3-(Substituted phenyl)imidazolidine-2-thiones (SPITs) and related compounds were synthesized by cyclizing monoethanolamine hydrogen sulfate with arylisothiocyanates in the presence of sodium hydroxide. The activity for stimulating adenylate cyclase prepared from thoracic nerve cords of the American cockroach, Periplaneta americana L., was examined with these compounds. A SPIT with a 2,6-diethylphenyl group (48) was the only full agonist, the other SPIT derivatives being partial agonists. Greater enzyme activation appeared to result from short-chain alkyl rather than halogen substitution at the 2,6-positions of the aromatic ring of SPITs. Increasing the chain length from methyl to ethyl in 2,6-disubstituted SPIT caused an increase in the enzyme activation. Meanwhile, further increase of the chain length from ethyl to isopropyl in 2,6-disubstituted SPIT caused a decrease in the enzyme activation. Superimposition of energy-minimized octopamine and 48 revealed structural and conformational similarities that account for the higher Vmax value of 48. There was a marked decrease in the enzyme activation after alkylating at C₄ or C₅ of the imidazolidine ring of the potent SPITs. Thus, a certain degree of bulkiness and hydrophobicity at the 2- and 6-positions on the phenyl ring of a SPIT and the N-terminal was favorable for activating adenylate cyclase.

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  An intramolecular hydrogen bond between NH•••O₂N in insecticide, imidacloprid (1), and its nitromethylene analog 15 was proved by NMR and IR spectra. That electron delocalization over their planar moieties was disrupted by alkylation at the imidazolidine nitrogen atom is demonstrated by the hypsochromic shifts in UV and deshielding effect in NMR spectra. Interestingly, the N-alkyl derivatives (C1-5) had greater water solubility than 1, although increasing alkyl chain length decreased the solubility. The hydrophilicity of the alkyl derivatives would result from remote charge heads being formed as a result of the conjugation disruption by alkylation, while the hydrophobicity of 1 could be ascribed to the charge distribution over the conjugated system coupled with the intramolecular H-bonding. The greater water solubility of 15 than 1 and contrastively small solubility of the cyanoimine analogue are discussed based on the difference in their steric crowding.

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  Regioselective mono-2-O-sulfonation of cyclomaltooctaose was conveniently achieved by using the combination of sulfonyl imidazole and molecular sieves in DMF. In this reaction, no 3-O- or 6-O-sulfonation products were produced. The reactions do not require strict anhydrous or basic conditions, or specific sulfonyl groups.

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  Both enantiomers of 15-hexadecanolide, a sex pheromone component of the stink bug (Piezodorus hybneri), were synthesized by using the Yamaguchi or Mitsunobu macrolactonization reaction of (R)-15-hydroxyhexadecanoic acid prepared from ethyl (R)-β-hydroxybutyrate in 5 steps.

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  A kinetic analysis of degradation of saturated oligoguluronates by poly(α-L-guluronate)lyase from Corynebacterium sp. ALY-1 strain was done. The saturated oligoguluronates were prepared by hydrolyzing poly α-1,4-L-guluronate from alginate with HCl, and then by gel filtration on a Bio-Gel P-6 column. The saturated pentaguluronate or above were rapidly degraded by the enzyme, while tetraguluronate was slowly degraded. From the dependency of the catalytic rate constant (k_cat) on the degree of polymerization of substrates, the enzyme was found to have a subsite size corresponding to hexaguluronate units. The action pattern of the enzyme on hexaguluronate suggested that the catalytic site of the enzyme was matched to the linkage between the second and third uronic residue from the non-reducing end, since the substrate was mainly split into a unsaturated tetramer and a saturated dimer from a HPLC analysis.

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  The gene that coded for a cellular oligo-1,6-glucosidase (dextrin 6-α-D-glucanohydrolase, EC 3.2.1.10) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51-82°C was expressed in Escherichia coli JM109. The enzyme had a half-life of 10 min at 89.2°C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 529 residues with a molecular weight of 61,469 deduced from the gene was 40-42% identical to the sequences of less thermostable oligo-1,6-glucosidases from Bacillus cereus ATCC 7064, Bacillus coagulans ATCC 7050, and Bacillus thermoglucosidasius KP1006.
  Sequence analysis showed that the B. flavocaldarius enzyme shared 14 proline residues at the same positions as in the three other enzymes, and that the B. flavocaldarius enzyme had 22 of 33 additional proline residues (cf. 1/5, 5/10, and 9/18 in the respective counterparts) in three long polypeptides constituting the active-site cleft, which connected the third, fourth, and eighth β-strands to the corresponding third, fourth, and eighth α-helices in the (β/α)₈-barrel.

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   Phosphopentomutase catalyzes the transfer of an intramolecular phosphate on ribose or deoxyribose, and is involved in the salvage pathway of nucleoside synthesis. We identified a sequence 5′-upstream of the genes for the nucleoside phosphorylases of Bacillus stearothermophilus as the phosphopentomutase (ppm) gene. The novel gene corresponded to an open reading frame of 1,179 nucleotides that is translated into a putative 393-amino acid protein with a molecular weight of 43,735. The gene product, partially purified from ppm-overexpressing Escherichia coli cells, was judged to be a monomer of a 44-kDa polypeptide. The phosphopentomutase was found to catalyze the phosphotransfer on not only ribose or deoxyribose but also arabinose or dideoxyribose.

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  Suppression of mitochondrial respiration and increased glycolysis are characteristic features of activated macrophages. We show here that antimycin A, a respiratory inhibitor, induced interleukin-1 synthesis and tumoricidal activity without inducing tumor necrosis factor or nitric oxide. The induction of tumoricidal activity was resistant to inhibitors of tyrosine-specific protein kinases and intracellular glycoprotein transport. The cognate interaction between macrophages and target cells was not a prerequisite for the tumoricidal activity. In contrast, lipopolysaccharide induced the production of interleukin-1, tumor necrosis factor and nitric oxide, the induction of tumoricidal activity being sensitive to genistein and brefeldin A. Antimycin A, like lipopolysaccharide, induced the release of a cytoplasmic enzyme and apoptosis of macrophages. Antimycin A showed anti-metastatic activity in vivo. These results suggest that the inhibition of oxidative respiration would induce apoptosis and the resultant release of soluble effector molecules of macrophages which inhibit tumor metastasis in vivo.

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  A sheathed bacterium, Sphaerotilus natans, was cultured with vigorous shaking in a medium containing peptone. Then the biomass was harvested and treated with lysozyme, sodium dodecyl sulfate, and protease. With treatment, 1.6 mg of sheaths was obtained from 15 mg of biomass. For the preparation of sheaths of high purity, cultivation must be in the absence of glucose with sufficient aeration to prevent poly(3-hydroxybutyrate) accumulation. Carbohydrate (54.1%), protein (12.2%), and lipid (1-3%) were detected in the sheaths by colorimetric reactions and solvent extraction. Gas-liquid chromatography showed glucose and galactosamine to be present in the molar ratio of 1:4. The most abundant amino acids in the sheath protein were glycine (49.2 mol%) and cysteine (24.6 mol%). The sheaths were resistant to agents that reduce disulfide bonds (dithiothreitol and 2-mercaptoethanol) and to protease. However, sheathes were degraded completely by hydrazine, and a heteropolysaccharide composed of glucose and galactosamine (1:4) was released. The weight-average molecular weight of the polysaccharide was estimated to be 1.2×10⁵ by gel filtration chromatography with a low-angle laser-light scattering photometer and a rotation index detector. A ladder of 1.5-kDa peptides separable by sodium dodecyl sulfate gel electrophoresis was obtained by partial hydrolysis of sheaths, suggesting the sheath protein has repeating units of 1.5 kDa.

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   A wound-inducible ribonuclease (RNase NW) was purified from leaves of Nicotiana glutinosa. The purified RNase NW has an optimum pH around 5 and 7, and its base specificity is suggested based on the relative rates of hydrolysis of homopolyribonucleotides to be a preference for guanine base. The complete amino acid sequence of RNase NW was deduced by a combination of protein and cDNA sequencings. The cDNA sequence includes the entire coding sequence for a polypeptide with 229 amino acids including a putative secretion signal peptide at the N-terminus composed of 25 amino acids. The amino acids identified by the protein chemical methods are unambiguously localized within the deduced amino acid sequence from the cDNA sequence. Comparison of the sequence of RNase NW with those of other known plant RNases showed that it was identical except for eight residues to that of N. alata RNase NE, which is present in the style and pollen under normal conditions and is induced in roots in response to phosphate starvation [Dodds et al., Plant. Mol. Biol., 31, 227-238 (1996)]. RNase NW shows considerable sequence similarity to other known RNases, sharing 57% to 84% identical residues. Northern blot analysis using an RNase NW cDNA fragment as a probe showed that the RNase NW transcript was not detected in leaves without wounding, but it was induced within 4 h after wounding and then gradually decreased during 20 h.

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  A protein-synthesis inhibitor, designated RPSI, was isolated from the seeds of rye (Secale cereale) using gel filtration and S-Sepharose column chromatography. RPSI is a basic protein with an isoelectric point of over 10, and the concentration of protein required for 50% inhibition of protein synthesis (IC₅₀) of purified RPSI was about ten-fold the concentration of ricin A-chain. The complete amino acid sequence of RPSI was discoverd by analyzing the peptides and fragments obtained from the proteolytic digests and by the cyanogen bromide- and hydroxylamine-cleavages of RPSI. RPSI consists of 280 amino acid residues and has a molecular weight of 30,171. RPSI has only 21% sequence identity with that of ricin A-chain, but all five amino acid residues involved in the active site of ricin A-chain are conserved in RPSI.

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  Alkaline metalloendopeptidase (metalloprotease) AP1 (48 kDa) from Vibrio sp. isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) was reported in our previous paper to produce AP2 (36 kDa) by releasing a peptide fragment (molecular mass of about 12 kDa) from the C-terminal end of AP1 by autodigestion.¹⁾ AP1 strongly agglutinated fish (flounder, Paralichthys olivaceus) and rabbit erythrocytes, and weakly chicken erythrocytes. In contrast, AP2 had no significant hemagglutinating activity toward any erythrocytes tested, except for weak activity on flounder erythrocytes, suggesting that the C-terminal region of AP1 may be required for the strong hemagglutinating activity. The optimum temperature for the hemagglutinating activity of AP1 was found to be lower than that for the proteolytic activity. At acidic pHs (below pH 7.5), the hemagglutinating activity of AP1 decreased, and its pH profile resembled that of the proteolytic activity. The hemagglutinating activity of AP1 was not observed in the presence of o-phenanthroline or synthetic and proteinous substrates, but different kinds of saccharides and lipids had no effect. While the proteolytic activity of AP1 was not affected by CaCl₂, the hemagglutinating activity of AP1 decreased with increases in CaCl₂ concentrations. These results suggested that the hemagglutinating activity of these proteases (AP1 and AP2) was most likely caused by their proteolytic action on erythrocyte cell surfaces.

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  Based on the soluble MTT tetrazolium/formazan assay, we evaluated the cytotoxicity of Erythrina variegata proteinase inhibitors in some tumor hematopoietic stem cell lines. Among the proteinase inhibitors, EBI, which belongs to the Bowman-Birk family of inhibitors, was cytotoxic in relatively differentiated cells such as Molt4 and Jurkat derived from acute T lymphoblastic leukemia (T-ALL) cells specifically, but ETIa and ECI, which are classified into Kunitz family inhibitors, did not. It was suggested that the differences in the cytotoxicity might be due to the molecular size of the inhibitors. The succinylation of lysine residue(s) of EBI led to about 50% loss of the trypsin inhibitory activity as compared with the authentic EBI. When Molt4 cells were incubated with this derivative, no significant cytotoxicity was observed. This suggests that the proteinase inhibitory activity might be involved in the cytotoxicity in human tumor cell lines.

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  Artificial N-glycopolypeptides carrying N-acetyllactosamine (LacNAc) or related compounds were synthesized. First, sugars were converted into their corresponding β-glycosylamines with ammonium hydrogen carbonate. Then, the β-glycosylamines were condensated with the carboxyl groups of poly(L-glutamic acid). N-Glycopolypeptides with different degrees of substitution of sugars were isolated by passage through a column of Sephadex G-25. These synthetic polymers were used as model compounds in the analysis of oligosaccharide-lectin interactions. Interactions with some lectins were investigated by agar-gel double-diffusion tests and in terms of inhibition of hemagglutination. A glycopolypeptide substituted with LacNAc reacted with Erythrina cristagalli agglutinin (ECA), peanut (Arachis hypogaea) agglutinin (PNA), Ricinus communis agglutinin-120 (RCA₁₂₀), wheat germ (Triticum vulgaris) agglutinin (WGA) lectins, which recognize either galactosyl or N-acetylglucosamine (GlcNAc) residues. Other synthetic glycopolymers carrying N-acetylisolactosamine, GlcNAc, N,N′-diacetylchitobiose, or N,N′,N″-triacetylchitotriose also reacted with WGA, and these last two polymers inhibited hemagglutination most. Of these five glycopolypeptides, only the one substituted with LacNAc reacted with ECA. These sugar-substituted glycopolypeptides interacted specifically with the corresponding lectins, no matter how much shorter the sugar side chains of the glycopolymers were than those of natural glycoproteins.

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  CEL-III is a Ca²⁺-dependent lectin from a marine invertebrate, Cucumaria echinata, which shows strong hemolytic activity toward human and rabbit erythrocytes. After binding to carbohydrate receptors, CEL-III oligomerizes in the erythrocyte membrane to form ion-permeable pores, leading to the colloid osmotic rupture of the cells. Since hemolysis was greatly increased in the alkaline pH, especially above pH 9, involvement of amino groups of CEL-III in its hemolytic activity was evaluated using chemical modification by succinic anhydride. After modification of 7 amino groups per protein molecule, the hemolytic activity of CEL-III was reduced to 23% of the native protein, but hemagglutinating and carbohydrate-binding activities were only slightly affected even after modification of 14 amino groups. A circular dichroism spectrum of modified CEL-III showed almost no change in the secondary structure from that of the native protein, indicating that the decrease of hemolytic activity was not caused by partial unfolding of the protein. Immunoblotting analysis of the erythrocyte membrane treated with modified CEL-III showed a decrease in the formation of CEL-III oligomer in the membrane in parallel with the decrease in hemolytic activity. These results suggest that amino groups of CEL-III are involved in its oligomerization in the cell membrane, and their modification leads to inactivation of the protein without much influence on the carbohydrate-binding activity.

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  Vitellogenin is a protein induced by estrogens, including environmental chemicals with estrogenic activity. To measure the effects of environmental estogens, we developed an effective and rapid one-step method of detecting and purifying fish plasma vitellogenin using a high-performance anion-exchange chromatography column, POROS-HQ. Vitellogenin in a plasma of estradiol-treated male fish (mummichog and red sea bream) was eluted as a single peak with a retention time of 10 minutes from the column, which gives an almost pure preparation as assessed by SDS-PAGE. The lowest detectable amount of vitellogenin was 2 μg per assay. The method was used to analyze the plasma vitellogenin level of aquacultured red sea breams caught in August, when the spawning season is over, and usually no vitellogenin is detected in either females or males, physiologically. However, the data showed that in addition to a few females, some male fish synthesized vitellogenin, suggesting that some chemicals or unknown factors with estrogenic activity have induced fish in the ocean to produce vitellogenin.

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  The structure of the N-linked carbohydrate chains of peptide isomerase from the venom of the funnel web spider (Agelenopsis aperta) has been analyzed. Carbohydrates were released from peptide isomerase by hydrazinolysis and reductively aminated with 2-aminopyridine. The fluorescent derivatives were purified by phenol/chloroform extraction, followed by size-exclusion HPLC. The structure of the purified pyridylamino (PA-) carbohydrate chains were analyzed by a combination of two-dimensional HPLC mapping, sugar composition analysis, sequential exoglycosidase digestions, and mass spectrometry. The peptide isomerase contains six kinds of N-linked carbohydrate chains of truncated high-mannose type, with a fucose α1-6 linked to the reducing N-acetylglucosamine in approximately 80% of them.

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  Partially purified hot-water extracts of the roots of plants of the Sophora family suppressed the increase in blood glucose concentration of rats in the oral sugar tolerance test. The extracts also inhibited rat intestinal sucrase and maltase. The most potent sample was about 15 times more active than catechin, a positive control, in these experiments.

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  We have previously identified glyceraldehyde-3-phosphate dehydrogenase as an immunoglobulin production stimulating factor (IPSF) which facilitated immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of this enzyme was suppressed by the coexistence of some sorts of nucleotides. We now report that the IPSF effect of GAPDH was suppressed by the coexistence of DNA, the inhibiting effect of degraded DNA being inferior to that of long-chain DNA. Both single-stranded and double-stranded synthetic polyribonucleotides also inhibited the IPSF activity of GAPDH. Moreover, nicotinamide adenine dinucleotide (NAD⁺) repressed the IPSF effect.

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  Calmodulin (CaM) from rice germ (Oryza sativa L) was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The protein showed a single spot by SDS-PAGE. This purified protein had multiple absorption maxima at 276~279, 268, 265, 258, and 253 nm. Like other plant CaM, the protein contained one mole of Tm₃Lys, cysteine, and tyrosine, and tryptophan was not detected. Hydrophobic properties of rice germ, spinach, and Neurospora crassa CaM were directly tested by an HPLC method using an ODS-120T column and by a hydropathy plotting method. Obvious hydrophobic differences with rice germ CaM>spinach CaM>N. crassa CaM, were observed among calmodulins from rice germ and others.

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   An archaeal geranylgeranyl diphosphate synthase was overexpressed in Escherichia coli cells as fusion proteins. These fusion proteins retained their thermostability and had higher specific activity than did a partially purified native enzyme Previously reported. We purified 24.3 mg of MBP (maltose-binding protein)-fusion protein and 5.4 mg of GST (glutathione S-transferase)-fusion protein from a one-liter culture of E. coli.
  The MBP-fusion proteins existed in dimer, tetramer, octamer, or dodecamer form, and their product specificities were altered according to the oligomerization. The MBP-fusion protein has protease-sensitive sites in the portion corresponding to geranylgeranyl diphosphate synthase.

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  Hybrids of a ligand protein crosslinked to a DNA binding protein have been developed as gene delivery vehicles mediated by receptors. To identify the effect of the crosslinks between the ligand and DNA binding protein on gene expression caused by an internalized hybrid-DNA complex, we prepared two kinds of transferrin-poly-L-lysine (TF-PL) hybrids: one was crosslinked by probably cleavable disulfide bonds (TF-ss-PL) and the other was linked by a probably uncleavable Schiff’s base (TF-Schiff-PL). The binding affinity of the hybrids to HeLa cells was not different. However, the expression of a reporter gene (for luciferase) bound to these hybrids in HeLa cells transfected with TF-Schiff-PL was greater than that of TF-ss-PL.

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  The complete amino acid sequences of tulip bulb chitinase-1 and -2 (TBC-1 and -2) were determined. The sequences of the TBC-1 and TBC-2 were solved by analysis of peptides derived by enzymatic digestions as well as by chemical cleavages with cyanogen bromide (CNBr), o-iodosobenzoic acid, and hydroxylamine. TBC-1 and TBC-2 both consisted of 275 amino acid residues and had molecular masses of 30,825 and 30,863, respectively. They shared 247 identical residues, that is 90% identity. Comparison of their sequences with that of gladiolus bulb class IIIb chitinase-a (GBC-a) showed that 63% of the residues of both TBC-1 and TBC-2 are identical to that of GBC-a. From these results, it was seen that TBC-1 and -2 are class IIIb chitinases. The characteristic difference in specific activity between TBC-1 and -2 was also discussed on the basis of their amino acid sequences.

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  Caenorhabditis elegans putative copper ATPase (CUA-1) had been functionally expressed in a yeast Δccc2 mutant (copper ATPase gene disruptant). We found that CUA-1 with Cys-Pro-Cys to Cys-Pro-Ala mutation could not rescue the yeast Δccc2 mutant, suggesting that the carboxyl terminal cysteine residue in the conserved Cys-Pro-Cys motif is essential for copper transport.

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  The effects of an activator, cardiolipin, on the three peptidase activities of the 20S proteasome of Xenopus oocytes were examined. The trypsin-like activity was activated when the enzyme was treated with cardiolipin before the addition of the substrate, but there was no appreciable activation when cardiolipin was added concomitantly with the substrate. On the other hand, the chymotrypsin-like peptidase and peptidylglutamylpeptide hydrolase (PGPH) were activated regardless of the sequence of addition. When very low concentrations of the substrate (e.g. 0.1-0.5 μM; about 1/100 of the K_m) were used, cardiolipin strongly activated trypsin-like peptidase by the simultaneous addition but not after substrate addition. These results suggest that the trypsin-type substrate produces a conformational change in the enzyme in a concentration-dependent manner which makes the activator sites inaccessible to cardiolipin.

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  The second lysine decarboxylase gene (ldc) is at 4.7 min on the Escherichia coli chromosome [Kikuchi et al., J. Baceriol. 179, 4486-4492 (1997)]. This report showes that the expression of ldc as well as cadA was induced at stationary phase in the wild type of E. coli. The ldc was not expressed in a rpoS deletion mutant of E. coli at any growing stage. In contrast, cadA was expressed in the rpoS mutant. Thus, we conclude that the expression of ldc but not cadA at stationary phase is regulated by a RpoS-dependent mechanism (s) in E. coli.

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  V_H (heavy-chain variable region) and V_L (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar V_H and identical V_L domains. Initially, the V_H and V_L genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified V_H and V_L genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.

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  Freeze-dried cells of acetic acid bacteria were prepared to use as an additive for manufacturing and processing foods. When the freeze-dried cells were stored for 1 week at 5°C, however, more than 50% of the original activity of aldehyde oxidase (AOX) was lost.
  It was found that this decrease in AOX was caused by damage to both the membrane-bound aldehyde dehydrogenase and terminal oxidase activities involved in the aldehyde oxidase electron transport system of acetic acid bacteria. The addition of 30% sucrose to the cell suspension prepared in a McIlvaine buffer (pH 6) before lyophilization was found to be effective for preventing the decrease of AOX activity. Cells freeze-dried in this way lost no AOX activity at all during first 3 weeks of storage at 5°C and, even after 9 weeks, 80% of the original activity remained.

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  Traditional vegetables in Kyoto are a unique group of vegetables that have been cultivated in limited areas near Kyoto city. We compared the traditional vegetables in Kyoto with common vegetables for the bio-antimutagenicity of their extracts against UV-induced mutation of E. coli B/r WP2. Among the traditional vegetables in Kyoto, Kamo eggplant (Solanaceae) and Katsura oriental pickling melon (Cucurbitaceae) showed higher bio-antimutagenicity and yield in the n-hexane, chloroform and ethyl acetate fractions than their common vegetable counterparts. Shishigatani pumpkin (Cucurbitaceae) possessed bio-antimutagenicity in the chloroform and ethyl acetate fractions, but common pumpkin did not. Polyphenolic compounds in the ethyl acetate fraction of plants are known to be related to antimutagenicity. However, the intensity of bio-antimutagenicity was not correlated with the polyphenol content in the ethyl acetate fractions of the present vegetables. In particular, Kamo eggplant possessed both polyphenolic and non-polyphenolic bio-antimutagenic sub-fractions in the ethyl acetate fraction. In the aqueous fraction, taro (Dioscoreaceae) was the most capable among our samples, whether being of common or traditional origin. Consequently, it is considered that some traditional vegetables in Kyoto are superior to common vegetables in their bio-antimutagenicity and that these could be used as starting materials to identify new bio-antimutagens.

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  An HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) is reported. The activity was evaluated by measuring the decrease of DPPH detected at 517 nm. By using this novel method, we determined the free radical-scavenging activity of several antioxidants: ascorbic acid, α-tocopherol, Trolox, and cysteine. The results gave good correlation between the radical-scavenging activity determined by HPLC and by conventional colorimetry. This methodology was applied to determine the free radical-scavenging activity of 8 beverages. The activity of coffee was the highest, followed by red wine, green tea, oolong tea, black tea, rosé wine, white wine, and orange juice. The results well agree with those of previous reports. This method is expected to be useful for a simple and rapid determination of free radical-scavenging activity in colored foods, because coloring substances in foods do not interfere with the measurement.

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  To examine whether albumin-2, a specific protein found only in amaranth seeds so far, is associated with protein bodies, we isolated protein bodies from Amaranthus cruentus seed embryos by rate-zonal centrifugation with a sucrose gradient. Most protein bodies in the final preparation were intact when observed by electron microscopy. Profiles by SDS-PAGE showed that the isolated protein bodies contained globulin and albumin-2.

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   The metabolites of the antirheumatic agent KE-298 in humans, (-)-(2R)-M-4 [(-)-(2R)-4-(4-hydroxymethylphenyl)-2-methylthiomethyl-4-oxobutanoic acid], (-)-(2R)-M-5 [diastereomers of (-)-(2R)-4-(4-hydroxymethyl-phenyl)-2-methylsulfinyl-methyl-4-oxobutanoic acid], (-)-(2R)-M-6 [(-)-(2R)-4-(4-carboxyphenyl)-2-methylthio-methyl-4-oxobutanoic acid], and (-)-(2R)-M-7 [di- astereomers of (-)-(2R)-4-(4-carboxyphenyl)-2-methyl-sulfinylmethyl-4-oxobutanoic acid] were synthesized based on microbial transformation. The substrate KE-748 (racemic form of (-)-(2R)- and (+)-(2S)-4-(4-methyl-phenyl)-2-methylthiomethyl-4-oxobutanoic acid: 7.5 g) was converted to (-)-(2R)-M-4 (1.84 g) using Rhizopus sp. TF0040 in a 50-l jar fermentor. Specific cytochrome P-450 inhibitors, SKF-525-A and metyrapone strongly inhibited the hydroxylation reaction. It was suggested that cytochrome P-450 is responsible for the microbial reaction. Furthermore, (-)-(2R)-M-4 (200 mg) was transformed to (-)-(2R)-M-6 (144 mg) by co-oxidation with n-hexadecane as a carbon source using Rhodococcus sp. TA0250 in a 1.4-l jar fermentor. Starting from (-)-(2R)-M-4 and (-)-(2R)-M-6 obtained as above, (-)-(2R)-M-5 and (-)-(2R)-M-7, respectively were chemically synthesized by m-chloroperoxybenzoic acid oxidation.

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   The gene xynA encoding an acid endo-β-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65°C, and is stable pH between 3.0 and 8.0. The K_m and V_max with oat spelt xylan as a substrate at pH 5.0 and 30°C are 3.5 mg/ml and 403 μmol/min/mg.

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  A soil bacterium with α-L-rhamnosidase was isolated from a cumulative mixed culture containing a polysaccharide of gellan as a carbon source and identified to be Sphingomonas paucimobilis, known as a potent producer of gellan. The isolate (designated Sphingomonas sp. R1) produced an unusual exopolysaccharide of sphingan (denoted HWR1) distinct from gellan. The rhamnose in gellan was replaced with mannose in HWR1. The bacterium had a peculiar cell surface covered with many complicated plaits. α-L-Rhamnosidase purified from Sphingomonas sp. R1 grown in the presence of naringin was a monomer with a molecular mass of 110 kDa and most active at pH 8.0 and 50°C. The enzyme required divalent metal ions for the activity and released L-rhamnose from various rhamnosyl glycosides.

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   The indole tolerance level of Pseudomonas sp. strain ST-200 was 0.25 mg/ml. The level was raised to 4 mg/ml when diphenylmethane was added to the medium to 20% by volume. ST-200 grown in this two-phase culture system containing indole (1 mg/ml) and diphenylmethane (0.2 ml/ml) produced a water-soluble yellow pigment, isatic acid, and two water-insoluble and diphenylmethane-soluble pigments, blue indigo and purple indirubin. The amounts of the water-insoluble pigments corresponded to 0.5% (indigo) and 0.2% (indirubin) of the indole added to the medium. Of the conditions tried, indigo and indirubin were formed only when ST-200 was grown in the two-phase system overlaid with organic solvents with appropriate polarity.

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  Sulfite ion (HSO₃^-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO₃^- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO₃^-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO₃^- on cytochrome c oxidase activity were studied with the plasma membranes of HSO₃^--resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO₃^-. To investigate the inhibition mechanism of HSO₃^- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO₃^-. In contrast, the same concentration of HSO₃^- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO₃^- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.

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  To study the early stages of concrete corrosion by bacteria, sulfur-oxidizing bacterium strain RO-1, which grows in an alkaline thiosulfate medium (pH 10.0) was isolated from corroded concreate and characterized. Strain RO-1 was a Gram negative, rod-shaped bacterium (0.5-0.6×0.9-1.5 μm). The mean G+C content of the DNA of strain RO-1 was 65.0 mol%. Optimum pH and temperature for growth were 8.0. and 30-37°C, respectively. When grown in thiosulfate medium with pH 10.0, growth rate of the strain was 48% of that observed at the optimum pH for growth. Strain RO-1 used sulfide, thiosulfate, and glucose, but not elemental sulfur or tetrathionate, as a sole energy source. Strain RO-1 grew under anaerobic conditions in pepton-NO₃^- medium containing sodium nitrate as an electron acceptor, and had enzyme activities that oxidized sulfide, elemental sulfur, thiosulfate, sulfite, and glucose, but not tetrathionate. The bacterium had an activity to assimilate ¹⁴CO₂ into the cells when thiosulfate was used as an energy source. These results suggest that strain RO-1 is Thiobacillus versutus. Strain RO-1 exuded Ca²⁺ from concrete blocks added to thiosulfate medium with pH 9.0 and the pH of the medium decreased from 9.0 to 5.5 after 22 days of cultivation. In contrast, Thiobacillus thiooxidans strain NB1-3 could not exude Ca²⁺ in the same thiosulfate medium, suggesting that strain RO-1, but not T. thiooxidans NB1-3, is involved in the early stage of concrete corrosion because concrete structures just after construction contain calcium hydroxide and have a pH of 12-13.

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  We cloned and characterized a gene, tgl, encoding transglutaminase in Bacillus subtilis. The tgl gene contained a open reading frame 735-nucleotides long that encoded a 245-residue protein with the molecular weight of 28,300. The deduced amino acid sequence had little sequence similarity with sequences of other transglutaminases from a Streptoverticillium sp. or from mammals. The -10 and -35 regions of a putative promoter resembled the consensus sequence for the σ^K-dependent promoter. In addition, a sequence similar to the consensus sequence for the GerE binding site was found upstream from this region. These findings suggested that tgl was transcribed in the mother cells during a late stage of sporulation. Evidence for this suggestion was that transglutaminase activity was detected in sporulating cells during the same stage. Transglutaminase activity was detected in Escherichia coli cells transformed with a plasmid for expression of the tgl gene.

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   Intracellular pools of three CoA molecular species of coenzyme A, CoASH, acetyl-CoA, and malonyl-CoA, in Escherichia coli K-12 cells were studied by acyl-CoA cycling method in replacement culture. The sizes and compositions of CoA pools starved for a carbon source changed within minutes after the addition of one of various carbon sources. A large acetyl-CoA pool formed after the addition of D-glucose, D-fructose, D-mannose, glycerol, or sorbitol, but there was little change when L-glucose, sucrose, maltose, succinate, or acetate was added. The β-anomer of D-glucose was assimilated 10 times faster than the α-anomer. Intracellular CoA pools also changed with stress: in the pH, incubation temperature, or with osmotic stress. The sizes and compositions of CoA pools were not affected by pH changing between 4 and 8, but the breakdown of acetyl-CoA and CoASH was greater at pH 9 than at pH 4 to 8. Production of acetyl-CoA was greatest at 40°C, and at 50°C, an acetyl-CoA pool did not form at all and the size of the CoASH pool declined. When the organism was stressed by the addition of NaCl at concentrations of more than 0.6 M, little acetyl-CoA was produced. The total CoA pool (the sum of the concentrations of CoASH, acetyl-CoA, and malonyl-CoA) remained within the limits of 0.83-1.40 nmol/mg of dry cell weight (0.30-0.52 mM). Whenever acetyl-CoA increased, CoASH decreased. Therefore, the acetyl-CoA/CoASH ratio is an important index of facultative anaerobes that reflects the state of carbon and energy metabolism in vivo.

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  Two muconate cycloisomerases (MC I and MC II, EC 5.5.1.1) were purified to homogeneity from an aniline-grown Frateuria sp. ANA-18. MC I and MC II were similar in molecular mass, optimal pH, and pH stability but different in thermostability, and some other enzymatic properties. NH₂-terminal amino acid sequences were different between the two isozymes, indicated that these are encoded by different genes. Different inducible production of MC I and MC II suggested that two catechol branches involved in the β-ketoadipate pathway function in Frateuria sp. ANA-18.

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  Polysaccharide (partially sulfated agarose) with macrophage-stimulation activity, derived from Gracilaria verrucosa, was decomposed by two types of β-agarase (agarases II and IV) from Pseudomonas sp. O-148. The hydrolysates were fractionated with ethanol precipitation and anion-exchange chromatography. The resulting anionic oligosaccharides with sulfate groups were investigated by ¹³C-NMR spectroscopy. While the spectra of oligosaccharides produced by agarase IV showed identical patterns with those by β-agarase I from Pseudomonas atlantica and indicated the location of a sulfated saccharide unit on the non-reducing end, another new type of saccharide was found in the products by agarase II. The novel oligosaccharides by agarase II had a neoagarobiose unit on their non-reducing end and had sulfated units internally. This indicated the novelty of agarase II in cleavage fashion.

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  The overexpression of red sea bream (Pagrus major) transglutaminase (TGase, E.C. 2.3.2.13) in Escherichia coli mostly leads to the accumulation of biologically inactive enzyme. Although the solubility of the gene products could be improved by cultivation at a lower temperature (26-28°C), most of the synthesized TGase was still in the form of insoluble aggregates. The effects of overproduction of molecular chaperones on the intracellular solubility of newly produced recombinant TGase were examined. The overexpression of dnaK or groES/EL did not improve solubility. However, DnaJ greatly increased the solubility of the recombinant TGase, resulting in active enzyme in the presence of calcium ions. Co-expression of dnaK along with dnaJ further increased the content of soluble TGase. Under our experimental conditions, supplementation with both DnaJ and DnaK elevated the TGase activity in the producer cells by roughly 4-fold, compared with the control strain cultured at 30°C. Thus, we found that DnaJ is important in controlling the solubility of protein overproduced in E. coli.

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  Polyclonal antibodies against an extracellular proteinase of Lactobacillus helveticus CP53 were raised. The antibodies reacted with a 170-kDa enzyme with activity and a 53-kDa protein that seemed to be a degradation product of the 170-kDa proteinase from results of immunoblotting. The antibodies reacted also with a 45-kDa extracellular proteinase of L. helveticus CP790. However, monoclonal antibodies to the CP790 proteinase did not react with the proteinase of L. helveticus CP53. Seventeen strains of L. helveticus were tested for immunological reactivity with the two kinds of antibodies. The strains all had the same reactivity as either strain CP53 or strain CP790. Eleven strains with the 45-kDa proteinase were identified as L. helveticus biovar jugurti because they did not ferment maltose, four other strains with the 170- and 53-kDa proteins were identified as L. helveticus biovar helveticus because they fermented maltose. The remaining two strains dit not fit this pattern; they had both the 170- and 53-kDa proteins, but classification by their sugar utilization showed them to be L. helveticus biovar jugurti.

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  The mechanism of the increased cell growth and cellulose production of Acetobacter xylinum subsp. sucrofermentans BPR3001E, a sulfaguanidine (SG)-resistant mutant, was investigated. We found that adding p-aminobenzoic acid (PABA) to cultures of the parent strain, BPR2001, led to increased levels of intracellular adenosine-related purine compounds and increased cellulose production. Furthermore, adding ATP increased the cellulose production by permeabilized BPR2001 cells. On the other hand, the intracellular levels of PABA and adenosine-related purine compounds in BPR3001E cells were higher than those in BPR2001 cells. These results suggest that SG resistance increases enhance cellulose production through increased levels of intracellular high-energy compounds caused by increased PABA biosynthesis, reflecting the promoted supply of cellulose precursors.

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   The bio-deposition of amorphous silica, which occurred in vitro by exposure to the extremely thermophilic bacterium Thermus spp. began from the latter part of the exponential phase of growth of the bacteria. The concentration with which the deposition occurred exceeded the solubility of amorphous silica of neutral pH at the temperature 60~85°C. Our observations suggest that Thermus spp. promotes the formation of siliceous minerals in a geothermal environment.

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