Six angiotensin I-converting enzyme inhibitory peptides were isolated from a bonito bowels autolysate. Their amino acids were sequenced as Tyr-Arg-Pro-Tyr, Gly-His-Phe, Val-Arg-Pro, Ile-Lys-Pro, Leu-Arg-Pro, and Ile-Arg-Pro. Peptides having corresponding amino acid sequences were synthesized by a solid-phase method and their inhibition of the activity measured. IC₅₀ of these peptides were estimated to be 320, 1100, 2.2, 2.5, 1.0, and 1.8μM, respectively. The role of carboxyl terminal proline residues on the inhibition is discussed.

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The wheat glycosphingolipids, O-(β-D-mannopyranosyl-[(1→4)-O-(β-D-mannopyranosyl)]_n-O-(β-D-glucopyranosyl)-(1→1)-(2S, 3S, 4R)-4-hydroxy-N-tetracosanoylsphinganine (n=1 and 2), were stereoselectively synthesized. Silver silicate promoted glycosylation when 4-O-acetyl-2, 3, 6-tri-O-benzyl-α-D-mannopyranosyl bromide was used for elongating the glycan chains, which were latter coupled with the ceramide derivative by the trichloroacetimidate method.

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Pseudomonas paucimobilis UT26 grows on γ-hexachlorocyclohexane (γ-HCH) as a sole source of carbon and energy. Tn5 mutation was introduced into UT26, and two kinds of mutants defective in γ-HCH degradation were phenotypically isolated; one (UT64) completely lacked the activity to degrade γ-HCH, while the other (UT61) retained a very low level of activity. Tagging and sequencing analysis showed that both mutants had a Tn5 insertion at the same site of the linA (γ-HCH dehydrochlorinase encoding) gene. However, UT61 had an additional rearrangement, which could be the cause of its retaining a low level of activity. An in vitro complementation test with a crude extract from UT64 plus partially purified LinA protein showed that LinA was essential not only for the first-step reaction (γ-HCH to γ-pentachlorocyclohexene; γ-PCCH), but also for the second-step reaction (γ-PCCH to compound B) of γ-HCH degradation in UT26.

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We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus. Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome. To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli. By Western blotting analysis of the cell extract of E. coli expressing the polyprotein, mature CP was detected. A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein. These results indicated that the polyprotein expressed in E. coli was cleaved depending on the proteolytic activity of PVY protease.

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In Chinese spring wheat (Triticum aestivum), GA₁, GA₃, GA₈, GA₉, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄, and GA₅₃ were identified in the mature leaves, GA₁, GA₃, GA₈, GA₁₉, GA₂₀, GA₂₉, GA₄₄, and GA₅₃ in the sheath segments, and GA₁, GA₃, GA₁₉, GA₂₀, GA₄₄, and GA₅₃ in the roots of seedlings, based on C₁₈ HPLC, GC-SIM and KRI data. Seven of these GAs were quantified by capillary GC-SIM, using deuterated GA₁, GA₃, GA₈, GA₉, GA₁₉, GA₂₀, and GA₅₃ as internal standards. The concentrations of these GAs tended to be highest in the sheath segments and lowest in the roots. In general, higher concentrations of GA₅₃, GA₁₉, and GA₃ were present in mature leaves, sheath segments, and roots of seedlings grown under vernalizing conditions (low temperature, short photoperiod, and low light intensity), while the concentrations of GA₂₀, GA₁, GA₈, and GA₉ were higher in seedlings grown under non-vernalizing conditions (higher temperature, longer photoperiod, and higher light intensity). The relative concentrations of GA₅₃, GA₁₉, GA₂₀, GA₁, and GA₈ from the two growing conditions lead us to infer that the conversion of GA₁₉ to GA₂₀ (C₂₀-GA to C₁₉-GA) would be a rate-limiting step for the biosynthesis of GA₁, this GA being apparently responsible for shoot elongation in wheat. This rate-limiting step may be associated with a short photoperiod and/or low light intensity.

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A purple nonsulfur bacterium, Rhodopseudomonas sp. No. 7, grew well in a salt medium containing levulinic acid (LA) as a source of carbon and an electron donor. When strain No. 7 was incubated in a medium containing glutamate as a nitrogen source, it continued to evolve hydrogen from 20 mM LA for about 200h with a yield of hydrogen of more than 75%. The consumption of LA by resting cells was strongly dependent on light. The level of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) in cells grown in the LA-medium increased about 2 to 5 times over that of cells grown in LA-free media. However, about 50% inhibition of the enzyme activity in cell-free extracts prepared from these cells was observed in the presence of 5 mM LA regardless of growth conditions. Rhodopseudomonas palustris and Rhodopseudomonas acidophila could use LA for growth like strain No. 7, but Rhodopseudomonas blastica, Rhodospirillum rubrum, Rhodobacter sphaeroides, Rubrivivax gelatinosus, and Rhodocyclus tenuis could not.

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The DNA-breaking and DNA-breakage-inhibiting activities of 23 steroids (bile acids, steroid hormones, neutral sterols, and oxidized cholesterols) were measured in vitro. No compounds examined broke DNA, but some bile acids such as taurocholic, lithocholic, ursodeoxycholic, chenodeoxycholic, and hyocholic acids inhibited DNA breakage by ascorbic acid. Taurocholic acid had the highest inhibiting activity at concentrations above 10 mmol, but its constituents, taurine and cholic acid, had no activity. On the contrary, glycine was an ihibitor although glycine-conjugated bile acids were not effective. Analysis of the structure-activity relationship of bile acids suggested that the H group but not the OH group in the 12-position of the molecule is required for the DNA-breakage-inhibiting activity of non-conjugated bile acid. Among the conjugated bile acids having the OH group in the 7, 12-positions, taurocholic acid had the DNA-breakage-inhibiting activity, but not glycocholic acid, although glycine, but not taurine, was effective.

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The metabolic fate of the non-physiological synthetic dipeptide, sarcosyl-[2-¹⁴C]glycine, was investigated in normal young rats in vivo and in vitro compared to that of seryl-[2-¹⁴C]glycine as a typical example of common physiological dipeptides. The radioactive dipeptides were synthesized from sarcosine or L-serine and [2-¹⁴C]glycine in our laboratory. When radioactive sarcosylglycine was given intraperitoneally, about 30% of the dose was excreted in the urine, and more than 90% of the urinary radioactivity was present in the sarcosylglycine fraction. The recovery of radioactivity in the the expired carbon dioxide and body protein was 8 and 50% of the dose, respectively, during a 24-h period. When the labeled serylglycine was given, the recovery of radioactivity in the urine was 8% of the dose, and 15% in the expired carbon dioxide. In slices of the kidney, liver and small intestine from normal rats, serylglycine was rapidly and almost completely hydrolyzed, and a large amount of free glycine was released. However, sarcosylglycine was hardly hydrolyzed to the corresponding amino acids in the liver and small intestine, and only slightly in the kidney. These results suggest that a considerable amount of sarcosylglycine given intraperitoneally was rapidly excreted into the urine of normal young rats, reflecting less sensitivity to hydrolysis by tissue peptidase(s) when compared to serylglycine.

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An extensive survey for Chlorella viruses was done across Japan and in nine countries of Asia, Europe, North America, and South America. Of 150 fresh-water samples collected from 77 distinct areas in Japan, 29 showed plaques on a lawn of Chlorella sp. (strain NC64A). Typically, 10 to 1000 PFU/ml of viruses were present at sampling sites located throughout Japan. Viruses were also detected in two samples from Brazil and China but not from other countries where water samples were screened. Several representatives of these viruses including ones isolated from Brazil and China were compared with respect to genome size, genome structure, and protein components. It was found that these viruses should belong to the same virus group consisting of large icosahedral particles with linear dsDNA genomes. Physiological and ecological factors affecting virus distribution are discussed.

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The reaction system for the bioconversion of dethiobiotin into biotin by resting cells and protoplasts of a Bacillus sphaericus bioB transformant was established. The reaction mixtures consisted of completely synthetic components, such as amino acids and metal salts. Among the sulfur compounds tested, L-Cys and L-cystine were effective in the biosynthesis of biotin from dethiobiotin both by resting cells and by protoplasts. The optimum concentrations of L-Cys were 2 to 3 mM and more than 0.25 mM for resting cell and protoplast systems, respectively. Vigorous shaking enhanced the biotin biosynthesis by protoplasts. The addition of yeast extract to the reaction mixture without a mixture of amino acids brought about a three-fold increase in the amount of biotin synthesized by protoplasts when compard to the case with the reaction mixture containing the amino acid mixture. The amount of biotin synthesized by protoplasts increased with the incubation time up to 6 h and reached about 2μg/ml. There was a clear correlation between the number of remaining protoplasts and their biotin-biosynthesizing activity during the incubation.

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Levan-assimilating microorganisms obtained from soil samples were screened for levan-degrading enzyme production. Among them, the strain F3-2 was found to produce an extracellular levan-degrading enzyme that hydrolyzed levan to a disaccharide identified as levanbiose. The strain F3-2 was identified as Streptomyces exfoliatus F3-2. This levan-degrading enzyme was specifically induced by levan, and the activity was increased to 2.58 units/ml by improvement of the culture conditions. The optimum temperature and pH for the enzyme reaction were 60°C and 5.5, respectively. The crude levan-degrading enzyme was stable within a pH range of 3.0 to 9.0 and at up to 50°C. Using this crude levan-degrading enzyme, 50 mg/ml of levan was converted to levanbiose in amounts of 42 mg/ml.

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A process was developed to produce a characteristic milk gel. Raw and market milk samples were freeze-concentrated using bacterial ice nuclei. The concentrates were kept at 5°C and compressed at 300-600 MPa for 5 min. The combination of the freeze concentration and the pressurization gave a milk gel without adding any gelling agents. The addition of sugar at 10% to the concentrated milk improved its gel strength and viscoelasticity. The gel was characterized by a phase transition at about 62-75°C.

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Intracellular interaction of the pyridoxal 5'-phosphate (PLP) form of aspartate aminotransferase (AspAT) with vitamin B-6 and antagonists of vitamin B-6 in rabbit erythrocytes was measured in situ. In the erythrocytes, about 75% of the total AspAT was saturated with PLP. On the basis of the concentration of PLP in the erythrocytes, the result showed that about 13% of the total PLP was bound to AspAT in the erythrocytes. The form of the residual approximately 25% of the total AspAT was not identified: the residual AspAT was not converted to the PLP form even when a high amount of PLP was accomulated in the erythrocytes. Neither the PLP-AspAT level nor concentrations of PLP and pyridoxamine 5'-phosphate (PMP) were changed by incubation of the erythrocytes with the rabbit plasma and crude extracts of liver and kidney which were dialyzed or treated with dinitrophenylhydrazine. The modified form of PLP-AspAT with D-cycloserine was converted to PLP-AspAT in the hemolysate but was not in the erythrocyte. In contrast, the modified form of PLP-AspAT with DL-penicillamine was converted to PLP-AspAT both in the hemolysate and the erythrocyte. The concentration of vitamin B-6 compounds in the erythrocytes and the effects of the antagonists on the concentration were also measured after the erythrocytes were incubated with free vitamin B-6 compounds.

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We analyzed mutation points of the biotin operon from biotin-overproducing mutants of Escherichia coli resistant to two biotin analogs, actithiazic acid and 5-(2-thienyl)-valeric acid, by DNA sequencing. The biotin operons cloned from these mutants were classified into three groups. One point mutation, which was a GC→AT change within the operator overlapping the -10 region of the rightward (bioB) promoter, was considered to result in disruption of operator structure and enhancement of promoter activity. Two other point mutations, which were both GC→AT changes just before and after the initiation codon of the bioB gene, were considered to activate the translation efficiency. These mutations significantly accelerated the biotin-forming activity from dethiobiotin in cell-free extracts.

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PI-220 was found in a culture broth of Streptomyces sp. A8059, which had been isolated from a soil sample collected in Okinawa Prefecture, Japan. PI-220 inhibited aggregation of rabbit platelets with an IC₅₀ value of 1.0-2.1×10^-3 M.

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Two types of cell lines, a red-cell predominant line and a yellow-cell predominant one, were selected from secondary callus of rabbiteye blueberry (Vaccinium ashei Reade) leaf, and subcultured in the light and in the dark, respectively. Cultures of the red-cell line produced anthocyanins and the composition was simple as compared with that of the parent plant. The yellow-cell line produced carotenoids which had a visible absorption spectrum similar to that of neochrome. Both cell lines produced proanthocyanidins at the same levels. Proanthocyanidins were detected as red pigment (anthocyanidins) upon incubation of cells with 1% HCl-methanol at 50°C for 18 h. Composition of the anthocyanidins was simple as compared with that of those formed from proanthocyanidins in leaves during incubation. The anthocyanidins showed a retention time very closely related to that of authentic cyanidin by high performance liquid chromatography and comprised about 64% of the total peak area. Amounts of anthocyanins and proanthocyanidins (as anthocyanidins) in cultured cells didn't exceed those in the parent plant.

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In order to trap the biosynthetic intermediate of violacein, a number of mutants were prepared. A novel tryptophan metabolite was produced by the administration of tryptophan to washed whole cells of the mutant of Chromobacterium violaceum. The chemical structure was determined to be 3, 4-di-(3-indolyl)-pyrrole 2, 5-dicarboxylic acid on the bases of ¹H- and ¹³C-NMR spectra, and MS data. This compound was named chromopyrrolic acid, and was also detected in the various metabolites produced by the parent strain, although the amount produced was somewhat smaller. The structure of chromopyrrolic acid was closely related to that of violacein and deoxyviolacein. The pyrrole dicarboxylic acid moiety of chromopyrrolic acid was biosynthesized by condensation reactions of the tryptophan side chains of two tryptophan molecules, as was found in the biosynthesis of violacein. However, the 1, 2-shift of the indole ring that was found in the biosynthesis of violacein did not occur. To confirm whether or not chromopyrrolic acid was a biosynthetic intermediate, a feeding experiment with radiolabeled chromopyrrolic acid was carried out. The incorporation into violacein analogues failed, indicating that chromopyrrolic acid was produced independently of violacein biosynthesis.

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The process conditions concerned with the production of D-β-acetylthioisobutyric acid were investigated. Methyl DL-β-acetylthioisobutyrate as an enzyme substrate was produced from methyl methacrylate in 96.6% yield by allowing 1.0 mol of methyl methacrylate to react with 1.6 mol of thioacetic acid at 80°C for 6 h. Through the study of hydrolytic reaction of methyl DL-β-acetylthioisobutyrate with the cells of Pseudomonas putida MR-2068, it was found that D-β-acetylthioisobutyric acid having more than 98% (e.e.) optical purity was obtained when the reaction was done below pH 7.5 and below 50°C. For example, 10% (w/v) methyl DL-β-acetylthioisobutyrate in the reaction mixture was asymmetrically hydrolyzed by 0.2% (w/v) cells at 45°C at pH 7.0 for 18 h to give DAT having 98.2% (e.e.) optical purity in 49.7% yield. To prevent the decompositon and racemization of D-β-acetylthioisobutyric acid in the purification process, the use of a thin-film distillation apparatus for a continuous distillation was proposed to be an effective purification method.

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Malformin B, produced by Aspergillus niger, was separated into six compounds by HPLC. Their structures were determined by amino acid analyses, and by mass spectral and two-dimensional NMR experiments to be cyclic pentapeptides structurally related to malformin A₁. Both the NMR and MS/MS experiments suggest cyclo-D-cysteinyl-D-cysteinyl-L-amino acid-D-amino acid-L-amino acid as the essential structure of malformins.

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Cyclization mechanisms of sesquiterpenes including cuparene, β-bisabolene, and α-curcumene have been studied by GC/MS analyses of the deuterated sesquiterpenes that were biosynthesized from deuterated mevalonates in callus of Perilla fruterscens Britton var. crispa Decne f. purpurea Makino (akachirimenshiso in Japanese). The labeling patterns of cuparene demonstrate a concurrent mechanism of a 1, 4-hydride shift, and a double 1, 3-hydride shift to form the cyclopentane ring, and the losses of two H-5 atoms and one H-2 atom of mevalonate to form the aromatic ring. It is postulated that a C=C bond in the cyclohexene ring of β-bisabolene might partly migrate to the neighboring C-C bond with the loss of one H-2 atom in mevalonate in its formation. The labeling patterns of deuterated α-curcumene indicate a 1, 2-hydride shift to form the aromatic ring.

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The nutritional and biological efficiency of 3-acetylpyridine, pyridine-3-aldehyde, pyridine-3-methanol, and β-picoline as a substitute for nicotinic acid (pyridine-3-carboxylic acid) was investigated by using weanling rats fed with a nicotinic acid-free, tryptophan-limiting diet containing various amounts of 3-acetylpyridine, pyridine-3-aldehyde, pyridine-3-methanol, or β-picoline. Judging from the growth response, the levels of NAD activity in the blood and liver, the level of total nicotinamide in the liver, and the urinary excretion of such nicotinamide metabolites as N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide, we conclude that 3-acetylpyridine and β-picoline were approximately 1/10 as active as nicotinic acid, and that pyridine-3-aldehyde and pyridine-3-methanol were approximately 1/1 as active.

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Six new 6-{N-[n-(N-alkoxy-N-alkylamino)alkyl]amino}purines were synthesized, and their cytokinin activity was tested by a rice leaf antisenescence bioassay and soybean hypocotyl bioassay. All six compounds were active in both bioassays. The most active compound in this study was 6-{N-[2-(N-methoxy-N-methylamino)ethyl]amino}purine (4a) which had the smallest side chain. In the rice leaf antisenescence bioassay, the activity of 4a was almost comparable to that of BA(6-benzylaminopurine) and somewhat higher than that of kinetin(6-furfurylaminopurine). However, in the soybean hypocotyl bioassay, 4a was about 10 times less active than BA. Unusually, 4a exhibited very high solubility in water.

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Proteases catalyze the coupling of N-protected L-amino acids and C-protected L-amino acids, but the enzymatic synthesis of aminomalonic acid (Ama)-containing peptides has not been reported. We undertook the synthesis of sweet (R)-Ama-(S)-Phe-OMe (800 times sweeter than sucrose) and (R)-Ama-(S)-Phe-OEt (50 times sweeter than sucrose) by coupling Z-(RS)-Ama(OBzl)-OH and H-L-Phe-OR (R, Me or Et) with thermolysin (EC 3.4.24.4) and subsequent hydrogenolysis. Thermolysin catalyzed the synthesis of Z-(R)-Ama(OBzl)-(S)-Phe-OR (R, Me or Et), which is the precursor of sweet (R)-Ama-(S)-Phe-OR (R, Me or Et). The Ama residues of Z-(R or S)-Ama(OBzl)-(S)-Phe-OR (R, Me or Et) and (R or S)-Ama-(S)-Phe-OR (R, Me or Et) were susceptible to facile racemization. Both Z-(R)-Ama(OBzl)-(S)-Phe-OMe and Z-(S)-Ama(OBzl)-(S)-Phe-OMe were sparingly soluble in water. The desired Z-(R)-Ama(OBzl)-(S)-Phe-OMe was more soluble than Z-(S)-Ama(OBzl)-(S)-Phe-OMe; consequently, the precipitated Z-(R)-Ama(OBzl)-(S)-Phe-OMe gradually dissolved in water, and changed to Z-(R, S)-Ama(OBzl)-(S)-Phe-OMe, because of racemization of the Ama residue. The less-soluble Z-(S)-Ama(OBzl)-(S)-Phe-OMe, which is the precursor of non-sweet (S)-Ama-(S)-Phe-OMe, remained after 24 h. On the other hand, Z-(R)-Ama(OBzl)-(S)-Phe-OEt, which is the precursor of sweet (R)-Ama-(S)-Phe-OEt, was less soluble than Z-(S)-Ama(OBzl)-(S)-Phe-OEt, and therefore remained throughout the reaction.

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Two coryneform bacteria, Arthrobacter globiformis IFO 12137 (ATCC 8010) and Brevibacterium helvolum IFO 12073, which have the arginine oxygenase pathway, could utilize L-ornithine, L-citrulline, and D-arginine. The cells of the bacteria grown on these amino acids contained high levels of guanidino-butyrase and induced levels of the enzymes of the preceding steps of the pathway. 4-Guanidinobutyrate induced guanidinobutyrase but failed to induce the other enzymes, indicating that it was the direct inducer of guanidinobutyrase. These amino acids and L-arginine also induced L-arginine : 2-ketoglutarate aminotransferase. 4-Aminobutyrate was formed on incubation of L-citrulline with L-citrulline-grown cells of A. globiformis in the presence of gabaculine; its amount was about 50% of the L-citrulline degraded. The L-arginine-grown cells produced 4-aminobutyrate and urea from L-arginine in the presence of aminooxyacetate or gabaculine; the amount of 4-aminobutyrate was 80% or more of that of the L-arginine degraded. When the oxygenase pathway was blocked with thioglycolate, the degradation of L-arginine and the formation of urea and 4-aminobutyrate were greatly suppressed. These results indicate that these amino acids are degraded via the arginine oxygenase and the arginine aminotransferase pathways and the major route is the former. Agmatine was degraded in these bacteria and induced agmatine deiminase, carbamoylputrescine hydrolase, putrescine oxidase, and aminobutyraldehyde dehydrogenase. None of the enzymes was induced by L-arginine.

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For use in a differential assay of human α-amylases, a variety of 6⁵-S-substituted p-nitrophenyl α-maltopentaoside derivatives (6-54) were systematically synthesized via the key intermediate, p-nitrophenyl O-(2, 3-di-O-acetyl-6-S-acetyl-4-O-benzoyl-6-thio-α-D-glucopyranosyl)-(1→4)-tris[O-(2, 3, 6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)]-2, 3, 6-tri-O-acetyl-α-D-glucopyranoside (4), which was easily prepared from p-nitrophenyl α-maltopentaoside (G5P) in four steps. The sulfoxide and sulfone derivatives were prepared by oxidizing the corresponding sulfides with m-chloroperbenzoic acid.

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When the cells of Escherichia coli K-12 were exposed to a field of direct electric current, a protein with a molecular mass of 23-kDa was detected in an electric shock-induced fluid. The protein was purified by DEAE-cellulose column chromatography to a homogenous state on SDS-polyacrylamide gel electrophoresis. The protein was designated ESP23, an electric shock-induced fluid protein.

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