We examined whether developing cotyledons of soybean seed had photosynthetic activities. The cotyledons evolved oxygen under illumination and the activity was inhibited by 3-(3', 4'-dichlorophenyl)-1, 1-dimethylurea. The rate of oxygen evolution decreased during the development of seeds; about 30 μmol O₂•mg chlorophyll^-1•hr^-1 at the early developing stage and about 10 μmol O₂•mg chlorophyll^-1•-hr^-1 at the late developing stage. The rate of oxygen uptake remained at an almost constant level of 40 μmol O₂•mg chlorophyll^-1•hr^-1 throughout the development. Photosynthetic ¹⁴CO₂-fixation by the cotyledon was observed. Ribulose bisphosphate carboxylase was immunochemically detected in the developing cotyledons. These results show that functional photosynthetic apparatus is present in the developing cotyledons of soybean seeds.

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Protein components in the hypocotyl-radicle axis of soybean seeds were compared with those in the cotyledon. The hypocotyl-radicle axis was rich in 7S and 2S proteins but poor in 1 IS protein on the profiles of analytical ultracentrifugation and of SDS-PAGE. Among the 7S-protein constituent polypeptides, α'-polypeptide was abundunt but α- and β-polypeptides were less abundunt than in the cotyledon. Several isomers of 7S protein were found and their main constituents were α-', α'-, or both polypeptides on two-dimensional PAGE. These results indicated that the hypocotyl-radicle axis contained different types of the 7S protein than the cotyledon.

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A method for the direct determination of carbamate pesticides and their formulation by gas chromatography (GC) was developed. Four causes for the thermal decomposition of carbamates during GC were found, these being tarry matter remaining in the injection port, the active site on the glass column wall, neutral or weakly alkalic column packings, and inert ingredients of the formulation. To prevent carbamate decomposition by these causes, a dimethyldichlorosilane (DMCS)-treated glass insert injection port was used, which was always cleaned to remove any tarry matter, together with a DMCS-treated glass column, weakly acidic column packing (Thermon^® 3000 on Shimalite^® WP), and formic acid with which the formulation samples were injected into the GC. With these improvements, all of 8 N-methylcarbamates, i.e., BPMC, MPMC, MTMC, propoxur, aminocarb, bendiocarb, carbofuran and NAC (carbaryl), were determined directly and quantitatively with a 0.2% to 0.4% of coefficient of variation (C.V.). The formulations of BPMC, MPMC, MTMC and NAC were also directly determined with 0.3% to 0.6%C.V.

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The metabolism of S-sulfocysteine was studied in Salmonella typhimurium. Nutritional experiments with sulfite- and sulfide-requiring mutants ascertained that this amino acid is converted into cysteine and sulfite, and served exclusively as a sulfur source. Two different NADPHdepending systems able to participate in the metabolism of S-sulfocysteine were detected in a crude extract. Both systems were purified and identified as glutathione-glutathione reductase and thioredosin-thioredoxin reductase. The latter system seems to function more efficiently.

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6PG dehydrogenase (EC 1.1.1.44), partially purified from Brevibacterium flavum, was specific to NADP⁺, stabilized by KCl, and had an optimum pH of 7.5 to 8.0. Its activity was inhibited by FBP, PRPP, GAP, oxaloacetate, E4P, acetyl-CoA, Ru5P, and NADPH, among which, FBP showed the.strongest inhibition. The concentrations of the latter giving 50 and 100% inhibition were 0.07 and 1.0 mM, respectively. Inhibition by FBP, PRPP, and E4P was competitive to 6PG but not to NADP⁺. Homotropic interaction of 6PG was observed in the presence of FBP and the Hill coefficient was estimated to be 1.6. Inhibition by the reaction products, Ru5P and NADPH, was mixed and noncompetitive to 6PG and noncompetitive and mixed to NADP⁺, respectively. Combination of acetyl-CoA with FBP or PRPP inhibited the enzyme synergistically and that of acetyl-CoA with GAP or Ru5P did cumulatively. The sensitivities of the enzyme to the FBP, PRPP, acetyl-CoA, oxaloacetate, and E4P inhibitions were lost with ammonium sulfate treatment. The enzyme was induced by gluconate. Cells grown on gluconate did not contain an FBP-insensitive and NAD-specific enzyme at all.

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The enantioselective hydrolysis of (R, S)-3-acetoxymethyl-7, 8-difmoro-2, 3-dihydro-4H-[l, 4]benzoxazine (I) with enzymes was investigated. Optically active I and its hydrolyzate, 7, 8-difluoro-2, 3-dihydro-3-hydroxymethyl-4H-[l, 4]benzoxazine (II), are the intermediates for preparing optically active ofloxacins, whose racemate is known to be an excellent antibacterial agent. Lipoprotein lipase from Pseudomonas fluorescens (LPL Amano 3) was found to predominantly hydrolyze (S)-I, giving (R)-I in 54% e.e. and (R)-II in 44% e.e. On the other hand, lipase from Candida cylindracea was found to predominantly hydrolyze (R)-I, giving (S)-I in 24% e.e. and (S)-II in 20% e.e. Since, the optical purities of I and II thus obtained were not particularly high, these optically active I and II were converted into 3-acetoxymethyl-7, 8-difluoro-2, 3-dihydro-4-(3, 5-dinitrobenzoyl)-4H-[1, 4]benzoxazine (IV). After recrystallizing IV from ethyl acetate-hexane, (S)-and (R)-II were obtained with high enantiomeric excess by removing the crystallized racemic IV and subsequently hydrolyzing the resulting optically active IV with alkali. The reduction of II afforded 7, 8-difluoro-2, 3-dihydro-3-methyl-4H-[1, 4]benzoxazine (III), for which the optical purity was estimated to be > 96% e.e. by HPLC analysis. (R)- and (S)-ofloxacin were prepared from (R)- and (S)-III with retention of their configuration.

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The optical resolution of (±)-α-cyclogeranic acid (4) with (-)-α-methylbenzylamine gave (-)-(S)-α-cyclogeranic acid (4). Epoxidation of (-)-(S)-methyl α-cyclogeranate (5), followed by reduction with lithium aluminum hydride, gave mainly (-)-(1R, 2R)-2-hydroxy-2, 6, 6-trimethyl-1-cyclohexanemethanol (7b), which was oxidized with pyridinium chlorochromate to give (-)-hydroxyaldehyde 8. The reaction of aldehyde 8 with 2-oxopropylidenetriphenylphosphorane gave (-)-(1'S, 2'R)-2', 3'-dihydro-2'-hydroxy-α-ionone (9). The thermal decomposition of (1'S)-2'-acetoxy-2', 3'-dihydro-α-ionone (10) gave (-)-(S)-α-ionone (2) and (+)-(S)-γ-ionone (1). The partial hydrogenation of (+)-(S)-γ-ionone (1) gave (+)-(S)-dihydro-γ-ionone (3).

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Approximately 0.14μmol of hematoporphyrin derivative (a preparation made by treating hematoporphyrin with sulfuric acid-acetic acid) per μmol of phospholipids was included in the lipid phase of reverse-phase evaporated liposomes which were composed of 4 μmol of phosphatidylcholine, 1 μmol of phosphatidylglycerol, 5 μmol of cholesterol and 1 μmol of α-tocopherol. Treatment of mouse carcinoma FM3A cells with liposomes of the above composition which encapsulated 25 mM methotrexate, a water-soluble antifolate drug, inhibited cell multiplication completely whereas the same amount of the drug encapsulate in liposomes that were devoid of hematoporphyrin derivative and α-tocopherol did not affect cell growth. Of the constituents of the hematoporphyrin derivative, O, O'-diacetylhematoporphyrin was the most effective in raising the efficacy of delivery of aqueous materials into FM3A cells.

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Four stereoisomers of sesaminol were artificially produced after the intermolecular transformation of sesamolin by the presence of acid clay during the decolorization process used to commercially produce sesame oil. After isolating and purifying, the Stereochemical structures of four stereoisomers of sesaminol were deduced mainly by the ¹H-NMR spectroscopic technique, and X-ray crystallographic analysis of the 3, 5-dinitrobenzoate of one of the sesaminol epimers confirmed the relative stereochemistry of all four sesaminol isomers.

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The reaction of gluten and its components, gliadin and glutenin, with lipid hydroperoxides was studied. The proteins were reduced and incubated with linoleic acid hydroperoxide and monolinolein hydroperoxide at various molar ratios to their sulfhydryl groups under anaerobic conditions. Half of these sulfhydryls were converted to the disulfide form and the other half seemed to be part of higher oxidation products. The formation of interchain disulfide linkages in gluten protein was shown by the increase in the intrinsic viscosity and the production of protein aggregates seen by SDS-polyacrylamide gel electrophoresis. Sulfhydryls in glutenin and gliadin behaved differently toward lipid hydroperoxide. Most of the sulfhydryls in glutenin seemed to be converted to interchain disulfide linkages and those in gliadin to intrachain disulfides at early incubation times and higher oxidation products appeared in both proteins during prolonged incubation. This suggests that the lipid hydroperoxides formed during dough mixing under air lead to the formation of disulfide linkage and build up gluten networks.

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Under the conditions of citric acid fermentation, Aspergillus niger WU-2223L possessed at least two respiratory systems. One system was sensitive to cyanide (CN) and the other was sensitive to salicylhydroxamic acid (SHAM). The respiration of mycelia was partially, inhibited by CN or SHAM, and was completely inhibited in the presence of both CN and SHAM. With culture time, the CN-seflsitivity of respiration decreased and the SHAM-sensitivity increased. The respiratory rate itself decreased suddenly at 4 days and thereafter remained at a low level. The SHAM-sensitive respiration was localized in the mitochondria. The alterations of respiration in the mitochondria were similar to those observed in mycelial cells.

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Multiple shoot cultures of Catharanthus roseus (Madagascar periwinkle) were directly induced, with high frequencies, from seedlings in the presence of 1.0mg/l benzyladenine. The resulting cultures consisted of unorganized tissue touching the solid medium and multiple shoots having several leaf tissues. Quantitative HPLC assay of the culture extracts showed that vindoline and catharanthine were mostly found in the shoot, especially in the leaf tissue, whereas ajmalicine was localized in the unorganized tissue. The contents of these alkaloids were much changed in the cultures; the concentration of catharanthine in the leaf tissue was several times higher than in the parent, while vindoline content of the leaf tissue was comparable to that of parent leaves.
Transfer of the cultures to a low-benzyladenine (0.1 mg/l) or a benzyladenine-free medium, stimulated the morphological development of the shoots and increased the production of vindoline and catharanthine, although growth and ajmalicine production were suppressed.

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An about, 4-fold H₂ evolution rate and a 5-fold H₂ molar yield (mol H₂/mol glucose) were obtained with a mixed culture of Chlamydomonas reinhardtii and Rhodosprillum rubrum, compared with in the case of an algal culture of C. reinhardtii alone. This increasing effect was due to the consumption of formate formed by C. reinhardtii; R. rubrum evolved hydrogen from formate via the formate hydrogen-lyase system under dark anaerobic (N₂) conditions. Maximum H₂ evolution by the mixed culture was observed with a ratio of 8:2 (alga: bacterium) at a total cell concentration of above 0.6mg dry wt/ml. Sustained H₂ production with an alternating light/dark cycle in a membrane reactor, in which this alga and bacterium were cultured in separate compartments, was performed for one week.

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Properties of proteins in the light (<d 1.03 on Ficoll and then <d 1.14 on sucrose) and heavy (d 1.03/1.05 on Ficoll and then <d 1.14 on sucrose) plasma membranes (PM) isolated from lactating bovine mammary gland were investigated. The PMs consisted of 57-62% of protein and 38-43% lipid. The lipid/protein ratio was 0.77 in the light PM and higher than 0.61 of the heavy PM. However, no appreciable differences were found between the light and heavy PMs with respect to polypeptide, amino acid, and carbohydrate compositions. The protein moiety contained approximate 5-6% carbohydrate: fucose 8-9, mannose 11-12, galactose 16, N-acetylglucosamine 32, N-acetylgalactosamine 12-15, and sialic acid 17-20 mol%. PM protein was high in the content of aspartic acid, glutamic acid, and leucine and low in proiine, cystine, methionine, and histidine. On double immunodiffusion both PMs formed precipitin lines against milk fat globule membrane (MFGM) antiserum. Electrophoretic analysis on sodium dodecyl sulfate-polyacrylamide gel revealed the presence of many minor polypeptides and three major glycopeptides (PAS-I, II, and III) of molecular weights of 115, 000, 94, 000, and 82, 000. The glycoprotein profiles of the PMs were different from those of MFGM, except that PAS-II and -III of PM corresponded to PAS-3 and -4 of MFGM, respectively.

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The degradation of nitrite and the inhibition towards formation of carcinogenic nitrosamines by melanoidins, produced from the glucose-glycine system were investigated at various conditions. The degradation of nitrite was highest at pH 1.2 (29%), when the ratio of melanoidins to nitrite was 1:3. The inhibition towards formation of nitrosamines by melanoidins had the same tendency as the degradation of nitrite, the inhibition also being highest at pH 1.2 (99%). In addition, melanoidins after nitrite treatment exhibited a little higher mutagenicity and much stronger desmutagenicity than those of the original melanoidins. The change of the structure of melanoidins after treating with nitrite was also investigated by HPLC and CP-MAS NMR.

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Forty-one 2-difluoromethylthio-1, 3, 5-triazines were synthesized and their herbicidal activities were evaluated under pot conditions. Some of them showed high potential as useful herbicides against weeds with little phytotoxicity toward crops. 2-Difluoromethylthio-4, 6-bis(isopropylamino)-1, 3, 5-triazine (exp. code No. SSH-108) showed a particularly high specific pre-emergence herbicidal activity. Their activities and selectivities were compared with those of prometryn.

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The microbial esterification of (±)-menthol with various fatty acids by Pseudomonas sp. NOF-5 strain was investigated. A water insoluble fatty acid higher than caproic acid was esterified with (-)-menthol with high enantioselectivity; especially, when lauric acid was used as the fatty acid, the yield of ester was satisfactory. The microbial esterification of various racemic terpene alcohols with lauric acid by the same microorganism was further studied. (-)-Isopulegol and (-)-trans-carveol were esterified from their racemates with high stereoselectivity. However, the esterification of (±)-cis-carveol was with poor enantioselectivity, and (±)-citronellol was entirely nonselective.

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Conversion of ergosta-5, 7-dien-3β-ol to ergosterol (ergosta-5, 7, 22-trien-3β-ol)-Δ²²-desaturation-by microsomes of Saccharomyces cerevisiae was inhibited by CO. The inhibition was partially reversed by illumination with white light. The Δ²²-desaturation was also inhibited by SKF 525A. In addition, the activity was inhibited by the antibodies against yeast NADPH-cytochrome P-450 reductase [EC 1.6.2.4]. These results together with our preceding reports [S. Hata, T. Nishino, M. Komori and H. Katsuki, Biochem. Biophys. Res. Commun., 103, 272 (1981) and S. Hata, T. Nishino, H. Katsuki, Y. Aoyama and Y. Yoshida, Biochem. Biophys. Res. Commun., 116, 162 (1983)] suggest the contribution of a cytochrome P-450-containing monooxygenase system to the Δ²²-desaturation, and a cytochrome P-450 (P-450_22DS) may be responsible for the oxidative desaturation as the terminal enzyme. When semi-anaerobically-grown yeast cells were aerobically adapted, the Δ²²-desaturation activity increased together with the increase in the cellular sterol content. The increment of Δ²²-desaturation activity was inhibited by cycloheximide, indicating that the increase in the Δ²²-desaturation activity was due to de novo synthesis of the enzyme protein(s). However, NADPH-cytochrome P-450 reductase, which is another component of the reaction, was nearly constant during the aerobic adaptation and was not affected by the cycloheximide. These results suggest that the component responsible for the induction of Δ²²-desaturation activity is P-450_22DS.

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Esters selected from the combination of tricycloalkanecarboxylic acids and some representative pyrethroidal alcohols were prepared, and their insecticidal activities were measured by topical application on housefly, mosquito and German cockroach. Both the exo- and endo-isomers of 2, 3-trimethylenenorbornane-2-carboxylic acid exhibited a high level of activity when esterified with m-phenoxymandelonitrile. Piperonyl butoxide showed a strong synergistic effect on some of the esters. Several structural resemblances were found among these active tricyclic acids and the chrysanthemummonocarboxylic acids, which were suggested to be related to insecticidal activity.

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A series of products with partial and complete hydrolyses of sweet-potato starch by β-amylase was prepared, and their structural properties were characterized. The hydrolysis of amylopectin components was near-linearly proportional to increasing β-amylase treatments on the starch at the range between 0 and 40% of the β-amylolysis. However, very little degradation of amylose components was shown to take place. An increased rate of amylose hydrolysis was found after 40% of β-amylolysis. The longer unit-chain fraction originating from amylopectin molecule was scarcely degraded during the β-amylolysis of starch. The results of this study demonstrate that most of the amylose component still remains even after the greater part of the starch is hydrolyzed by β-amylase.

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The mycotoxin citrinin had antifungal activity under acidic conditions. At the minimum inhibitory concentration, it completely inhibited cellular respiration and partially inhibited the incorporation of radioactive precursors into macromolecules in Saccharomyces cerevisiae. It had no effect on cell permeability. In mitochondrial preparations, it significantly inhibited succinate oxidase and NADH oxidase. Rhizopus chinensis was more sensitive than S. cerevisiae', its growth and mycelial respiration at acidic pH were completely inhibited by lower concentrations of citrinin. The pH-dependent antifungal activity of citrinin seems to be associated with its uptake by fungi. Approximately half of the citrinin taken up was found in mitochondria. The main site of the antifungal action of citrinin, therefore, appears to be the mitochondrial electron transport system.

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(-)-Invictolide [(3R, 5R, 6S, 1'R)-3, 5-dimethyl-6-(1'-methylbutyl)-tetrahydro-2H-pyran-2-one] was synthesized in 16 steps from 2-methylpentanal.

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Expression of human interleukin 2 (IL-2) at high levels has been achieved in human cells by cotransfection and subsequent coamplification of the transfected sequences. IL-2 production by these cells reached 2700 U/ml of culture medium. This productivity was higher than the 100U/ml attained by concanavalin A (ConA) stimulated human leukaemic Jurkat, T cells. A plasmid, pSDI, containing human IL-2 cDNA and mouse dihydrofolate reductase (DHFR) cDNA was constructed, and cotransfected with plasmid pSV2neo at a 10:1 molar ratio, into Hela (human epitheloid carcinoma) cells by the DNA-calcium phosphate coprecipitation technique. Transformants producing IL-2 were easily obtained among antibiotic G418 resistant colonies. Cells resistant to increased levels of methotrexate (MTX) secreted a large amount of IL-2 as a result of coamplification of DHFR cDNA and IL-2 cDNA. Expression of IL-2 cDNA was stimulated by removing a G-C stretch which was present between Simian Virus 40 (SV40) early promoter and IL-2 cDNA. Plasmid pSDI was rescued from the transformed Hela cells and shown to be present in the intact form among the amplified cellular DNA.

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The stimulating effects of monoterpenes in pine trees on the molting and the multiplication of the pine wood nematode were investigated. The results indicated that the molting of the dispersal 4th stage larva (L_IV) to the adult nematode was stimulated by these monoterpenes, especially β-myrcene. The population of the nematode cultured on fungal mats of B. cinerea, A. kikuchiana and C. miyabeanus increased considerably in the presence of a small amount of β-myrcene. This compound seemed to play a hormone-like role for both the molting of L_IV and the multiplication of the nematode.

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Three new antibiotics were obtained from the culture filtrate of an actinomycete identified as Streptomyces violaceolatus, and were named kerriamycins A, B and C, respectively. The structures of these kerriamycins were elucidated by NMR spectral analysis and chemical degradation, which revealed that each kerriamycin was composed of a common chromophore identified as aquayamycin and two or three mols of hexoses. The kerriamycins inhibited the growth of Gram-positive bacteria and prolonged the survival periods of mice bearing Ehrlich ascites carcinoma.

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Synthesis of the spoVE gene product (a 32 kilodalton protein) of Bacillus subtilis was studied using a DNA-directed protein-synthesizing system from B. subtilis. The spoVE gene product was synthesized specifically by T₂ (2 hours after the end of logarithmic growth) cell extracts but not by vegetative cell extracts. A dot blot hybridization experiment with a spoVE-specific probe showed that the spoVE mRNA was synthesized by vegetative as well as by T₂-cell extracts, and was present in most spo0 mutants. These results suggest that the transcription of the spoVE gene is not dependent on the spo0 gene function and the synthesis of the SpoVE protein is somehow controlled at the level of translation. Heterologous combination experiments using supernatant fractions from the vegetative and T₂ cells, and ribosomes from the vegetative and T₂ cells showed that T₂-specific synthesis of the SpoVE protein is dependent on the T₂-cell supernatant. The addition of the T₂-cell supernatant to the vegetative cell extract induced SpoVE protein synthesis, suggesting the presence of a positive factor in the T₂-cell supernatant. The heat inactivation profile for the stimulatory activity in the T₂-cell supernatant and the preparative method for the supernatant fraction suggest that the factor(s) in the supernatant may be a protein(s).

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The nutritive values of protein, expressed as a modified chemical score, in two kinds of rations were compared with those of the rumen contents of faunated and defaunated goats given the two rations singly. The first, second and third limiting amino acids in the rations were lysine, methionine and histidine, respectively. In the case of the rumen contents of faunated animals, histidine and methionine tended to be the first and second limiting amino acids, respectively, whereas in those of defaunated animals lysine tended to be the second limiting amino acid. The improvement in the quality of protein in the rations in the rumen and the strict requirement of histidine for ruminants were discussed.

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Phosphatidylethanolamine N-methyltransferase, which catalyzes all the three-step methylation from Phosphatidylethanolamine to phosphatidylcholine, was purified to homogeneity from the membrane fraction of Zymomonas mobilis. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42, 000 on comparison with those of marker proteins. The three activities dependent on phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N, N'-dimethylethanolamine of the purified enzyme showed similar pH profiles with an optimum of pH 8.5, and were enhanced in the same manner by Triton X-100 and L-cysteine. The maximal velocities of the three reactions for S-adenosyl-L-methionine were 0.04, 1.36 and 0.69nmol/mg protein/min with apparent Michaelis constant values of 3.6, 1.9 and 3.9μM, respectively, indicating that the first-step methylation is rate-limiting for the pathway in the organism.

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