Mutants of the yeast Saccharomyces cerevisiae that are partially deficient in aldehyde dehydrogenases (ALDHs) were isolated. A mutant, AK-9, was shown to be especially defective in the NADP⁺-linked ALDH. The K⁺-activated ALDH of a mutant AK-25 was found in the cytosol, but not in the mitochondria. The strain AK-25 continuously accumulated acetate even in the phase of ethanol consumption in batch aerobic cultures. A mutant, AK-26, was defective in two isozymes of ALDH, particularly in the K⁺ -activated ALDH. The aldh mutants of AK-9 and AK-25 were more sensitive to the growth inhibitory effects of ethanol and NaCl, but the mutant AK-26 was only sensitive to ethanol. All aldh mutants stimulated more acetaldehyde production than the wild-type strain during conditioning in media containing ethanol. The mutants except for AK-26 almost never produced acetaldehyde during conditioning in media containing 2+6% NaCl. The mutant AK-26 produced glycerol almost the same as the wild-type strain during conditioning in NaCl. These results suggested that two isozymes of ALDH were related to ethanol tolerance of which the key substance was acetaldehyde induced by ethanol stress, and that glycerol production induced by salt stress was closely related to K⁺ -activated ALDH.

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Cellulolytic enzyme activities in 11 strains of B. cinerea and their pathogenicity against apples, grapes, and lettuce were tested. A positive correlation was found between the β-glucosidase activity of B. cinerea cultured in PYA-medium and the pathogenicity, as expressed in the area of lesions. Further, this correlation was found to hold for the β-glucosidase activity of the apple fruit lesion caused by B. cinerea. Since it was suggested that pathogenicity of B. cinerea depended on its β-glucosidase activity, the β-glucosidase was purified and characterized. The enzyme had its optimum pH at 3. 0, and was very stable in a wide range of pH. These properties were extremely advantageous for B. cinerea to infect the apple fruit.

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We have cloned a genomic DNA encoding the alkaline protease (Alp) of Fusarium sp. S-19-5 from a genomic DNA library and sequenced the nucleotides. Complementary DNA encoding Alp was also isolated from the cDNA library after amplifying the gene by PCR using partial sequences of the Alp genomic DNA as primers. The Alp gene has an open reading frame of 1137 nucleotides containing three introns. A TATA box (TAAATA) was observed 112 base pairs upstream from the translation initiation codon in the 5'-non coding region. The Alp protein has a pre region consisting of 14 amino acids and a pro region of 85 amino acids preceding the mature region, which consists of 280 amino acids. The amino acid sequence of Fusarium Alp has 52% homology with that of Aspergillus oryzae and 51% homology with that of Acremonium chrysogenum. The entire cDNA encoding Fusarium Alp was introduced into Saccharomyces cerevisiae, which then secreted enzymatically active Alp into the culture medium.

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We transformed Acremonium chrysogenum with the genomic DNA of the alkaline protease (Alp) from Fusarium sp. S-19-5 including its promoter. Most of the transformants thus obtained produced a large amount of Alp. PCR and Southern hybridization analysis of genomic DNAs from these transformants showed chromosomal integration of the full-length Alp gene. SDS-PAGE analysis of the supernatant from the transformants showed the presence of Fusarium Alp. The amino terminus of the Alp produced in A. chrysogenum was identical to that of native Fusarium Alp. These results indicate that the Alp promoter, signal sequence, and introns functioned correctly in A. chrysogenum. 0ne of the transformants produced more than 4g of the Alp per liter in a jar fermentor.

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Wheat-germ extract for cell-free protein synthesis was condensed with ultrafiltration membranes of which the molecular cut-off values were l0kDa, 100kDa, and 300kDa. Reaction conditions of the cell-free system were optimized for the condensed extracts, which needed a higher concentration of creatine phosphate than the uncondensed one, probably due to the increased activity of degradation of ATP and GTP. By using the condensed extract and optimized reaction conditions, the rate of protein synthesis was increased 2- to 3-fold compared with using an uncondensed extract, and about 10-fold compared with conventional conditions. Condensation of the extract with the 300-kDa membrane showed the highest productivity, which was about 30μg dihydrofolate reductase protein ml^-1 h^-1. The final amount of synthesized protein was one third of that of a continuous-flow cell-free (CFCF) system reported by Endo et al. [J. Biotechnol., 25, 221-230 (1992)] but the productivity was 5-fold higher than that obtained by the CFCF system.

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Corynebacterium glutamicum mutants lacking ATP phosphoribosyl transferase (PRT) were selected by complementation with the Escherichia coli PRT gene. The recombinant plasmid pCH13 carrying a wild type PRT gene from C. glutamicum T106 was obtained in one of the mutants, LH13. Transformants, LH13/pCH13 and T106/pCH13, had three times higher PRT specific activity than T106. The plasmid pCH99 specifying the PRT, which was desensitized to feedback inhibition by L-histidine fifty-fold higher than the wild type PRT, was derived from pCH13. L-Histidine productivity of C. glutamicum F81, was markedly decreased by pCH13, but increased twice by pCH99. In cultivation in jar fermentors, F81/pCH99 continued to accumulate L-histidine through fermentation and yielded to the titer of 22.5g/liter, while F81accumulated only 11.5g/liter due to production retardation halfway through fermentation. Moreover, F81/pCH99 had a larger production rate than F81 even in its production phase. These results indicate that the yield improvement results from amplification of the highly desensitized PRT provided by pCH99.

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Two new compounds of 1-β-D-arabinofuranosylcytosine (ara-C) were found to be produced in a high yield in a culture filtrate of Sporobolomyces singularis, when grown on a medium containing lactose and ara-C. The compounds I and II were obtained as white needle crystals from the culture filtrate by preparative paper chromatography, gel-filtration on Sephadex G-10 and Toyopearl HW-40S, lyophilization, and ethanol treatment. The compounds I and II were identified as 3'-O-(β-D-galactopyranosyl)-ara-C and 3'-O-[β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl]-ara-C, respectively, on the basis of the various experimental results, viz., elementary analyses, UV, IR, 1^H-, and <13>^C-NMR spectra, and products by hydrolysis with α- and β-galactosidases. Also, the yeast produced a large amount of 3'-O-β-galactosyl compounds of adenosine and inosine in the culture filtrate when grown on a medium containing lactose and their ribonucleosides.

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The volatile flavor components from the fruits of S. borneensis were isolated by two different procedures. The purge and trap injection (PTI) method showed that the volatiles consisted of ethanal, methyl methylthiomethyl disulfide (I), and some other sulfur-containing compounds. Steam distillation (SD) under reduced pressure revealed that an additional sulfur compound, bis(methylthiomethyl) disulfide (II), was also a significant volatile component. The odor thresholds of synthesized I and II in an aqueous solution were determined. At a dilute concentration, I and II exhibited the fresh and long-standing smell of sliced fruit, respectively. II also possessed relatively strong antifungal activity.

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Twenty four tetrahydropyran esters were synthesised and tested as attractants toward Blattella germanica and Supella longipalpa in the laboratory by using the choice-chamber method. In general, carboxylates were superior to propionates and acetates. Among the 2-, 3-, or 4-substituted esters, substitution at the 4-position was better than at the other two positions. These results are explained in terms of the receptor model proposed earlier by others.

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Transgalactosylated derivatives of cyclodextrins (CDs) and glucosyl and maltosyl CDs (Gl- and G2-CDs) were synthesized by α-galactosidases from coffee bean and Mortierella vinacea (M. vinacea). The structures of the transfer products were analyzed by FAB-mass, <13>^C-NMR and methylation. Coffee bean α-galactosidase transferred a galactosyl residue not only to side chains of G1-CDs and G2-CDs, but also directly to CD rings. M. vinacea α-galactosidase transferred a galactosyl residue only to side chains of G2-CDs.

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Microorganisms were isolated from fermented milk manufactured by Masai men in northern Tanzania. The lactic acid bacteria isolated were identified as Lactococcus lactis ssp. lactis, Lactococcus garvieae, Enterococcus faecium, Streptococcus bovis, Lactobacillus confusus, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus viridescens. The dominant strains were Lc. lactis ssp. lactis and Lb. confusus. Yeasts identified as Saccharomyces sp. and Candida sp. were isolated from several samples as minor strains. The binding ability of 21 strains of lactic acid bacteria to amino acid pyrolysates was investigated. 3-Amino-1, 4-dimethyl-4H-pyrido[4, 3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2), and 1-methyl-9H-pyrido[4, 3-b]indole (Harmane) were bound to cells of all bacterial species. The growth of Staphylococcus aureus and Escherichia coli in the cultured milk was inhibited by Lc. lactis ssp. lactis and Lb. confusus.

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Resting cells of Pseudomonas fluorescens TN5 catalyze the hydroxylation at the 6-position of nicotinic acid to produce 6-hydroxynicotinic acid. We optimized the reaction and culture conditions for the production of 6-hydroxynicotinic acid from nicotinic acid by using P. fluorescens TN5 resting cells. The addition of molybdenum and iron ions and nicotinic acid as an inducer to the culture medium significantly enhanced the formation of the hydroxylation enzyme. The supply of oxygen was important for the enhancement of hydroxylation activity. Under the optimum conditions, 98.7% of the added 1.4M nicotinic acid was converted to 6-hydroxynicotinic acid, and the highest yield was 191g of 6-hydroxynicotinic acid per liter of reaction mixture containing 7.43g dry weight of cells in 45 h at 35°C

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Bialaphos, a glutamine synthetase (GS) inhibitor, is an effective inducer of rice sterility. L-Phosphinothricin (L-PTC), a metabolite of bialaphos, is the real inhibitor of GS from the results of an enzyme assay of GS from rice plants. Bialaphos and L-PTC induced a high level of sterility in rice at a concentration of 0.1 mM at the meiosis stage. The complete inhibition of GS activity was induced by 24-h treatments with 0.1 mM of bialaphos and L-PTC, respectively. These inhibitions continued until anthesis. In proportion to the inhibition of GS activity, the free ammonia content was increased in the panicles after treating with 0.1 mM of bialaphos and L-PTC. This accumulation of ammonia continued until anthesis. These results suggest that ammonia accumulating in the panicles as a result of GS inhibition directly caused sterility in rice.

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Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KYl0894 resulted in a marked increase (54%) in yield of tryptophan production (43g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C. glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901 : In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50g/liter of tryptophan in a 61% increased yield relative to strain KY10894.

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Pectin shows various physiological effects as dietary fiber. However, it is difficult to add pectin to food and drink an amount sufficient to give the physiological activities because of its high viscosity and poor solubility in water. Therefore, we examined a method for preparing low-molecular-weight pectins which would retain the activities. Using a culture fluid of Kluyveromyces fragilis as a crude enzyme solution, low-molecular-weight pectin (LP) was prepared by incubating with commercial citrus pectin at 40°C for 24h. The molecular weight and galacturonate content of LP were 66, 000 and 83.6%, respectively. LP showed low viscosity, high solubility, and a repression effect on liver lipid accumulation.

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Various microorganisms were screened for their ability to produce 2-hydroxybutyric acid (2-HBA) from 1, 2-butanediol (1, 2-BD). Strain TB-42, which was newly isolated from a soil sample, was selected as the best strain. From taxonomical studies, the strain was concluded to belong to the genus Rhodococcus. The effects of various carbon sources of the culture medium on the 2-HBA production from 1, 2-BD by the resting cells of this strain were tested. The addition of ethanol as a carbon source in the growing medium was found to be most effective. The resting cells of the strain grown on the ethanol as the carbon source yielded 16.7mg/ml of 2-HBA from 1, 2-BD for 12h incubation at 35°C in the reaction mixture under optimal conditions (20mg/ml of 1, 2-BD and 2% of CaCO₃).

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The glycoside fractions of a methanol extract from fresh tea leaves of Camellia sinensis var. assamica clone DT-1 (DT-1) and of C. sinensis var. sinensis cv. Yabukita (Yabukita) were obtained by Amberlite XAD-2 adsorption and methanol elution, respectively. These fractions were incubated with each of four enzymes, α-glucosidase, β-glucosidase, glycosidase and acetone powder (AP), at 37°C for 24h. After enzymatic hydrolysis by β-glucosidase, the formation of (Z)-3-hexenol and benzyl alcohol was observed by GC. (Z)-3-Hexenol, linalool, linalool oxides, methyl salicylate, geraniol, benzyl alcohol and 2-phenylethanol were observed after hydrolysis by the glycosidase and by acetone powder. A comparison of the calculated terpene index (TI) of the aglycones between the two varieties indicated the TI value for Yabukita to be lower and that for DT-1 to be as high as that of black tea manufactured from DT-1.

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D-Xylose isomerase was purified to homogeneity from cell-free extracts of Bifidobacterium adolescentis by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and Butyl-Toyopearl. The molecular weight of the purified enzyme was estimated to be 168, 000 by gel filtration on TSKgel G-3000SW, and 53, 000 on SDS-polyacrylamide gel electrophoresis. The optimum pH was around 7 and the enzyme was stable at pH 7-8. The enzyme required bivalent cations, Mg²⁺, Co²⁺, or Mn²⁺ for the activity, particularly Mn²⁺ to be best. The enzyme had a pI of 4.3, and the K_m for D-xylose was 4mM. The N-terminal amino acid sequence of the enzyme was not similar to those of D-xylose isomerases from other sources such as Clostridium thermosulfurogenes, Escherichia coli, or Bacillus subtilis.

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Dietary oils with a definite P/S ratio and different n-3/n-6 ratios (n-3 sources : α-LnA and EPA) were given to hypercholesterolemic rats for two weeks. Total cholesterol and phospholipids in serum decreased significantly in rats given α-LnA when the n-3/n-6 ratio was 0.1 or higher and in rats given EPA when the ratio was 0.05 or higher. But serum triacylglycerols decreased significantly only in the EPA group when the n-3/n-6 ratio was 0.2. Hardly any significant changes were observed in either group in the concentrations of liver lipids or neutral sterols and bile acids in feces. A significant increase of lipid peroxide concentrations was observed only in the liver of the EPA group when the n-3/n-6 ratio was 0.2. These results show that in rats fed on high cholesterol diets, the n-3/n-6 ratio of the dietary oil should be 0.1 when the P/S ratio is 1 and EPA is used as the n-3 source in view of significant decreases of serum TC and TG and suppression of the liver LPO formation.

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The rates of triacylglycerol and fatty acid synthesis in hepatocytes in suspension isolated from rats fed dietary soybean phospholipid were compared with those from the animals fed soybean oil. The rats were fed the experimental diets containing either soybean phospholipid or soybean oil (the dietary levels correspond to 3% fatty acids, 5.1 and 3.2% on weight bases for the phospholipid and the oil, respectively) for 10 days. Dietary soybean phospholipid compared to the oil decreased the rate of incorporation of [2-³H]glycerol into triacylglycerol in isolated hepatocytes. However, the difference disappeared when the incubation mixture was fortified with oleate substrate (1mM). The dietary phospholipid compared to the oil greatly decreased the rate of incorporation of [1-¹⁴C]acetate into fatty acids while it increased that of the tracer into cholesterol. The rate of ketone body production from oleate substrate was decreased in rats fed soybean phospholipid compared to those fed the oil. Thus, dietary soybean phospholipid compared to soybean oil appears to reduced the availability of fatty acids for triacylglycerol synthesis in rat liver through the depression in the rate of fatty acid synthesis.

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Glycinin is one of the most abundant storage proteins in soybean seeds. We earlier reported the preparation of proglycinins modified by protein engineering to improve food functions. Crystals of the modified proglycinins (ΔI, ΔV8, IV + 4Met, V + 4Met, Gly12, and Ser88) expressed in Escherichia coli were grown, each under different suitable crystallization conditions. The crystals of ΔI, V + 4Met, Gly12, and Ser88 diffracted X-rays sufficiently for crystallographic analysis. ΔI, Gly12, and Ser88 crystals were tetragonal, space group P4₁ or P4₃, and with unit cell dimensions a = b = 114.3-l15.9Å and c = 145.1-146.1Å. V + 4Met crystals were monoclinic, space group P2, and with unit cell dimensions a = 118. 7Å, b = 78.1Å, c = 109.9Å, and β = 119°. The number of protomers per asymmetric unit of all of the crystals of these four modified proglycinins was about 3. This value is consistent with proglycinins being trimers. These data indicated that most of the modified proglycinin crystals examined here could be studied by X-ray crystallography to elucidate the relationships between the structure and the functional properties of glycinin at molecular level.

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8'-and 9'-Methoxyabscisic acids (methoxy-ABAs) were synthesized as antimetabolic analogs of abscisic acid (ABA). The racemic mixtures were resolved by HPLC. (+)-8'-Methoxy-ABA inhibited the elongation of rice seedlings four times more strongly than (+)-ABA did, and (+)-9'-methoxy-ABA inhibited the germination of lettuce seeds and the induction of α-amylase by gibberellin A₃ in barley seeds seven and two times more strongly, respectively. These methoxy-ABAs are the first analogs reported to have higher activity than ABA. (+)-8'-and (+)-9'-Methoxy-ABAs were less active in causing stomatal closure than (+)-ABA. These biological activities and 1^H-NMR analysis suggest that the activity of methoxy-ABAs involved delayed metabolism, as was expected. The activity of (-)-isomers of ABA and methoxy-ABAs was also tested.

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Ricin A-chain, a protein that inactivates ribosomes by a specific RNA N-glycosidase activity, has been shown to be inactivated by chemical modification of a few arginine residues. When two or fewer arginine residues in the A-chain were modified with [¹⁴C]phenylglyoxal, arginines at positions of 193, 196, 213, and 234/235 were found to be modified, from amino acid compositions and radioactivities of the modified peptides that were obtained by cyanogen bromide cleavage followed by tryptic and chymotryptic digestion. All these arginines have side chains outside the active site cleft ; the side chain of Arg213 is adjacent to the edge of the cleft, while other modified arginines are located on the opposite side of the cleft. Kinetic analysis showed that the modification of two arginine residues caused a 8-fold loss in k_cat with a 3-fold increase in K_m, suggesting that this modification mainly decrease the rate of depurination with an additional effect on the affinity for ribosomes. Neither the environment of tryptophan 211 at the bottom of the cleft nor an interaction of adenine with the cleft was changed by this modification, as judged by fluorescence spectroscopy, suggesting that a conformational change of the catalytic site does not occur upon the modification. These results, taken together with other works, suggest that some of the above arginine residues outside the active site cleft may additively contribute to the catalysis of depurination and/or the initial formation of the A-chain/ribosome complex.

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The agglutination of bacterial cells in Clostridium innocuum, Bifidobacterium longum, and Micrococcus luteus by lactoferrin (Lf) and ovotransferrin (OTf) was observed by measuring the absorbance of the cells in suspension and also under a phase-contrast microscope. Lf had the ability to agglutinate C. innocuum and M. luteus, but not B. longum. However, OTf and other proteins from bovine and human milk and from hen's egg white did not affect the agglutination of these bacteria. Lf, when the lysine or arginine residues were chemically modified, completely lost the ability to agglutinate the bacterial cells, while desialylated Lf enhanced the agglutination of the bacterial cells. The absorbance of the cells in suspension by deglycocylated Lf was the same as that by untreated Lf after 1 min, but was markedly decreased after 30min.

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It has been reported that pellagra is caused by abnormal metabolism of tryptophan, although it is generally believed that pellagra is caused by a deficiency of niacin. The administration of 6-aminonicotinamide (6-AN) to rats induces central nervous lesions being similar those seen in pellagra. So, we investigated the effects of 6-AN on the metabolism of tryptophan to nicotinamide. The threshold of the toxicity of 6-AN was around 0.001% when it was incorporated in the diet. In enzyme activities involved in the metabolism of tryptophan to nicotinamide, the activities of tryptophan oxygenase and aminocarboxymuconate-semialdehyde decarboxylase were greatly increased, while that of kynureninase was decreased by feeding of 6-AN diets. In enzymes involved in catabolism of nicotinamide, the activity of nicotinamide methyltransferase was increased and those of N1-methyl-2-pyridone-5-carboxamide (2-Py)-and N1-methyl-4-pyridone-3-carboxamide (4-Py)-forming N1-methylnicotinamide (MNA) oxidases were decreased by feeding of 6-AN diets. These changes were similar those seen in alloxan- and streptozotocin-induced diabetic rats. The urinary excretion ratio of (2-Py+4-Py)/MNA, which is reported to be lower in pellagrins than in normals, was decreased, and the conversion ratio of tryptophan to nicotinamide was also decreased. These results demonstrated that the metabolism of tryptophan to nicotinamide was greatly changed by intake of 6-AN.

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Setosol, a biologically active natural product, was extracted from Pleiochaeta setosa, an organism isolated from volcanic rock in the Canaries. With molecular formula C₁₅H₁₆O₅ and a melting point of 139°C, this pale yellow and amorphous compound inhibited the growth of Pyricularia oryzae, Drechslera oryzae, and Gerlachia oryzae. Isolation of the new compound was performed by repeated centrifugal TLC on silica gel, and its structure was established as 2, 8-dimethyl-4-methoxy-6, 10, 11-trihydroxy-benzooxaonin by detailed spectral studies, including COSY, 2D ¹H-¹³C direct chemical shift correlation (XHCORR), 2D ¹H-¹³C correlation via long-range coupling (COLOC), heteronuclear-gated decoupling (GATEDEC), and single-frequency heteronuclear-gated decoupling (SFDEC) NMR techniques, as well as UV, IR, and MS.

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Phosphatidylcholine was irradiated in the state of a film or liposome with gamma-rays or electron beams, and the amount of peroxide was determined to compare the effects of the two types of radiation. The amounts of peroxide formed in both the film and liposome with gamma-rays were significantly larger than those with electron beams, when the samples were irradiated at the same dose. Proteins such as bacteriorhodopsin reduced the degree of peroxide formation in liposome, and the effect of gamma-rays was much larger than that of electron beams, even in the presence of protein. The results of the present investigation indicate that the effects of gamma-rays on peroxide formation in phosphatidylcholine were significantly larger than those of electron beams, irrespective of the state of the lipid.

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The total synthesis of natural (+)-phomopsolide B (2) is described. A 5, 6-dihydro-2H-pyran-2-one framework was constructed by coupling chiral aldehyde 4 and 4-oxygenated enaminoester 5. The resulting 5, 6-dihydro-4-pyrrolidyl-2H-pyran-2-one (3) was converted to natural (+)-phomopsolide B (2) in several steps.

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A selective inactivating enzyme for the m-subunit of lactate dehydrogenase (LDH) was found in the culture filtrate of Penicillium citrinum KE-1, newly isolated from soil. The enzyme was purified from the culture filtrate by ammonium sulfate fractionation, column chromatography on CM-Sepharose CL-6B, and gel filtration on Sephadex G-100. The purification was 124-fold with an activity yield of 81%. The purified enzyme gave a single band, corresponding to a molecular weight of 32, 000, on SDS polyacrylamide gel electrophoresis, and the isoelectric point was 9.5. The enzyme specifically inactivated the m-subunit of LDH but showed no activity on the h-subunit of LDH. The enzyme, named KE-1 proteinase, proved to be a serine-type proteinase. Limited proteolysis of native m-subunit of LDH was assumed to result in a loss of enzyme activity.

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An intracellular carboxylesterase from Pseudomonas sp. was overproduced in E. coli, and purified to homogeneity by a combination of hydrogen bond chromatography, gel filtration, and hydrophobic interaction chromatography. Gel filtration and SDS-PAGE suggested that the purified enzyme consisted of two subunits of molecular mass of 28kDa. Its isoelectric point was 5.9. The enzyme was thermolabile, and showed its maximum activity at 22°C (pH 7.5). Methyl propionate was hydrolyzed at the highest rate among the fatty acid methyl esters tested. PMSF, DFP, PCMB, and HgCl₂ inhibited the enzyme markedly, suggesting that serine and/or cysteine is in or near the active site.

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Streptozotocin-diabetic rats were maintained on a stock diet for 16 weeks and some were given conduritol A (10mg/kg/day). The administration of conduritol A, having a hypoglycemic effect, markedly prevented the diabetic rats from getting cataracts. In practice, conduritol A inhibited aldose reductase (EC 1.1.1.21) that is capable of catalyzing the conversion of aldoses to sugar alcohols in the polyol pathway. In an in vitro assay, lens aldose reductase was most effectively inhibited by conduritol A when α, β-D-glucose was used as a substrate. Some enzymes from the rats were not inhibited by conduritol A. Neither an intraperitoneal injection nor oral dosage of conduritol A caused acute toxicity in the rats. These findings suggest that the inhibition of lens aldose reductase by conduritol A may be responsible for its cataract-suppressing effect.

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In this report, we have found that fatty acid-free bovine serum albumin stimulated the expression of a transfected foreign gene in Cos1 cells in serum-free medium and that its activity was as same as that of fetal calf serum. This will simplify the purification of the gene product from the culture medium.

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(+)-Haedoxan A, an insecticidal sesquilignan, inhibited the specific binding of the noncompetitive GABA antagonist [³⁵S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes in concentration-dependent and noncompetitive manners. The data show haedoxan's interaction with a site coupled to the TBPS-binding site on the GABA_A receptor in rat brain, while the physiological significance of the interaction remains to be discovered.

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An ice nucleation activity gene, named inaU, of the bacterium Erwinia uredovora KUIN-3 has been sequenced. This gene encodes a protein of 1034 amino acid residues, and its expression product, inaU protein, has an 832-amino acid residue segment consisting of 52 repeats of closely related 16-amino acid motifs (R-domain), flanked by N- and C-terminal sequences (N- and C-domains, respectively). The primary structure of the inaU protein is similar to those of the inaA, inaW, and inaZ gene products of Erwinia ananas, Pseudomonas fluorescens, and Pseudomonas syringae, respectively, but is smaller than any of these products in terms of the size of the R-domain.

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Murine monoclonal antibodies were prepared against fumonisin B₁-carrier protein conjugates, such as fumonisin B₁-ovalbumin and fumonisin B1-keyhole limpet hemocyanin. Five stable hybridoma clones were obtained. The response ranges of these monoclonal antibodies for fumonisin B₁ were from 10 to 1000-5000 ng/ml in the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA).

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When a Brevibacterium lactofermentum L-threonine producer that accumulated L-lysine as well, was transformed with a recombinant plasmid carrying the indigenous hom, thrB, and thrC genes, delayed growth and plasmid instability were observed. Addition of organic nutrients, vitamins (thiamine : HCl and d-biotin), and NaCl under the optimal culture conditions solved these problems and further increased threonine production in a small jar fermentor.

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Two strains of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, were examined for their pyrite-oxidizing ability. S. acidocaldarius ATCC 33909 was shown to possess iron-oxidizing activity by ferrous sulfate oxidizing experiments, but S. acidocaldarius No. 7 did not have it. Pyrite-oxidizing rate of S. acidocaldarius ATCC 33909 was 1. 6-fold higher than that of strain 7 though they had a similar level of sulfur-oxidizing ability. These results show that the iron-oxidizing activity accelerates pyrite oxidation.

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When Trichosporon fermentans WU-C12 was cultivated for 3-4days in media containing 3% (v/v) kerosene and 0.8% (v/v)Tween 85, 3% (v/v) gas oil and 0.6% (v/v) Triton X-405, and 3%(v/v) liquid paraffin and 0.6% (v/v) Span 80, the extracellular lipase activities reached 58-62 U/ml, approximately 2-3 times as much as that without any addition of surfactant. The extracellular and total (intra- plus extracellular) lipase activities were affected by the combination of surfactants and petroleum products as the carbon sources.

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Most fermented milk prepared by strains of Lactobacillus helveticus showed significant antihypertensive effect in spontaneously hypertensive rats (SHR) by oral administration. However, milk fermented by other species of lactic acid bacteria did not show significant antihypertensive effects. Most of the whey fractions of the milk fermented by L. helveticus or Lactobacillus delbrueckii subsp. bulgaricus showed higher angiotensin I-converting enzyme (ACE) inhibitory activity than the activity of milk fermented by other species. Proteolytic activity in cell wall and peptide content of the fermented milk were higher in L. helveticus strains than other species.

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We examined the secretion of human growth hormone in yeast cells with the artificial signal sequence L8LP, which is functional for human lysozyme secretion. The precursor was cleaved etliciently and the mature protein was secreted into the periplasmic space, but the protein aggregated. These results suggest that L8LP is also functional for human growth hormone. pl precipitation might be responsible for the aggregation.

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The effects on cholesterol absorption of oxidized cholesterols were studied in lymph-cannulated rats. When a single emulsitied lipid meal containing a tracer amount of [¹⁴C]cholesterol with either 50 mg cholesterol or 50mg oxidized cholesterol mixture was administered intragastrically, the lymphatic absorption of cholesterol for 24h was 33.1±6.3 and 21.7±82.6%, respectively. Moreover, oxidized cholesterols also inhibited the absorption of triolein (85.9±3.2% and 59. 5±18.8%). The observation may be of use since the ingestion of oxidized cholesterols tends to be increasing.

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Two phenolic compounds were isolated from a filtrate of Valsa ambiensis. Their structures were determined to be a new compound (1) and scytalone (2) by spectroscopic methods. Both had an inhibitory effect on the growth of lettuce roots and hypocotyls.

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Two cDNA fragments of terminal deoxynucleotidyl transferase, which contained new insertion sequences, were found from bovine thymus cDNA. One of the clones encoded 18 new amino acids and the other encoded 9 new amino acids. In the results of genomic structure analysis around the new insertion sequences, alternative splicing of the bovine terminal deoxynucleotidyl transferase gene was suggested.

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Syntheses of a fluorobenzoxazine derivative, fluorobenzothiazine derivative and fluoroquinoxaline derivative are described. These compounds were synthesized by reductive cyclization of the corresponding fluorodinitrobenzene derivatives. The fluorobenzoxazine derivative and its analogues are useful intermediates for agrochemicals.

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Kojibiose and nigerose were found in a reaction mixture of sucrose phosphorylase from Leuconostoc mesenteroides using _α-D-glucose-1-phosphate (G-1-P) as a donor and sucrose as an acceptor, and confirmed by ¹³C-NMR and HPLC analyses. The glucobioses were also detected using combinations of sugars such as sucrose and D-glucose, sucrose alone, or G-1-P and _D-glucose, while none of them was detected using G-1-P alone or _D-glucose alone. The efficiencies of the substrare on the formation of the glucobioses were as follows : sucrose and _D-glucose > sucrose > G-1-P and _D-glucose > G-1-P and sucrose.

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In the course of our search for new indophenol-reducing substances, a strain of Streptomyces was found to produce USF-142A, a novel 1, 3-dicarbonyl compound which reduces 2, 6-dichlorophenol indophenol and exists in keto-enol tautomers in solution. This compound showed antioxidative activity by the Rhodan iron method.

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The amino acid sequence of copper pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 20, 77, and 113 for Lady Amherst's pheasant lysozyme and seven amino acid substitutions at positions 3, 15, 20, 41, 113, 121, and 124 for hen lysozyme. Phenylalanine at position 20 was newly detected in avian lysozyme.

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Single-stranded plasmid DNA of pPF1 from Phormidium foveolarum that was specifically degraded by S1 nuclease was detected by Southern hybridization. This is also the case of the homologous plasmid pPB1 from Plectonema boryanum. These observations suggest that such small cryptic plasmids as pPF1 and pPB1, both from Gram-negative and filamentous cyanobacteria, replicate by a rolling circle mechanism in their living cells.

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