Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley α-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker's asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.

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Since the cell wall microfibril xylans from Caulerpa sp. were not assumed to be constituted from β-1, 3-straight chains of homoxylan like Bryopsis maxima but were heteropolysaccharides as glucoxylan or branched structures, identification of its fine structures was desired. Although the results for the linkage analysis were discovered from chemical methods, complementary confirmations were attempted by detection of a series of the β-1, 3-xylooligosaccharides prepared as partial acid hydrolysates. Complete separation of the higher oligosaccharide in each was difficult through high performance liquid gel permeation chromatography (GPC), so the composition of oligosaccharides in roughly sub-fractionated samples was detected using matrix-assisted laser desorption ionization/time of flight/mass spectrometry (TOF-MS). By these procedures, it was obvious that cell wall xylan from Caulerpa brachypus was constituted from at least 25 xylose residues of linear oligosaccharides with β-1, 3-linkages. Thus the successive methods of repeating GPC procedures and TOF-MS spectrometric detection were powerful techniques for the identification of incompletely separated oligosaccharides and it was available to obtain a series of purified β-1, 3-oligomers from cell wall xylan.

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The process of degradation of sulfhydryl (SH) groups in soymilk was investigated by using Glycine max var. Suzuyutaka (wild type) and the following seven mutant lines lacking lipoxygenase(s): L-1-, L-2-, L-3-, L-1, -2-, L-2, -3-, L-1, -3-, and L-1, -2, -3-null. The soymilk prepared from the L-1, -2, -3-null line of all the mutants had the highest SH content. The content of SH groups was the lowest with the L-1, -3-null line of the three double-null lines and the highest with the L-2-null line of the three single-null lines. These results show that lipoxygenases strongly participated in the degradation of SH groups in soymilk and that the L-2 isozyme had the greatest SH-degrading capability. When these soybean samples were ground under low-temperature conditions and in a nitrogen (N₂) atmosphere to inhibit the degradation of SH groups caused by lipoxygenases, SH degradation of the L-2, -3- and L-1, -3-null lines was strongly inhibited at low temperature, while that of the L-1, -2-null line was strongly inhibited in the N₂ atmosphere. In view of the strong inhibition of SH degradation in an N₂ atmosphere with Suzuyutaka (wild type), which has three L-1, -2, -3 isozymes, these results suggest that not only the L-2 isozyme but also that the L-3 isozyme of the three in Suzuyutaka played an important role in SH degradation during soybean grinding.

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To discover whether the protoplast fusion method is useful or not for interspecific breeding, some methods were devised, and the appearance of various hybrids with different characters and the change of antibiotic activities in the recombinants obtained by the protoplast fusion were investigated. The purification of protoplasts, the choice of parental natural characters as selection markers, and the adoption of a replica method for selecting all types of recombinants were devised and used for these experiments. Protoplast fusion was done between S. griseus KCC S-0644 and each strain of 5 species that were clearly different species from S. griseus, in addition to being streptomycin sensitive (SM^s) and capable of L-arabinose utilization for growth (Ara⁺). Recombinants (SM^r, Ara⁺) obtained by protoplast fusion displayed a great variety of hybrids in their taxonomic characters, e.g., 21 recombinant strains obtained by the cross between S. griseus and S. griseoruber consisted of 14 types of hybrids. Antibiotic productivity was examined in all recombinants obtained. Although both parental species produced their respective antibiotics, 60% of the recombinants did not produce any antibiotic and 24% produced different antibiotics from those of their parents. Among those recombinants, it was also found that the distribution of the productivity of each antibiotic among the recombinants was entirely different from that of the allelo-character in each taxonomic feature.

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A bacterium that produced a large amount of poly(γ-glutamic acid) (PGA) when it was grown aerobically in a culture medium containing ammonium salt and sugar as sources of nitrogen and carbon, respectively, was isolated from soil. The bacterium, strain TAM-4, was classified as Bacillus subtilis. The maximum PGA production (22.1 mg/ml) was obtained when it was grown in a medium containing 1.8% ammonium chloride and 7.5% fructose at 30°C for 96 h with shaking. Some properties of the PGA obtained at different times of cultivation were investigated by gel permeation chromatography, SDS-PAGE, and measurement of viscosity, and calculation of the D/L ratio of glutamic acid constituting PGA. The results suggested that PGA was elongated with no changes in the diastereoisomer ratio in the molecule.

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Respiration of a facultative alkaliphile, Bacillus lentus C-125, was increased around alkaline pH near the upper pH-limit for growth of the organism. O₂-Uptake rates of the cells grown in a complex medium at pH 7-9 and 9.5-10 were 0.98-1.4 and 2.4 μmol O atom/min/mg cell protein, respectively. Membrane vesicles from the cells grown at pH 7-9 and 9.9 incorporated O₂ at rates of 1.1-1.4 and 2.5 μmol O atom/min/mg envelope protein, respectively, using exogenous NADH as an electron donor. In the presence of menadione as an exogenous electron acceptor, the membrane vesicles from the cells grown at pH 7-8.5 and 9.9 oxidized NADH at rates of 1.4-1.7 and 6.3 μmol NADH/min/mg envelope protein, respectively. Levels of respiratory and NADH-oxidation activities of the organism are dependent on the growth pH, and higher than those reported previously in alkaliphilic Bacillus spp.

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A divergent and highly enantioselective synthetic methodology for producing chirally β-deuterated L-amino acids was developed. This method is based upon the chirality transcription approach, using diacetone-D-glucos-3-ulose (1) as a template, 3-C-[2-²H₁]-Ethenyl-3-O-(N-benzyl)methylthioformimidoyl-D-allo-derivatives (3b and 3c), which are easily accessible from 1, were subjected to halonium ion-assisted cyclization to afford highly diastereoselectively and efficiently versatile 5-membered cyclic carbamate synthons having a stereochemically defined deuterated halomethyl group (4c and 4d, respectively). Subsequent straightforward transformation of these synthons gave rise to (3R)- and (3S)-[3-2H₁]-L-serine. Further transformation of the crucial halomethyl group of 4a-c was also pursued to extend this methodology.

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The cloned gene was composed of 1638 bp for coding plus promoter like and SD-like sequences ahead of it. The deduced amino acid sequence had high similarity with known β-amylases. The N-terminal sequence of the cloned β-amylase seemed to be a signal peptide. The gene was introduced into Bacillus subtilis 1A289 using pHY300PLK as a vector and the expressed protein was recovered from the culture media. The enzyme fraction produced was divided into two components upon the DEAE column chromatography. The amino acid sequence of one fraction (FrI) was the same as the mature enzyme, and the other (FrII) lacked the N-terminal amino acid residue (Ala) of the mature enzyme. The kinetic parameters of the hydrolysis catalyzed by the enzyme component fir were measured, and the subsite affinities of the enzyme were evaluated. In conclusion, it was shown that the recombinant enzyme was the same as the mature enzyme functionally and proteochemically.

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Protein phosphatases are classified into types 1 (PP1) and 2 (PP2A, 2B, and 2C). We have already established a new analysis method for PP2A inhibitors by using the firefly bioluminescence system for detection. This method was successfully applied to determine the PP1_Y inhibitory activity of known inhibitors, i.e., calyculin A, microcystin-LR, and tautomycin, requiring less than 10 pmol of a sample.

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Studies have been done on the inhibition and inactivation of the β-glucosidase and β=fucosidase enzyme from Thai Rosewood (Dalbergia cochinchinesis Pierre). The enzyme was inhibited by Tris with similar K_i of 11.7 mM and 14.3 mM for the hydrolysis of p-nitrophenyl β-D-glucoside (PNPG) and p-nitrophenyl β-D-fucoside (PNPF), respectively. Conduritol B epoxide inhibited both β-glucosidase and β-fucosidase activities to similar extents, with a pseudo-first-order rate constant (k_obs) of inactivation of 5.56×10<-3>s<-1>, and binding stoichiometry of 0.9 mol per subunit. Partially inactivated enzyme showed similar kinetics with PNPG and PNPF as substrates. Moreover, Tris at 300mM protected both β-glucosidase and β-fucosidase activities from inactivation by 6mM CBE. The data support the idea that the Dalbergia cochinchinensis Pierre enzyme has a common active site for the hydrolysis of PNPG and PNPF.

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Based on the wheat glutenin IgE-binding epitope, Gln-Gln-Gln-Pro-Pro, a practical method is proposed for the production of hypoallergenic wheat flour. Bromelain was found effective for decomposing the epitope structure. In practice, soft flour was mixed with water dissolving bromelain and the mixture was incubated at 37°C for 4 h. The result of IgE-ELISA (enzyme-linked immunosorbent assay) suggested negative allergenicity. A mixture of bromelain-modified flour, glucose, citric acid, a surfactant and sodium hydrogen carbonate was baked to produce hypoallergenic bread, resembling English muffins.

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Six tyrosinase isozymes were purified from the browned gill of the fruiting body of Lentinus edodes by ammonium sulfate fractionation, DEAE-Sephacel and Q-Sepharose column chromatography, and partially denaturing SDS-PAGE. At the stop of Q-Sopharose column chromatography, two active fractions (A and B) were obtained. Each fraction was separated to three further fractions, A1, A2, and A3, and B1, B2, and B3, respectively, by partially denaturing SDS-PAGE. All these isozymes consisted of two types of polypeptides: α polypeptide (Aα or Bα) and either β (Aβ or Bβ) or γ polypeptide (Aγ or Bγ). The αpolypeptide contained the consensus amino acid sequence of the active site of known tyrosinases, which is considered to act as a catalytic subunit. From the results of peptide mapping and the amino acid composition, Aα and Bα polypeptides were considered to be different proteins. The kinetic properties of the purified tyrosinase isozymes differed greatly according to whether they contained β or γ polypeptide, indicating these polypeptides to be a possible regulatory subunit.

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A Pseudomonas strain, named BC-18, which can convert racemic phenylacetaldehyde-cyanhydrin (3-phenyllactonitrile) enantioselectively to S-(-)-3-phenyllactic acid (S-PLA), was isolated from soil. Although PLA produced with intact cells contained the S enantiomers of approximately 75% enantiomeric excess (% e.e.), repeated crystallization gave a higher purify (99.8% e.e) of the S configuration product. Production of S-PLA was significantly increased when 2.0% (w/v) of calcium chloride were added to the reaction mixture for precipitation of S-PLA. Chemical mutagenesis yielded a mutant strain, named BC348-9, with 16 times higher activity (40mU/OD⁶³⁰), compared with that of the parent strain (2.5mU/OD⁶³⁰). When the mutant strain BC348-9 was used, approximately 18 g/OD⁶³⁰ was produced, which is 12 times higher than that of the parent strain. The final accumulation of PLA exceeded 6.0%, 1.2 times higher than that of the parent strain.

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The gene (aprI) encoding alkaline serine protease (AprI; subtilase) from Alteromonas sp. strain O-7 was cloned and sequenced. The nucleotide sequence of aprI has been identified. The deduced amino acid sequence indicated that aprI codes for a precursor of 715 amino acids and the precursor is composed of four regions including a signal peptides an N-terminal pro-region, a mature protease region and a C-terminal extension region of 215 amino acids as previously described for aprII [H. Tsujibo et al., Gene, 136, 247-251(1993)]. The amino acid sequence of the mature AprI (AprI-M) showed high sequence homology with those of other class I subtilases. The C-terminal region was characterized by a repeat of 94 amino acids residues, which showed about 50% similarity with those of the C-terminal pro-region of several known proteases from Gram-negative bacteria.

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The merC gene of Thiobacillus ferrooxidans was overexpressed in Escherichia coli under the control of the tac promoter. MerC protein synthesized in E. coli has a N-terminal amino acid sequence of S-A-I-X-R-I-I-D-K-I-G-I-V-G-, which agrees with the amino acid sequence deduced from its nucleotide sequence except that an initiating methionine residue was removed. The MerC protein was localized in the particulate (membrane) cell fraction, and not in the soluble cytoplasmic fraction. E. coli cells carrying a plasmid containing the tac promoter-directed merC showed ²⁰³Hg²⁺ uptake in an isopropyl-1-thio-β-D-galactopyranoside (IPTG)-dependent manner.

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Eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) were distributed mainly in the sn-1 and 3 positions of seal oil triglyceride and in the sn-2 position of fish oil triglyceride. In Expt. 1, the structural distribution of EPA and DHA in lymph triglyceride of rats given seal or fish oils was similar to the distribution in the administered oils. In Expt. 2, seal oil-rich or fish oil-rich fats having constant polyunsaturated/monounsaturated/saturated fatty acids and n-6/n-3 polyunsaturated fatty acids ratios were fed to rats for 3 weeks. Seal oil more effectively reduced plasma and liver triglyceride than fish oil. Ratio of the productions of aortic prostacyclin and platelet thromboxane A₂ stimulated by thrombin was significantly higher in rats fed seal oil than in those fed fish oil. The results suggested that the different intramolecular distribution of EPA and DHA in dietary fat affected lipid metabolism differently in rats.

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Soluble egg shell membrane protein (SEP) was prepared from egg shell membrane by the combined treatment of performic acid oxidation (4 or 25°C for 24 h) and pepsin digestion (25°C for 24-72 h) before dialyzing against water and lyophilizing. The yield of SEP was about 16-39%. SEP had high contents of acidic amino acids (320-340 residues/1000 residues) containing cysteic acid (101-108 residues) converted from cystine, a small amount of saccharides, a main molecular weight of 12, 000-22, 000 and pI 4.2-4.8. SEP accelerated the reconstruction of collagen matrix with ordered molecular rearrangement and reduced redissolution of the collagen matrix under several solvent conditions. SEP elevated the denaturation temperature of the matrix.

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Eight strains of Saccharomycopsis species were examined for their partial base sequences of 18S and 26S rRNAs. The base sequences on the fingerprint segment showed three kinds of ACUAU, UCUAU, and AUAU. In the partial base sequences (positions 1451-1618, 168 bases) of 18S rRNA, all the strains of Saccharomycopsis species examined had 1-0 base difference except for the strains of Saccharomycopsis fibuligera (base differences, 5-4). In the partial base sequences on the positions 1611-1835 region (225 bases) of 26S rRNA, however, S'copsis vini had 15-11 base differences. The other species had 7-2 base differences. In the partial base sequences on the positions 493-622 region (130 bases) of 26S rRNA, the calculated percent similarities were 62-77. Discussion was made phylogenetically and taxonomically, especially on the separation of S'copsis fibuligera (≡Endomyces fibuliger) from the genus Saccharomycopsis.

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A hexaosyl fragment of the major component of lipooligosaccharide HS-142-1, O-(3-O-caproyl-β-D-glucopyranosyl)-(1→6)-[O-(β-D-glucopyranosyl-(1→6)-O-(3-O-caproyl-β-D-glucopyranosyl)-(1→6)₂-D-glucopyranose (1), was efficiently synthesized by block synthesis. More stable analogs, O-(3-O-hexyl-β-D-glucopyranosyl)-(1→6)-[O-(β-D-glucopyranosyl)-(1→6)-O-(3-O-hexyl-β-D-glucopyranosyl)-(1→6)]₂-D-glucopyranose (4) and O-(3-O-caproyl-2, 4, 6-tri-O-methyl-β-D-glucopyranosyl)-(1→6)-[O-(2, 3, 4-tri-O-methyl-β-D-glucopyranosyl)-(1→6)-O-(3-O-caproyl-2, 4-di-O-methyl-β-D-glucopyranosyl)-(1→6)]₂-2, 3, 4-tri-O-methyl-D-glucopyranose (2), were also designed and synthesized in a similar manner.

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Human plasma proteins were subjected to affinity chromatography with (-)-epigallocatechin gallate (EGCg)-agarose, and the bound proteins were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A molecular weight evaluation of the protein bands suggested the presence of three proteins, fibronectin, fibrinogen, and a 75-kDa protein. When human serum was used, the 75-kDa protein dominated the bound fraction. The determination of the partial amino acid sequence of a peptide derived by endopeptidase digestion of this fraction suggested the 75-kDa protein to be histidine-rich glycoprotein (HRG). The presence of these proteins in the bound fraction was confirmed by the immunoblotting method. Affinity chromatography of the individual proteins indicated that fibrinogen and HRG had direct affinity for EGCg. Dot binding assays demonstrated the interaction of EGCg with these proteins. The method also showed that only gallate-containing catechins were bound by these proteins. These data suggest that when EGCg is absorbed in the body through the digestive system, it may interact with these proteins in blood plasma.

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An enzyme that doacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190, 000 Da and was predicted to be composed of four identical subunits with molecular masses of 45, 000 Da. The purified enzyme hydrolyzed N-acetylglucosamine, N-acetylglucosamine 6-phosphate, and N-acetylglucosamine 6-sulfate, but not chitin oligosaccharides, and N-acetylgalactosamine. The deacetylase activity was completely abolished by N-ethylmaleimide, p-chloromercuribenzoate, EDTA, and Cu²⁺. On the other hand, the activity was activated by Co²⁺. The amino-terminal amino acids of the purified enzyme were sequenced. Among the 22 N-terminal amino acid residues, 12 residues of Vibrio deacetylase were identical with that of Escherichia coli GlcNAc 6-phosphate deacetylase.

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The ATPase bound to the inner membrane of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1) was extracted with Triton X-100 and purified by fractionation with polyethylene glycol, sucrose density gradient centrifugation, and DEAE-Toyopearl 650M column chromatography. The molecular masses of subunits constituting the purified ATPase were estimated as 54, 49, 33.5, 27, 23.5, 18.5, and 15 kDa by SDS-PAGE. The composition and molecular masses of the subunits of the purified ATPase were similar to those of Escherichia coli F₀F₁-ATPase (EF₀F₁). The 54-, 49-, and 18.5-kDa polypeptides of the Vibrio ABE-1 ATPase strongly cross-reacted with the antibodies against the EF₀F₁ α, β, and b subunits, respectively. However, the Vibrio ABE-1 ATPase contained no cross-reactive polypeptide with the antibodies against A and B subunits of V-type H⁺-ATPase from mung bean tonoplasts. The ATPase activity showed two pH optimum peaks at pH 5.3 and 8.0 and was strongly inhibited by N, N'-dicyclohexyl carbodiimide (DCCD) and NaN₃. It hydrolyzed ATP, GTP, and ITP at similar rates. These properties confirm that the purified ATPase is a F₀F₁-type. The optimum temperature for the ATP-hydrolyzing activity of the enzyme was observed at 50°C, but the DCCD-sensitivity of the enzyme was markedly decreased above 30°C, suggesting that the F1-moiety is released from the enzyme complex at high temperatures. This characteristic is compatible with the psychrophilic nature of Vibrio ABE-1.

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A xylan-degrading enzyme produced by yeast Cryptococcus sp. S-2 was isolated and purified, and characterized as an endoxylanase (1, 4-β-D-xylan xylanohydrolase [EC 3.2.1.8]). We estimated the molecular weight and isoelectric point of purified xylanase (xyn-CS2) to be 22, 000 and 7.4, respectively. This low-molecular-weight xylanase had an unusual pH optimum of 2.0, and showed 75% of maximal activity even at pH 1.0. An open reading frame of the cDNA specified 209 amino acids, including a putative signal peptide of 25 amino acids. The deduced amino acid sequence of xyn-CS2 shared significant similarities with the family-G xylanases of B. pumilus, C acetobutylicum, T reesei, and A. kawachii. Xyn-CS2 included two unique cysteine residues in a putative catalytic region, raising the possibility that these residues are at least partially responsible for its acidophilic nature.

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We have found a factor that induces neurite outgrowth of rat PC12 cells in the culture supernatant of the cell line MLEL15A2. This factor was designated as MDDF. The factor was sensitive to protease, dithiothreitol, and high-temperature treatments. The apparent molecular mass was 80 kDa on Superdex 200 gel filtration. No significant tyrosine phosphorylation was detected after MDDF stimulation in Western blotting analysis with anti-phosphotyrosine antibody, suggesting that the signal transduction may not be mediated by a tyrosine kinase cascade that is involved in signaling of most of the known factors. Activation of MAP kinase was very weak and was seen only 5 min after stimulation, suggesting that prolonged activation of MAP kinase was not required for neurite outgrowth induced by MDDF. Because the biochemical characteristics of MDDF are different from those of any known peptide factors that induce neurite outgrowth of PC12 cells, MDDF may be a novel differentiation factor for PC12 cells.

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Two cDNAs encoding class I chitinases of rice were expressed in Escherichia coli. The cDNAs were fused to the MS2-polymerase gene in an expression vector, pEx31. The fusion proteins, expressed under the control of the λP_L-promoter, showed the chitinase activity independent of the existence of the hevein domain. The enzymatic hydrolysis of colloidal chitin by the fusion proteins showed that the proteins were endo-type enzymes.

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The gel-setting and gel-melting temperatures of aqueous gelatin solutions (0.5 and 1.0 wt% ) were measured by a steady state hot-wire method during treatment under high hydrostatic pressure up to 200MPa. The high-pressure treatment caused both the gel-setting and gel-melting temperatures to increase with increasing pressure. The hot-wire method was proved to be effective for assessing the coagulation or gelation of food materials under pressurized conditions.

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Compared with the enzyme activity of a recombinant Taka-amylase A (wild TAA) at 25°C and pH 5.3, those of two mutants (E230Q and D297N) were 1/10, 000 and 1/16, 000 for amylase activity, and 1/920 and less than 1/20, 000 for maltosidase activity, respectively. This indicates that all residual activities of E230Q are not due to contamination by wild-type TAA. The results from difference spectroscopy suggested that E230Q retains amylose binding ability.

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Autolysis of squid liver and mantle muscle homogenates, and blends of both, each yielded inhibitory activity toward the angiotensin I-converting enzyme. The inhibitory activity and the amount of solubilized protein in each of these autolysates were examined over a period of 24 h. Inhibitory peptides were isolated from the mixed autolysate, their structures (IC₅₀) being Tyr-Ala-Leu-Pro-His-Ala (9.8 μM) and Gly-Tyr-Ala-Leu-Pro-His-Ala (27.3 μM).

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The mechanism of Fe²⁺ production by a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was studied. Strain TI-1 produced H₂S outside of the cells when the following five L-amino acids, aspartic acid, glutamic acid, serine, arginine, and histidine, were added to the medium. The activity of H₂S production was completely inhibited by uncouplers and inhibitors of cytochrome c oxidase or sulfite reductase. When Fe³⁺ was added to the H₂S production medium, it was chemically reduced by the H₂S produced by the strain to give Fe²⁺, the sole energy source of this bacterium.

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New water-soluble fullerene carboxylic acids (1 and 2) derived from C₆₀ and C₇₀ fullerenes, respectively, were examined for photocytotoxicity toward Raji cells (B lymphocyte). These compounds did not show any photocytotoxic effect even at 50 μM, while pheophorbide a showed significant photocytotoxicity at 0.5 μM. Therefore, fullerene derivatives derived from C₆₀ and C₇₀ would not be practical agents for photodynamic therapy.

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The effects of oxidized cholesterols on proliferation and IgM production of human lymphoblastoid Namalwa cells were examined. An oxidized cholesterol mixture, in contrast to cholesterol, was a potent cytotoxin to Namalwa cells. Among oxidized cholesterols examined, 25-hydroxycholesterol was the most cytotoxic. However, no oxidized cholesterol examined suppressed IgM production, although cholestanetriol and 7-ketocholesterol did suppress it. Thus, oxidized cholesterols are cytotoxic to lymphocytes, while the influence on the immunoglobulin production may be marginal.

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An expression plasmid carrying an alkaline lipase gene from Proteus vulgaris was constructed. The lipase content in Escherichia coli cells harboring the expression plasmid reached about 22% of total soluble protein. The purified enzyme displayed a partial interfacial activation toward p-nitrophenyl butyrate (PNPB).

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Rhodanine (1), rhodanine-3-acetic acid (2), methylrhodanine (3), and aminorhodanine (4) inhibited the growth of plants. Among them, 4 was the strongest inhibitor of the roots of all the tested plants. On the other hand, N-acetylaminorhodanine (5) and N-benzoylaminorhodanine (6) greatly decreased the inhibitory activity. The results suggest that the free amino group at N-3 of 4 is essential to the greater inhibitory activity of rhodanine derivatives. The plant-growth inhibition of 1-6 is related to the chlorophyll content of the plant treated with them and their acute toxicities in mice.

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Sporogen AO-1 is a sporulation-stimulating substance isolated from Aspergillus oryzae. We speculated that ring B would be important for the activity and designed a compound without functional groups in ring A. We synthesized the compound from 2-methylcyclohexanone via chiral octalone. The compound showed activity at 200 μg/disc. The functional groups on ring B are suggested to be important for the activity.

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Aspyrone (1) was isolated from a culture filtrate of Aspergillus melleus, and the structure was determined by a comparison of spectroscopic data with those of an authentic sample. 1 showed nematicidal activity toward Pratylenchus penetrans by 80.8% at a concentration of 300 mg/liter.

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For characterization of the biosynthetic pathway of cellulose in a cellulose-producing Acetobacter xylinum strain BPR2001, the activities of several enzymes were measured. The activity of phosphoglucose isomerase catalyzing the conversion of fructose-6-phosphate into glucose-6-phosphate was greatly increased by fructose in the medium. The UDP-glucose pyrophosphorylase activity catalyzing the synthesis of UDP-glucose was very high in strain BPR2001, consistent with the idea that this is the key enzyme in cellulose biosynthesis. Strain BPR2001 was found to have a fructose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).

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Two pokeweed lectins, designated PL-D1 and PL-D2, have been isolated from the roots of pokeweed (Phytolacca americana) using chitin affinity column chromatography followed by gel filtration on a Sephacryl S-200 column and fast protein liquid chromatography on a Mono-Q column, and their amino acid sequences have been analyzed. PL-D1 consists of 84 amino acid residues and has a molecular mass of 9317, while PL-D2 has an identical sequence with PL-D1 except lack of the C-terminal Leu-Thr. PL-D is composed of two chitin-binding domains, A and B, with 50% homology with each other. Both PL-Ds did not agglutinate native rabbit erythrocytes, but showed about 0.1% of the agglutinating activity of wheat germ agglutinin toward trypsin-treated erythrocytes. In the presence of β(1→4) linked oligomers of N-acetyl-D-glucosamine, which inhibit the hemagglutination, PL-D1 had an ultraviolet-difference spectrum with maxima at 292-294 nm and 284-285 nm, attributed to the red shift of the tryptophan residue, suggesting the location of tryptophan residue(s) at or near saccharide-binding site of PL-D1.

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The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman-Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIa inhibited plasma kallikrein. Neither ETIa nor ETIb exhibited any inhibitory activity toward β-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the Fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence.

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A mutant of a Brevibacterium lactofermentum L-leucine producer was obtained that was resistant to high concentrations of D-α-aminobutyric acid (D-ABA) and the production by which of L-valine decreased by an order and that of L-leucine increased by 24% compared to its parent, No. 34. In this mutant strain, No.12-6, the activities of both acetohydroxy acid synthase and isopropylmalate synthase were higher than those of the parent.

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We found a novel proteinaceous Kex 2 proteinase inhibitor, named kexstatin, in the culture supernatant of Streptomyces platensis Q268.The purified kexstatin was homogeneous by SDS-PAGE and the molecular weight was estimated to be 13, 000. The N-terminal amino acid sequence of kexstatin has high similarity to Streptomyces subtilisin inhibitor (SSI), suggesting that kexstatin belongs to the SSI family. Kexstatin was a strong inhibitor of Kex 2 proteinase and subtilisin but not thermolysin, trypsin, or chymotrypsin. The IC₅₀ value of kexstatin against 1 μg of Kex 2 proteinase was 1.4 μg.

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