Phormidium-Plectonema (LPP). We found a thermostable neutral α-glucosidase with optimum pH 6.5 in the LPP. A polysaccharide isolated from the TCA-soluble fraction of the LPP was characterized as glycogen that resembled animal glycogen in structure. We also recognized the presence of the TCA-insoluble glycogen at 32-38% of the total amount of glycogen, most of which was bound non-covalently to protein and had a similar iodine absorption spectrum to that of the TCA-soluble glycogen.

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High enantioselectivity was observed for the Pseudomonas cepacia lipase-catalyzed hydrolysis of (E)-allylic acetate l and propargylic acetate 2 in an acetone-water solvent system, while (E)-homoallylic acetate 3 and homopropargylic acetate 4 gave poor enantioselectivity.

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Peptidylarginine deiminase (PAD) catalyzes the conversion of arginyl residues in proteins to citrullyl residues in the presence of Ca²⁺ . Recently, we obtained a monoclonal antibody, EH7, which reacted only with mouse PAD type II. Here, we describe immunohistochemical findings on the cellular localization of PAD type II in mouse fetus by using the monoclonal antibody. PAD type II is expressed in yolk-sac erythroid cells and the level of the enzyme in these cells decreases as the cells differentiate.

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Studies were done on fermentative production of hydroxyproline, a useful chiral source of the side chain moiety of carbapenems. Various microorganisms were screened using an automatic screening-robot system, AminoQuant (Hewlett-Packard), and we found that one fungus strain produced 13.8μg/ml of trans-4-hydroxy-L-proline(t-Hyp). The strain was identified as Clonostachys cylindrospora SANK 14591. Addition of L-proline or glycine to the culture medium stimulated the production of t-Hyp by C. cylindrospora. A ¹³C-NMR study found that L-proline but not glycine was incorporated into t-Hyp.

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