Germination and growth inhibiting activities of surface lipids from 54 Nicotiana species were investigated. Almost half of the extracts were found to have such activities. Among them the surface lipids of N. glutinosa, N. bigelovii, N. sylvestris, N. repanda, N. stocktonii, and N. nesophila were rather strong. Guided by a bioassay using the inhibitory effects on tobacco seed germination and growth, two types of sucrose esters were isolated and identified from the surface lipids of N. glutinosa. The ester positions of each compounds were identified by ¹³C-NMR using deuterium exchange of HO ^←_→ OD. The structures were (2, 3, 4-tri-O-acyl)-α-D-glucopyranosyl)-(3-O-acetyl)-β-D-fructofuranqside (M1) and (2, 3, 4-tri-O-acyl)-α-D-glucopyranosyl-β-D-fructofuranoside (M2). The main fatty acids of M1 and M2 were acetic (only in M1), propionic, 2-methylbutyric, 4-methylpentanoic, 4-methylhexanoic, 5-methylhexanoic, and octanoic acids. These sucrose esters obtained from N. glutinosa inhibited not only tobacco seed germination and growth but also other plants' growth.

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Ten acylnornicotines (mainly N'-(S-hydroxyisotetradecanoy1)nornicotine) and acylanatabine(N'-(3-hydroxyisotetradecanoyl)anatabine) were identified from the surface lipids of Nicotiana species in the section Repandae. Synthetic acylnornicotines (formyl to linolenyl nornicotine, and acylnornicotines containing hydroxylated fatty acids) were tested in a tobacco seed germination assay to study the structure-activity relationships of acylnornicotines. Among acylnornicotines without hydroxy groups, N'-hexanoyl, N'-octanoyl, and N'-decanoylnornicotine had high inhibiting activity. Among hydroxy acyl nornicotines, N'-(4-hydroxyundecanoyl), N'-(4-hydroxydodecanoyl) and N'-(5-hydroxydodecanoyl)nornicotine had high inhibiting activity.

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D-Glucoside 3-dehydrogenase [EC 1.1.99.13], which oxidizes validoxylamine A to the 3-keto derivative, was purified from a soluble fraction of Flavobacterium saccharophilum by ion-exchange chromatography and gel filtration. The purified enzyme had a single protein band on disk gel electrophoresis, and FAD, nonheme iron, and acid-labile sulfide were shown to be the prosthetic group. The molecular weight of the enzyme was 65, 000 by gel filtration with a Sephacryl S-200 column and 66, 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, indicating that the enzyme is a monomer. The optimum pH for 2, 6-dichlorophenolindophenol, phenazine methosulfate, and purified cytochrome c551 reductase activity were 6.0, 8.0-9.0, and 7.0, respectively. D-Glucose and methyl α- and β-D-glucoside were converted to the corresponding 3-keto sugars. D-Glucoside 3-dehydrogenase reduced the membrane-bound cytochrome c₅₅₁ with methyl α-D-glucoside, D-glucose, sucrose, or validoxylamine A. This shows that the membranebound cytochrome c₅₅₁ may be the in vivo hydrogen acceptor of a soluble D-glucoside 3-dehydrogenase.

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Formation of transfer products from soybean arabinogalactan and glycerol by endo-1, 4-β-Dgalactanase from Penicillium citrinum was described. The amount of transfer products depended on the glycerol concentration. About 50% of the galactose residues which could be liberated from the polysaccharide by the enzyme were transferred to glycerol at an acceptor concentration of 2.5% (w/v). Transfer products with various polymerization degrees were accumulated at the beginning of the reaction and then those with higher polymerization degrees were degraded gradually. At a final stage of the reaction, two transfer products in addition to two hydrolysis products (galactose and galactobiose) were mainly accumulated. The two transfer products were isolated and their structures were examined. They were 2-O-β-D-galactosyl glycerol and 0-β-D-galactosyl-(1 → 4)-O-β-D-galactosyl-(1 → 2)glycerol.

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Of 52 algal cultures isolated in the Seto Inland Sea area, one cyanobacterium produced large quantities of H₂. This organisms, isolate 108, was a freshwater, nonheterocystous, ensheathed and filamentous cyanobacterium, and was morphologically identified as a Lyngbya species. The optimum conditions for hydrogen production by it were: pH, 6.5; temperature, 30°C; and light intensity, 1, 000 lux, under fluorescent light. Low concentration of potassium nitrate (0.05 g/1) and yeast extract (0.01%) stimulated its growth and hydrogen production. Of the mineral salts tested, FeSO₄ markedly stimulated the growth of isolate 108. The highest rate of hydrogen production was 124ml/g cells/day. The carbohydrate content of cultures was decreased, by 85%, during hydrogen production.

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L-Arabinose-induced bulge formation in Escherichia coli IFO 3545 was observed in L-ribulose5-phosphate (L-Ru5P) 4-epimerase deficient mutants such as ara-207 and ara-208 as well as the wild type in hypertonic medium with 5% sodium chloride but not in the other L-arabinose-negative mutants. Unlike the case with the wild type strain, bulge formation of ara-207 and ara-208 could be induced with 1% L-arabinose without sodium chloride. These results indicated that L-arabinose must be metabolized to L-Ru5P for induction of bulge cells and the presence of 5% sodium chloride was thought to regulate the activity of L-Ru5P 4-epimerase to cause accumulation of L-Ru5P as an effector molecule for bulge formation. Also, L-arabinose-induced morphological change was suggested to proceed via a mechanism similar to penicillin-induced inhibition of peptidoglycan metabolism.

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Using three kinds of polyesters, polycaprolactone-diol (I), poly(hexamethylene adipate) (II), and a copolyester of I and II, the effects of the molecular weight (M_n) of polyester on their enzymatic hydrolysis were examined. The degrees of hydrolysis of the copolyester by Rhizopus arrhizus and R. delemar lipases were much higher than those of the two homopolymers over a wide range of M_n. On the other hand, the degrees of hydrolysis of the copolyester by Candida cylindracea and hog pancreas lipases and hog liver esterase were between, or near, those of the two homopolyesters.
R. delemar lipase could hydrolyze the polyester moiety of polyurethane which hog pancreas lipase could not attack. However, no difference in the hydrolysis products between R. delemar and hog pancreas lipases could be detected.
The effects of the melting points of polyesters on their hydrolysis by lipases are discussed.

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Glycerol-negative mutants were derived by UV irradiation from a methylotrophic yeast, Candida boidinii No. 2201. The finding that mutant strain SK-71 was defective in glycerol 3-phosphate dehydrogenase confirmed the involvement of the phosphorylative pathway in glycerol utilization in the wild type strain and showed that the extracellular accumulation of glycerol formed from methanol through dihydroxyacetone in the mutant strain was due to repression of further utilization. On methanol-fed cultivation of the mutant strain, 0.6g per liter of glycerol was produced in 7 days.

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Correlation between the activity of dihydroxyacetone reductase (DHAR) and the intracellular content of glycerol in a methylotrophic yeast, Hansenula ofunaensis, was observed when it was grown on methanol. The DHAR activity and the glycerol synthesis were low in the presence of a high concentration of NaCl or KC1. DHAR activity increased with growth under less-aerobic conditions in the methanol medium. The less-aerobic conditions after aerobic cultivation increased the glycerol synthesis. Methanol was the most effective carbon source for DHAR synthesis and glycerol did not repress the synthesis. From these results, it was concluded that DHAR in methanol-grown cells of H. ofunaensis might be responsible for the growth under less-aerobic conditions, but not for that under salt stress conditions or for glycerol synthesis itself.

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A new assay for aminopeptidase P was established by coupling with proline iminopeptidase using Gly-Pro-chromogen (e.g. Gly-Pro-β-naphthylamide, Gly-Pro-p-nitroanilide, or .Gly-Pro-4-methyl coumarin amide) as the substrate. With each substrate, a linear relationship was established between the enzyme amounts and color development or fluorescence due to the chromogen released. This assay method did not suffer from interference by materials in culture broth. By using this assay method, aminopeptidase P was partially purified from Escherichia coli HB101 by chromatographies on DEAE-Sephadex and high performance liquid chromatography (HPLC). On the chromatogram with a DEAE-Sephadex column, two peaks of aminopeptidase P were observed and were named APP-I and APP-II. APP-I was further purified by HPLC using DEAE-5PW and Phenyl-5PW columns. Optimum pHs of APP-I and APP-II were 8.0 and 9.0, respectively. In contrast to APP-I which was stable around pH 10, APP-II was stable at pH 8 to 9. After incubation for 30min at pH 8.0, fifty percent of the remaining activity of APP-I and APP-II were observed at 60°C and 50°C. APP-I and APP-II were activated 3-fold by the addition of 5 and 30μM Mn²⁺. They were inhibited by EDTA, and reactivated by adding Mn²⁺. The molecular weights of APP-I and APP-II were 350, 000 and 210, 000, respectively. Each enzymes released the ammo terminal amino acid when proline is at the penultimate position. The velocity of hydrolysis by the enzymes was not significantly different for most X-Pro bonds (X=amino acid) of peptides except for Pro-Pro bond. APP-II hydrolyzed penta-(Pro-Pro-Gly) at a much higher rate than APP-I, suggesting the aminopeptidase P reported by Yaron and Mlynar (BBRC, 32, 658 (1968)) to be APP-II.

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Four water-soluble arsenic compounds were isolated from the brown alga S. divaricata by a combination of chromatographic methods. They were all identified by mainly ¹H NMR spectroscopy to be arsenic-containing ribofuranosides; 2, 3-dihydroxypropyl 5-deoxy-5-(dimethylarsinoyl)-β-ribofuranoside (1a), 2-amino-3-[5-deoxy-5-(dimethylarsinoyl)-β-ribofuranosyloyl]propane-1-β-ribofuranoside sulfonic acid (1b), 1-'glycerophosphoryr'-2-hydroxy-3-[5-deoxy-5-(dimethylarsinoyl)-β-ribofuranosyloxy]propane (1c), and 3-[5-deoxy-5-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropane1-sulfonic acid (1d). These compounds (1a-d) accounted for 75% of the arsenic in the algal extract.

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Deamidation of lysozyme was observed during storage in a buffer solution and in egg white. The peak corresponding to native lysozyme from Bio-Rex 70 column chromatography was gradually decreased, while the peaks corresponding to deamidated lysozyme were increased during storage in 0.1M carbonate buffer at pH 9.5. A similar change was observed during storage in egg white, but the change in egg white was larger than that in the buffer solution. A detailed analysis of the elution peaks from the Bio-Rex 70 column suggested that one to three residues of amide in lysozyme were mainly deamidated during storage in the buffer solution, and that more than three residues in lysozyme were deamidated during storage in egg white. There were significant differences in lysozyme activity between native and deamidated lysozyme, the activity being decreased in proportion to the degree of deamidation.

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The respiratory chain system and respiration-linked proton translocation sites (coupling sites) of Brevibacterium lactofermentum were studied. The H⁺/O ratio of the bacterium oxidizing endogenous substrates was approximately 4g ions proton translocated per g atom oxygen consumed. The measurement of absorption spectra showed that the bacterium contained A-, B-, and C-type cytochromes. The H⁺/O ratios of cells loaded with specific substrates suggested that protons were translocated at the site 1 (NADH-Q) and the site 2 (Q-Cyt. c). Cytochrome oxidase (site 3) of the bacterium seems not to pump protons, although this bacterium has an oxidase of the cytochrome aa₃-type.

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A new breeding technique for Aspergillus awamori var. kawachi and Aspergillus oryzae involving protoplast fusion was studied to obtain more useful interspecific hybrids for making spirits for drinking. A yield of protoplasts from mycelia of these fungi was obtained using a Trichoderma viride BIA lytic enzyme system. Interspecific hybridization between these fungi was brought about by protoplast fusion, in the presence of 35% polyethylene glycol as a fusogen. A strain obtained from the heterokaryon using d-camphor showed high stability and numerous sporulation, producing the same levels of amylase and citric acid as those of the original strains. The strain also showed a 2-fold DNA content per nucleus and about 1.5-fold conidial diameter compared to those of the original strains. The new strain was assumed to be a heterozygous diploid. Some strains considered to be recombinants were spontaneously obtained at low frequency from the heterozygous diploids. The existence of a parasexual cycle in these fungi was also discovered using protoplast fusion.

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After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet for 12 days, 70mg of streptozotocin/kg body weight (STZ group) or 70mg of Streptozotocin and 500mg of nicotinamide/kg body weight (STZ-Nam group) was injected intraperitoneally into the rats. The rats were kept for 21 more days on the 20% casein diet and killed by decapitation. Urine was collected for the last 2 days. The level of blood glucose was 2-fold higher in the STZ group than in the STZ-Nam group. Urinary excretion of large amounts of glucose was observed only in the STZ group. Extremely reduction of urinary excretion of nicotinamide was observed in the STZ group, but, urinary excretion of N¹-methylnicotinamide (MNA) and N-¹-methyl-2-pyridone-Scarboxamide (2-py) was about the same in the two groups and that of N¹-methyl-4-pyridone-3-carboxamide (4-py) was higher in the STZ group than in the STZ-Nam group. The sum of urinary excretion of nicotinamide, MNA, 2-py, and 4-py was higher in the STZ group than in the STZ-Nam group. The levels of NAD in liver, pancreas, and blood in the STZ group tended to be higher, or rather not to decrease compared to the STZ-Nam group. For enzyme activities concerned with the tryptophan-NAD metabolism, a marked increase was observed in the activities of aminocarboxymuconate-semialdehyde decarboxylase, 3-hydroxyanthranilic acid oxygenase, and nicotinamide methyltransferase, on the other hand, the activity of NAD⁺ synthetase decreased in the STZ group compared to the STZ-Nam group. The activities of tryptophan oxygenase, kynureninase, NMN adenylyltransferase, and MNA oxidase were about the same in the two groups. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to NAD is lower in the Streptozotocin diabetic rats than normal rats, but the tryptophan metabolites such as NAD and 4-py were higher in the STZ group than in the STZ-Nam group. This might be due to the higher food intake and the lower body weight gain in the STZ group than in the STZ-Nam group. Similar phenomena have reported in alloxan diabetic rats.

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Candida species YK 11 and YK 92 and Geotrichum candidum YK 57, which were isolated as nitrite-resistants, converted nitrite in the culture medium to nitrate stoichiometrically during growth. The nitrite-oxidizing reaction was confirmed under aerobic conditions in the intact cell system with 15mM nitrite, 150mM glucose, and 100mM Tris-HCl buffer (pH 7.0). Glucose or other carbohydrate which supported the microbial growth was indispensable for the reaction. The rate of oxidation (0.9-1.3 × 10⁵ μg-N/g of YK 92 cells day) and the maximum amounts of nitrate formed in the culture medium (200mM, 2800 μg-N/ml) were much larger than those of other heterotrophic nitrifiers and almost the same as those of Nitrobacter.
The nitrite-oxidizing activity was demonstrated in many types of yeast species.

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Linoleic acid, and its hydroperoxides and secondary autoxidation products were orally administered to rats (400mg/rat). Their effects on hepatic lipid metabolism were examined. Linoleic acid reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase. It decreased the CoASH level and caused the accumulation of long-chain acyl-CoA. Hydroperoxides changed the compositions of unsaturated fatty acids in the hepatic lipids and lowered the content of neutral lipids. Secondary products stimulated carnitine palmitoyltransferase and decreased the content of neutral lipids. They reduced the activities of de novo synthesis of fatty acids and acetyl-CoA carboxylase, and the levels of CoASH and acetyl-CoA. Thus, the effect of secondary products was apparently different from those of linoleic acid and its hydroperoxides.

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The reactive species for inactivating the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by copper salt in the presence of an ascorbic acid were investigated. Strain KUIN-1, ascorbic acid and copper salt were allowed to react for 3hr at 18°C, the ice-nucleating activity of this strain being markedly inactivated in the ascorbic acid-Cu(II) ion system. The scavengers for hydrogen peroxide prevented inactivation of the ice-nucleating activity of strain KUIN-1 by the ascorbic acid-Cu(II) ion system. The ice-nucleating activity of strain KUIN-1 was inactivated in a hydrogen peroxide-generating system, but not in a hydroxyl radical-generating system. The superoxide anion radical had a small effect on inactivating the ice-nucleating activity of strain KUIN-1. The degree of freezing injury to corn treated with the ascorbic acid-Cu(II) ion system and strain KUIN-1 was lower than that of corn treated with only strain KUIN-1.

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Versatile plasmids for use of a host-vector system with the yeast, Saccharomyces cerevisiae, were constructed. They contain the ARS1-TRP1 region of YRp7 as the replication origin and a selectable marker, the STB region of 2 μ circle for the plasmid mitotic stability, and a multiple cloning sequence for cloning foreign DN A. They do not contain the "poison sequence" of pBR322. These plasmids are very easy to handle because of their small size and mitotic stability in yeast cells.

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Benzyl 2-benzamido-2, 5-dideoxy-α-D-ribofuranoside was synthesized via benzyl 2-amino-3-C-(branched-chain)-2, 3, 5-trideoxy-α-D-furanoside from an optically active 3-oxa-6-azabicyclo[3.2.0]heptan-7-one derivative.

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We found that the expression vector previously constructed using the promoter of the GAL7 gene of Saccharomyces cerevisiae lacked the transcription terminator. The addition of the GAL7 terminator resulted in a more than 3-fold increase in the production of the protein encoded by cDNA of early region la (Ela) of human adenovirus, without affecting the amount of its mRNA. The positive regulatory gene, GAL4, when amplified with a vector of a high copy number, caused an about 5-fold increase in Ela production. We also found that medium containing a mixture of galactose and glucose can be used for the efficient production of the Ela protein.

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A novel synthesis of the enone 12 starting from (+)-dihydrocarvone (3) and its transformation into (+)-7-hydroxycostal (1) are described. The ketone 10, obtained from 4 through a four-step sequence was converted to 12 by acid-catalyzed elimination and subsequent regioselective hydrogenation. Alternatively, the methoxyhydroperoxide 13 generated by the ozonolysis of 4 was subjected to the Criegee rearrangement, providing a mixture of 10 and 14, which on acid treatment, gave 11. Transformation of 12 into 19 was accomplished via a five-step reaction sequence. The reaction of 19 with the lithium alkoxide of 2-lithio-2-propenol afforded (+)-7-hydroxycostol (2), those oxidation with manganese dioxide gave rise to (+)-7-hydroxycostal (1).

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New functionalized 2-oxabicyclo[2, 2, 1]heptane derivatives were synthesized for their potential biological activities by way of the intramolecular ether bond formation of plinol C that was obtained from linalool by a thermal ene reaction. The mechanism for the ether-forming reactions is discussed.

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2-Mono- and 2, 3-diacetate derivatives of (22R, 23R)- and (22S, 23S)-24-epibrassinolides, and (22R, 23R)- and (22S, 23S)-2-deoxy-24-epibrassinolides were synthesized. They were found to possess weak biological activity in a rice-lamina inclination test.

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The effects of an extract of Daisaikoto (a traditional Chinese medicine) on biliary constituents was studied in mice fed with a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate (the control diet) and in rats fed with a cholesterol-free diet. The Daisaikoto extract was added to the control diet at a level of 0.5%. A high incidence of cholesterol gallstones were found in the control mice, but not in the mice given the Daisaikoto extract. This difference was concluded to have been due to the absolute concentration of bile acid in the bile being significantly higher in the mice given the Daisaikoto extract than in the control mice. The result from rats fed with the cholesterol-free diet also demonstrated that the Daisaikoto extract caused an increase in the absolute concentration of bile acid in the bile.

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The effects of feeding with a histidine-excess diet and subsequent starvation on liver and muscle glycogen, and on serum glucose were investigated in young and adult rats.
Feeding with a histidine-excess diet resulted in the accumulation of liver glycogen in both young and adult rats. The hepatic glycogen continued to decrease during starvation, and the liver became almost totally depleted of glycogen after starvation for 48 hr. Glycogen in the liver of young rats starved for 24 hr after previous feeding with a histidine-excess diet was significantly higher than that of young rats starved for 24 hr after previous feeding with a basal diet.
Muscle glycogen after feeding and subsequent starvation was not affected by the types of diets fed previously, muscle glycogen during starvation showing a slight decrease in young rats and a slight increase in adult rats.
Feeding with a histidine-excess diet caused a significant decrease of serum glucose in young rats, but not in adult rats. Serum glucose in young rats was markedly reduced by starvation after previous feeding with a basal diet, but not after previous feeding with a histidine-excess diet. In adult rats, there were no changes in serum glucose between rats starved after feeding with either a basal diet or a histidine-excess diet, and serum glucose was decreased slightly by starvation after feeding with the test diets.
The overall results indicate that the maintenance of serum glucose in young rate even during starvation after previous feeding with a histidine-excess diet might be partially concerned with the export of glucose from the accumulated glycogen in the liver due to the diet.

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The stereochemistry of (+)-(2Z, 4E)-trans-r, 4'-dihydroxy-γ-ionylideneacetic acid, a major metabolite from Cercospora cruenta, a fungus found to produce (+)-abscisic acid, was reexamined as to its ¹H-¹H-Cosy and Noesy 2D-NMR spectra, and it was proved to have a chair conformation with an axial pentadienoate moiety. Further, the metabolism of (+)-[¹⁴C]-1'4'-dihydroxy-γ-ionylideneacetic acid in tomato plants suggested the possibility of it being a biosynthetic intermediate of ABA in plants.

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