As a part of studies on the mechanism of action of antibiotics polyoxins, effects of various N-aminoacyl derivatives of polyoxin C and other polyoxins on chitin-UDP acetylglucosaminyltransferase (EC 2. 4. 1. 16, chitin synthetase) prepared from phytopathogenic fungus Piricularia oryzae were investigated. It was found that they inhibited the enzyme in competition with the substrate UDP-N-acetylglucosamine. Inhibitor constants, Ki, for these polyoxins were determined and the values of binding affinity, -ΔG_bind of the inhibitors to the enzyme were calculated from the Ki values. In addition, by using these -ΔG_bina values the values of partial binding affinity, -Δg for the several atoms and atomic groups or the several moieties contained in the polyoxin J molecule were estimated. From the results obtained, it was concluded that the carbamoylpolyoxamic acid moiety of polyoxins helps to stabilize the polyoxin-enzyme complex through the contributions of its oxygen atom at C-1'', amino group at C-2'', hydroxyl groups at C-3'' and C-4'', aliphatic carbon chain and terminal carbamoyloxy group.
The results obtained by the kinetic investigation using various nucleotides and nucleotide sugars suggested that there was a specific binding site on the enzyme corresponding to the uridine moiety of UDP-N-acetylglucosamine, and that the pyrimidine nucleoside moiety of polyoxins was also bound to this site.

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Various derivatives of polyoxin C, other polyoxins and several uridine analogues have been known as competitive inhibitors of chitin-UDP acetylglucosaminyltransferase (EC 2. 4. 1. 16, chitin synthetase). Their inhibitory activities were more or less dependent on pH. The variation of inhibitor constants Ki or Michaelis-Menten parameters Km and V with pH was investigated and the data obtained were plotted according to the method proposed by Dixon et al. From the results of the pKi-pH plots for the above competitive inhibitors, it was concluded that the ionized amino group at C-2'' position acted a very important role for the binding of polyoxins to chitin synthetase. The carbonyl oxygen atoms at C-1'' and of the carbamoyloxy group probably participated in the hydrogen bond formation with the enzyme. And pH scarcely influenced on the interaction between the carboxyl group at C-5' and the enzyme. The results of the Dixon plots for variations of Km and V with pH suggested that an un-ionized imidazole group (pK_a=6.3) and an ionized amino group (pK_a=7.7) of chitin synthetase were concerned in the enzyme reaction.

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The conditions for biotin production were investigated. Urea was a more effective nitrogen source than ammonium chloride and ammonium sulfate. About 60% conversion from dl-cis-tetrahydro-2-oxo-4-n-pentyl-thieno-(3, 4-d)-imidazoline (dl-TOPTI) to biotinol and biotin occurred using Corynebacterium sp. B-321. Strain M-6318 which derived from B-321 as a mutant incapable of assimilating n-alkane produced large amounts of dl-biotin from dl-TOPTI. The inability of the microbe to assimilate n-alkane resulted in repression of biotin degradation. Maximum conversion (80%) was obtained by growing cultures of strain M-6318 in the constant presence of n-paraffin.

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The localization of tritium-radioactivity in dwarf kidney bean plants (Phaseolus vulgaris) of ³H-gibberellin A₃(³H-GA₃) applied in a large quantity was investigated in advance of the study on GA₃ metabolism in this plant. Immediately after the application of ³H-GA₃, the radioactivity was distributed uniformly in the top of this plant; no further transportation of the radioactivity into the growing apical region from mature leaves and stems was the observed as the growth stage proceeded. An investigation on the intracellular localization of the radioactivity demonstrated that most part of the radioactivity was found in the cellular soluble fraction, while no radioactivity was detected in such subcellular particles as nuclei, mitochondria and microsomes. Examinations of the occurrence of GA₃ bound with such macromolecules as RNA and protein gave negative results.

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Five radioactive substances were isolated from dwarf kidney bean plants (Phaseolus vulgaris) treated with radioactive gibberellin A₃. These compounds were identified respectively as original gibberellin A₃, 2-O-β-glucosyl-gibberellin A₃ (PIIIA), 2-O-β-glucosyl-isogibberellin A₃ (PII), 2-O-β-glucosyl-gibberellenic acid (PIIIB) and the β-glucoside of an unknown gibberellin-like substance (PI). PI, PII, PIIIA and PIIIB were drived directly from gibberellin A₃; the conversion of PIIIA to PI and PIIIB also occurred. PI, PII, and PIIIB were not further metabolized.

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Active substances which increased RNA content and RNA productivity in yeast culture without affecting the growth rate of yeast were investigated.
The remarkable effect of zinc ion on RNA accumulation was found in flask cultures of Candida utilis.
The active substance of culture of Saccharomyces cerevisiae was isolated from the culture filtrate of Streptomyces sp. S-22 and it was identified as anisomycin, an antiprotozoal and antifungal antibiotic. The effect of anisomycin on the enhancement of yeast RNA formation was shown only with the Saccharomyces genus, which was more sensitive to the antibiotic than other genus. This phenomenon was exhibited only in the case of anisomycin and cycloheximide, whose modes of action were similar among various antibiotics. The ratio of four nucleotides in RNA fraction was almost equal to that of ribosomal RNA.

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The conversion of (+)-carvone by Pseudomonas ovalis, strain 6-1, was investigated.
(+)-Carvone was found to be reduced to (-)-isodihydrocarvone, (-)-isodihydrocarveol, (-)-neoisodihydrocarveol, (-)-dihydrocarvone, (-)-neodihydrocarveol, and (+)-dihydrocarveol, of which the former three were the major products.
From these results, it was postulated that Pseudomonas ovalis, strain 6-1, has different pathways for (+)-carvone and (-)-carvone, respectively; (+)-carvone is converted via (-)-isodihydrocarvone to (-)-isodihydrocarveol and (-)-neoisodihydrocarveol, whereas (-)-carvone is converted via (+)-dihydrocarvone to (-)-dihydrocarveol.
Stereochemical structures of four isomers of dihydrocarveols were also discussed on the basis of PMR results.

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The conversion of (-)-carvone and (+)-carvone by a strain of Aspergillus niger was studied as one of the series of biochemical reduction of terpenes.
(-)-Carvone was found to be reduced essentially to (+)-neodihydrocarveol, although (+)-dihydrocarvone and (+)-isodihydrocarvone were also formed in small amounts, whereas (+)-carvone was converted to (-)-isodihydrocarvone, (-)-isodihydrocarveol, (-)-neoisodi-hydrocarveol, (-)-dihydrocarvone, (-)-neodihydrocarveol, and (+)-dihydrocarveol, of which the former three were the major products.
The metabolic pathways for (-)-carvone and (+)-earvone by the strain of Aspergillus niger are discussed and the results on microbial and chemical reductions of carvone and dihydrocarvone are summarized.

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Urinary peptides were roughly fractionated by combined columns of cation and anion exchange resins, and the peptides eluted from each column were further fractionated by a combination of various ion exchange resins and DEAF-cellulose column chromatography, paper chromatography and other methods. From the fractions adsorbed on cation exchange resin, 13 homogeneous peptides could be isolated, and from the ones adsorbed on anion exchange resin, 8 glycopeptides could be found. Their amino acid compositions were analyzed.
Although some fractions remain univestigated, an outline of the whole aspect of main urinary peptides has been clarified by this study.

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A lytic enzyme was isolated from phage HM 7-induced lysate of Clostridium saccharoperbutylacetonicum, and purified about 200-fold by precipitation with ammonium sulfate, gel filtration with Sephadex G-75 and ampholine isoelectric focusing. The purified lytic enzyme had an apparent homogeneity on disc-electrophoresis, and the character of acidic protein showing isoelectric point at pH 4.0. The molecular weight of lytic enzyme was estimated to be about 100, 000 from the result of SDS-polyacrylamide gel electrophoresis. The optimum pH for the lytic enzyme activity was 6.5. Maximum activity occurred at 30 to 35°C, and at the ionic strength of 0.04M or above. The lytic enzyme activity was stimulated about 140% by 10^-3M EDTA. The lytic enzyme lysed the living cells, but it had a narrow specificity which was restricted to a certain species of Clostridium such as Cl. saccharoperbutylacetonicum, Cl. butyricum, Cl. botulinum, Cl. sporogenes, and Cl. thiaminolyticum.

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The action of Clostridium phage HM 7-induced lytic enzyme on the cell wall peptidoglycan of Clostridium saccharoperbutylacetonicum was investigated. The cell wall peptidoglycan of this strain contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:2.08:0.97:0.92:0.68. It was strongly digested when incubated with the lytic enzyme. This digestion was accompanied by the release of NH₂-terminal L-alanine without a concomitant release of COOH-terminal amino acids and reducing groups. Chromatography of the lytic enzyme digest resulted in only two fractions, each of which was chromatographically homogeneous. One was a polysaccharide consisting of glucosamine and muramic acid in molar ratios 1.00:0.78, and other was a peptide composed of glutamic acid, alanine and diaminopimelic acid in molar ratios of 1.00:2.09:1.05. These results indicate that phage HM 7-induced lytic enzyme is N-acetylmuramyl-L-alanine amidase, which cleaves the linkage between N-acetylmuramic acid and L-alanine.
A possible structure for the cell wall peptidoglycan was also proposed.

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Male rats weighing about 200g were killed after 1, 2, 4, 10, and 20 days on a protein-free diet, and in vitro synthesis of protein was measured by the incorporation of ¹⁴C-glycine into protein of liver slices and isolated soleus muscle. The incorporation value was corrected for the differences in specific radioactivity of intracellular free glycine, and protein and RNA contents of tissue were determined.
Muscle protein synthesis began to decrease from the 1st day of depletion, fell rapidly until the 4th day, and then was reduced gradually to about 30% of the initial control by the 20th day. This reduction was due in a major part to a fall in the rate of protein synthesis per unit of RNA and in a minor part to a decline in RNA content. In the liver, protein synthesis increased in the early period of protein depletion, but declined with prolonged depletion, and was reduced greatly by severe depletion. These alterations were caused mainly by the changes in incorporative activity per unit of RNA.
From these results, it was suggested that different patterns of adaptive response to protein depletion occurred in both cases of early and prolonged depletion in connection with protein metabolism in rats.

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A nuclease was purified about 1500-fold with a recovery of 20% from an aqueous extract of culture of a pigmentless mutant VI-10-14 of Penicillium citrinum on wheat bran. The purified preparation was homogeneous on the basis of the criteria of ultracentrifugation and disc gel electrophoresis. The preparation was essentially free of 5'-nucleotidase, nonspecific phosphomonoesterase, non-specific phosphodiesterase and 3'-monoester forming nuclease. The preparation hydrolyzed phosphodiester bonds in RNA and DNA to yield 5'-mononucleotides, and also the phosphomonoester bond in 2'- and 3'-AMP to yield nucleoside and inorganic phosphate. The enzyme activities toward these substrates were not separated and relative ratio of their specific activities remained constant throughout the purification, suggesting that a single enzyme was responsible for these activities.

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Enzymatic properties of a purified Penicillium nuclease (designated as nuclease P₁) were investigated. The enzyme activities for RNA, heat-denatured DNA, native DNA, 3'-AMP and 2'-AMP showed a great degree of similarity with respect to the following properties: a) Range of stable pH (5_??_8), b) temperature optima (at around 70°C), c) thermostability (about 50% inactivation at 67°C, pH 6.0 for 15min, d) effect of metal ions and SH inhibitors, e) requirement of Zn²⁺, f) protection from the heat-inactivation by albumin and Zn²⁺, g) inactivation on standing in the cold and reactivation on heating, h) sensitivity to protease, and i) competitive relationship between substrates in the enzyme reaction. Moreover, the ratio of enzyme activities in several mutants of Penicillium citrinum was constant. From these results, together with constant ratio of the specific activities throughout purification, it is concluded that a single enzyme might be responsible for both phosphodiesterase and phosphomonoesterase functions.

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The biological and biochemical studies of the effect of tenuazonic acid on plant cells and seedlings were carried out. Tenuazonic acid exhibited a conspicuous stunting effect on the seedling-growth of rice plant, mung bean, radish and turnip, and on the growth of suspension cultured cells of soybean and rice plants. Tenuazonic acid exhibited no effect on the O₂-uptake and the activity of SH-enzyme of the plant, but inhibited the incorporation of ¹⁴C-leucine into the protein fraction and that of ¹⁴C-adenine into nucleic acid fraction of suspension cultured soybean cells as well as these uptake into the cells. And then it has been proved that these incorporation-inhibitions were not merely due to the inhibition of ¹⁴C-leucine and ¹⁴C-adenine uptake into the cells but based on the intrinsic inhibition of protein and nucleic acid syntheses, respectively.

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Selenium dioxide oxidation of methyl α-ionylideneacetate (IIb) in ethanol afforded methyl 1'-and 4'-hydroxy-α-ionylideneacetate (IIIb and IV), methyl 3'-hydroxy-β-ionylideneacetate (V) and crude dihydroxy-ionylideneacetate (VI). The latter was oxidized with active manganese dioxide to give methyl abscisate (Ib). The growth and germination-inhibitory activity of compounds related to abscisic acid on Azuki bean seedlings and some species of seeds were examined.

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The molecular weight of crystalline acetylesterase (AcE) from Sclerotinia libertiana was determined to be 78, 500 by both Archibald and gel filtration methods. The sedimentation coefficient, s°_{20, w}, was determined to be 5.05S. The amino acids and carbohydrate composition of AcE was determined as follows: Lys₁₂ His₁₄ Arg₁₈ Asp₇₆ Thr₅₂ Ser₆₂ Glu₆₀ Pro₄₈ Gly₉₀ Ala₅₇ Cysteine₄ Val₃₆ Met₈ Ile₃₇ Leu₄₂ Tyr₃₈ Phe₃₅ Trp₁₉ (carbohydrate as glucose)₁₆. The amino and carboxyl terminals of AcE were found to be threonine and serine, respectively. Isoelectric focusing electrophoresis with carrier ampholite revealed that AcE has an isoelectric point at around 3.95. Optical rotatory dispersion measurements showed that [α]D, a₀, b₀ and λ_c, were -22, -110, -75 and 234.5nm, respectively, suggesting that the enzyme contains 11% of the α-helix structure.

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Changes in chemical, physicochemical and biological properties of Serratia piscatorum polysaccharide, PLS N-I, by ultrasonic irradiation were investigated. Ultrasonication decreased the viscosity of an aqueous solution of PLS N-I to give two degraded polysaccharide fractions, PLS F-I and PLS F-II, as separated each other by column chromatography on Sepharose 4B. PLS F-I was different from PLS F-II in their physicochemical and biological properties as well as in their chemical compositions.

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Six antifeeding active diterpenes having a clerodane skeleton, clerodin (I), caryoptin (II), dihydroclerodin-I (V), dihydrocaryoptin (VI), clerodin hemiacetal (VII), and caryoptin hemiacetal (VIII), were isolated from Caryopteris divaricata Maxim. Antifeeding activities of six compounds and these derivatives against the 3rd instar larvae of Spodoptera litrrra F. were tested by the leaf disk method. In addition, the terms of “relative antifeedant” and “absolute antifeedant” were proposed for the antifeeding substances, and the latter term was used to the above six diterpenes.

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Four optically active bisnorchrysanthemic acids (dimethylvinylcyclopropanecarboxylic acids) were prepared from chrysanthemic acids, and the toxicities of their allethronyl esters towards houseflies were measured and compared with those of chrysanthemic acids. Some data were obtained on the correlation between chemical structure and biological activity.

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A β-glueoside of D-pantothenic acid was formed from D-pantothenic acid and β-glucosyl donors such as cellobiose, phenyl-β-D-glucoside, salicin, and 4-methylumbelliferyl-β-D-glueoside and naphthol AS-BI-β-D-glucoside by various β-glucosidases, i.e., almond β-glucosidase, cellulase type II and III, naringinase, and hesperiginase. The compound was isolated from a reaction mixture of almond β-glucosidase by treatment with active charcoal, Amberlite CG-50, and DEAE-cellulose column chromatography, paper chromatography, and Sephadex G-10 gel filtration. Then, the compound was characterized as 4'-O-(β-D-glucopyranosyl)-D-pantothenic acid by various analytical methods including bioassay, paper chromatography, NMR and specific optical rotation. The microbiological activities of the compound were also determined.

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The histidine productivity of 1, 2, 4-triazole-3-alanine (TRA)-resistant histidine producer, Corynebacterium glutmnicum KY-10260 was improved by successive additions of such markers as purine, pyrimidine, histidine and tryptophan analog-resistance to the mutant. A selected mutant AT-83, multi-resistant to 6-mercaptoguanine, 8-azaguanine, 4-thiouracil, 6-methylpurine, TRA and 5-methyltryptophan, accumulated twice as much histidine as KY-10260 in the culture medium. The level of histidine production by this mutant reached 15mg/ml or 10% (w/w) of the initial suagr in the medium containing 6% (as glucose concentration) cane molasses and 9% sucrose as carbon sources. 2-Fluoroadenine-resistant mutants produced adenine in addition to histidine.

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A new peptide, which has a marked effect on the growth of Bacillus species, was purified from soybean cake by a procedure employing ammonium sulfate fractionation, and active charcoal-, DEAE-Sephadex A-25- and Sephadex gel-column chromatographies. The purified peptide gave a single band on paper electrophoresis. The peptide possessed the following properties: specific extinction (E^1%_1cm at 280nm), 11.9; N-terminal amino acid, glutamate; molecular weight, 8058; total number of amino acid, 70; chemical composition (%), C, 49.12; H, 6.26; N, 16.44; and S, 2.34; isoelectric point, pH 4.3. The hydrolyzed peptide had no stimulative effect on the growth of Bacillus species. Especially, those microorganisms belong-ing to the genera of Bacillus, Arthrobacter and Xanthomonas were remarkably sensitive to the peptide.

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Bundlins A and B, antibiotics elaborated by Streptomyces sp. 6642 GC₁, have the unique structures which possess a seventeen-membered carbon skeleton fused with an unusual β-keto-δ-laetonie system and a pyruvamide side chain. In the course of the structural studies of the antibiotics, we found that these compounds showed the interesting fragmentations in their mass spectra and in consequence, the investigation about the interpretation of the principal peaks together with their formation mechanism was undertaken by the aid of high resolution mass spectrometry and the measurement of meta stable ions. In addition to the two antibiotics, the mass spectra of related compounds designated T-2636 D and T-2636 F were also investigated.

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