The cultural conditions for the production of bactobolin, an antitumor antibiotic produced by Pseudomonas yoshitomiensis, were studied using a mutant strain, C-17-20. The following cultural conditions were found to be suitable: 2.0% mannitol, 0.25% dried yeast, 0.5% (NH₄)₂SO₄, 0.4% KCl, 0.02% K₂HPO₄ and 0.8% CaCO₃; temperature, 25°C. The highest bactobolin production under the conditions was 189μg/ml, although the wild strain, Y-12278, produced only 4μg/ml in the basal conditions.

View full abstract

The reduction of 2, 6-dichlorophenolindophenol (DCIP) by aromatic reductones was studied in the range of pH 2.3 to 8.6, using stopped-flow apparatus. The apparent first-order rate constant, k_app, and the second-order rate constant, k, of the reaction were determined. The k-pH profile was considerably different from that of L-ascorbic acid. The logk of DCIP reduction in a solution of 50%ethanol was found to depend linearly on Hammett's σ_p value.

View full abstract

β-Amylase produced by Bacillus cereus BQ10-S1 Spo II was purified by salting out with ammoniumsulfate, and column chromatography on Sephadex G-100 and CM-SephadexC-50. The purified enzyme was homogeneouson disc electrophoresis and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enzymeshowed a single band suggesting no subunit structure. The sedimentation coefficient was 4.8S_{20, w} and the molecular weight was estimated to be 6.0×10⁴ by SDS-PAGE, 6.2×10⁴ by gel filtration in the presence of6m guanidine HCl, 6.25×10⁴ by sedimentation equilibrium and 5.5×10⁴ by amino acid analysis, respectively. The Kmvalue for soluble starch was 0.4%. The enzyme was remarkably inhibited by 5×10^-9m PCMBbut not by DTNBat the same concentration. Only one sulfhydryl group was detected by amino acid analysis, by Elluman's and Riordan's methods. The isoelectric point of the purified enzyme was 8.3.

View full abstract

The glucomannan isolated from larch holocellulose was hydrolyzed by a purified endo-D-β-mannanase. The products were fractionated by gel filtration on a polyacrylamide gel in water and partition chromatography on ion exchange resins in 80%ethanol. The following oligosaccharides were isolated and identified: (a) 4-O-β-D-Manp-D-Man, (b) 4-O-β-D-Glcp-D-Man, (c) 4-O-β-d-Glcp-D-Glc, (d) O-β-D-Manp-(1→4)-O-β-D-Manp-(1→4)-D-Man, (e) O-β-D-Glcp(1→4)-O-β-D-Manp-(1→4)-D-Man, (f) O-β-D-Manp-(1→4)-O-β-D-Glcp-(1→4)-D-Man, (g) O-β-D-Manp-(1→4)-O-[α-D-Ganp-(1→6)]-D-Man, (h) O-β-D-Manp-(1→4)-O-β-D-Manp-(1→4)-O-β-D-Manp-(1→4)-D-Man, and (i) O-β-D-Manp-(1→4)-O-β-D-Manp-(1→4)-O-β-D-Manp-(1→4)-D-Man.

View full abstract

Extracellular endoxylanase (1, 4-β-D-xylan xylanohydrolase [EC 3.2.1.8]) produced by Bacillus pumilus IPO, an isolate from soil in Thailand, was purified to homogeneity. The optimum pH and temperature of the purified xylanase for hydrolysis of larchwood xylan were 6.5 and 40°C. Its molecular weight was estimated to be 24, 000 dalton by SDS-polyacrylamide gel electrophoresis and 20, 000 dalton by equilibrium centrifugation. The number of amino acid residues per molecule was calculated to be 185, including one cysteine.
The maximumdegree of hydrolysis of larchwood xylan by the purified xylanase was 25%; xylobiose, xylotriose, xylotetraose and xylopentaose but not xylose were detected as end-products. trans-Xylosidase activity was indicated by the formation of xylobiose and xylotetraose from xylotriose and of xylopentaose and hexaose in addition to biose and triose from xylotetraose. Quantification of hydrolysis products indicated that the enzyme's greatest affinity was for the 2nd to 6th β-xylosidic linkages from the terminal of larchwood xylan.

View full abstract

Taxonomiccharacteristics of a strain of thermophilic acidophilic bacillus, Bacillus sp. 11-1S, which had the ability to produce thermophilic acidophilic amylase and thermostable xylanase were examined. Cells of the organism were aerobic, heterotrophic, Gram-positive, spore-forming rods. It grew at temperatures between 45 and 70°C (optimum 65°C) in media of pHs ranging from 2.0 to 5.0 (optimum 3.5-4.0). Physiological and biochemical characteristics were identical with those of Bacillus acidocaldarius, and % GC of DNA(59%) was close to that of the latter (61-62%). From these results it was concluded that the organism belongs to B. acidocaldarius Darland and Brock.

View full abstract

Pyruvate was proved to be utilized as a sole carbon substrate for vitamin B6 (B6) biosynthesis in an intact cell system. Glucose, fructose, glycerol, oxalacetate, succinate and α-ketoglutarate also increased the rate of the biosynthesis. Possible participation of inducible enzymes to convert glycerol into triose phosphates was shown when glycerol was used as the substrate. On the other hand, no inducible enzyme was required to synthesize B6 from pyruvate. Induction of phosphoenolpyruvate synthase, which leads pyruvate into the gluconeogenetic pathway, scarcely enhanced the potency of pyruvate. These results indicate that the biosynthetic pathway probably branches from pyruvate and/or acetyl-CoA on the main metabolic pathway.

View full abstract

Cuticular membranes (ca. 0.04 mg/cm²) were isolated from fresh and flue-cured tobacco leaves (Nicotiana tabacum) as clear, colorless and thin films by oxalic ammoniumtreatment and successive cellulase-pectinase digestion. GC-MS analyses, after hydrogenolysis and trimethylsilylation, revealed that the membranes are mainly built up of 10, 16-dihydroxy hexadecanoic acid in a polyester network structure which survives postharvest-treatments.
The fruit skins of solanaceaes; sweet pepper (Capsicum annuum), eggplant (Solarium melongena) and tomato (Solarium lycopersicum), have similar membranes which are essentially composed of 9, 16- and 10, 16-dihydroxy hexadecanoic acid.

View full abstract

L-Glutamate was taken up and concentrated into washed cells of Brevibacterium flavum, a glutamate-producing bacterium. Potassium ions specifically promoted the uptake rate. The uptake activity in the presence of K⁺ was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone but not by N, N'-dicyclohexylcarbodiimide, NaN₃, ouabain, valinomycin or gramicidin. The activity was specifically and competitively inhibited by L-aspartate among L-amino acids but not so strongly by other structural analogues including D-glutamate. L-Aspartate uptake was also competitively inhibited by L-glutamate. Michaelis constants for glutamate and aspartate were 19 and 25jum, respectively. The glutamate uptake activity of the cells increased whenNH₄⁺ in the culture medium was reduced. Five out of six mutants which were unable to grow on glutamate and produced more glutamate than the parent showeda lower maximumrate of uptake than the parent.

View full abstract

New devices for resolution of DL-phenylalanine by an enzymatic method have been developed by using ammonium N-acetyl-DL-phenylalanate as a substrate. In this procedure, crystals of lphenylalanine and ammonium N-acetyl-D-phenylalanate are separated alternately or simultaneously from reaction mixtures containing acylase, as first crops. The whole resulting solution including acylase can be reused. Ammoniumacetate formed as a by-product was found to inhibit the enzyme.

View full abstract

Callus tissues capable of producing anthocyanin pigments in the dark were induced from grape vines and cultured both in static and suspension conditions. Six kinds of pigments were extracted from the callus and suspension-cultured cells, and three of these pigments were identified as peonidin-3-monoglucoside, cyanidin-3-monoglucoside and peonidin-3, 5-diglucoside.

View full abstract

The ethanolic reaction mixture of dehydroascorbic acid (DHA)and an amino acid contained a significant quantity of a yellow product. During the reaction, this yellow product was found to appear only after the formation of scorbamic acid (SCA) and the red pigment. The reaction of SCA with DHAor with the red pigment (the oxidized form of bis(2-deoxy-2-L-ascorbyl)amine) also produced the same yellow product. Its formation by the DHA-aminoacid reaction was supposed to be the direct result of the above SCA-red pigment reaction. The isolated yellow product was examined mainly by spectroscopy, and a possible structure (1) containing one molecular portion each of hydrated DHA, cyclic DHAand AsAresidues, and two nitrogen atoms was proposed. The yellow product is probably a condensation and oxidation product of the reaction between SCAand the red pigment. The formation of the yellow product is probably a key intermediate in this kind of browning reaction. This supposition was as also supported by the highest degree of browning shown by the yellow product among other intermediates.

View full abstract

Bacillus sphaericus produced larger amounts of biotin when its precursor was added to the mediumafter 24-hr cultivation than when it was added before cultivation. Actithiazic acid (ACM)-and/or 5-(2-thienyl)-valeric acid (TVA)-resistant mutants were induced from B. sphaericus by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. ACM-and TVA-resistant mutant, AB12, excreted 7. 1 times more biotin than the wild type strain. The TVA-resistant mutant, A 16, excreted 1 1.9 times more total biotin than the wild type strain. The reaction conditions for biotin production using resting cells of strain AB12 were investigated. After 48-hr reaction, strain AB12 accumulated 9.5μg/ml of biotin under the optimum conditions.

View full abstract

Phenacylation of 5-aryl-3-mercapto-1, 2, 4-triazoles (I) furnished 5-aryl-3-phenacylthio-1, 2, 4-triazoles (II) which reacted with CS₂ and aryl isothiocyanates to give 5-aryl-1, 2, 4-triazolo[3, 4-c]-1, 2, 4-dithiazole-3-thiones (III) and 5-aryl-3-arylimino-l, 2, 4-triazolo[3, 4-c]-1, 2, 4-dithiazoles (IV), respectively. (IV) on refluxing with CS₂ yielded (III) which, when heated with aryl isothiocyanates, regenerated (IV). Compounds (II)-(IV) were compared with Dithane M-45 for their fungitoxicity against Helminthosporium oryzae and Fusarium oxysporium. The screening results have been correlated with the structural features of the tested compounds.

View full abstract

A computerized system that we designed allowed the fast tracing of the pedigree of cultured Euphorbia millii cells. Weobtained two distinct outputs for which the parentage of the cells were printed. These outputs were obtained by two distinct search routines; one traced the pedigree from ancestry, and the other traced the pedigree from progeny. These outputs proved that pedigree tracing is useful for genetic studies of repetitive selection of cultured E. millii cells that produce high levels of anthocyanin.

View full abstract

The effects of pressure on thermal denaturation and in vitro fibril formation of acid soluble collagen from calf skin were studied with various solvent conditions under high pressure of up to 4, 000 atm. For all the solvent systems, the thermal denaturation was depressed by pressure, e.g., the apparent denaturation temperature at pH 4.0 increased from 37.5°C at 1 atm to 44.5°C at 1, 400 atm. The pressure-enhanced thermal stability of collagen indicates that the volume change due to the helix-to-coil transition of this protein is positive in water. The fibril formation in both the nucleation and growth phases was extensively retarded by pressure in a relatively low region of up to 500 atm, indicating the positive activation volume of this fibril formation process. These results seemedto support a hypothesis that the protein-water interaction plays a dominant role in the structural stability of the triple helix and fibrils of collagen.

View full abstract

A series of mutants of Brevibacterium ammoniagenes selected for increased productivity of 5'-inosinic acid (5'-IMP) after successive mutational works were analyzed for the level of sensitivity toa variety of drugs. It was found that the series of mutants exhibited variations in sensitivity tovarious drugs such as antibiotics, detergents, dyes and lysozyme. The most notable alterations werefound in a Mn-insensitive mutant, KYI3171, and a guanine auxotrophic mutant, KYI3184, compared to each parent mutant. Furthermore, it was found that increased 5'-IMP productivity was always accompanied by increased sensitivity to deoxycholate and lysozyme, and that both the increased level of 5'-IMP production and increased degrees of sensitivity to these two drugs in all steps of mutation were not so drastic, but progressive.

View full abstract

The Brdicka current (polarographic catalytic hydrogen evolution current produced by proteins in the presence of cobalt salt) of human immunogloblin G (IgG) and sheep antihuman IgG antiserum (anti-IgG) was studied by the d.c. and differential pulse polarographic techniques. The Brdicka current for a mixture ofIgG and anti-IgG was smaller than the sumof the currents for IgG and anti-IgG. This difference was attributed to the complex formation between IgG and anti-IgG. Anti-IgG could be titrated with IgG (or vice versa) by polarographic techniques based on the Brdicka current. The average intrinsic dissociation constant of the IgG-anti-IgG complexes was estimated from the titration curve. The polarographic methodbased on the Brdicka current and coupled with immunoreaction is useful to determine trace amounts of antigen (or antibody) down to 10^-10 m using the differential pulse polarographic technique.

View full abstract

Rats starved for 48 hr were refed on a high carbohydrate diet and killed at various intervals (3-24hr) in order to examine structural changes in liver glycogen. Glycogen was isolated by the HgCl₂ method and thereafter treated with dithizone-chloroform to eliminate HgCl₂. The extracted glycogen revealed typical α-particles besides β-particles but the KOH-extracted glycogen showed only β-particles. The tissue glycogen level began to increase after refeeding and reached a maximum at 18 hr. However, the iodine adsorption capacity of the HgCl₂-extracted glycogen (but not the KOH-extracted one) increased by 30% in 6 hr-refed rats compared with control rats fed ad libitum, and then decreased to the control level. In the HgCl₂-extracted samples, changes in glycogen S_{20, w} after refeeding were coincident with the changes in the iodine adsorption capacity and β-amylolysis limit. Onthe other hand, these structural changes were not observed in the KOH-extracted samples.
These findings supported that the HgCl₂ method is useful for isolating native glycogen and suggested that the liver glycogen structure can be temporarily altered so as to have a high molecular-weight together with high iodine affinity and long outer chains.

View full abstract

Carotenoids and ubiquinone in aerobically grown cells of Erythrobacter species OCh1 14 were identified, which contained a considerable amount of bacteriochlorophyll α mediating light-driven electron flows. Spheroidenone was found to be the major component of carotenoids. Also small amounts of 2, 2'-diketospirilloxanthin and OH-spheroidenone were found. Since symmetrical ζ-carotene (together with phytoene and phytofluene) was accumulated in diphenylamine-inhibited cultures, these 3 keto carotenoids of the alternative spirilloxanthin-series are considered to be synthesized mainly via symmetrical ζ-carotene and not through asymmetrical ζ-carotene which is knownto be the intermediate in the biosynthesis of carotenoids in somespecies of non-sulfur purple bacteria. Onthe other hand, the major (or the sole) respiratory quinone of this strain was found to be ubiquinone-10.

View full abstract

The survival curves ofconidia of A. versicolor isolated from animal feeds were sigmoidal both in the wet and dry states. The D₁₀ values and induction doses of three isolates were 37 and 17-18 krad in the wet condition, and 50-51 and 25-48 krad in the.dry state, respectively. From these results, it was indicated that A. versicolor could be sterilized below 0.7 Mrad. The radiosensitivity of other osmophilic Aspergillus isolated from animal feeds was similar to those of A. versicolor.
Two of the three isolates of A. versicolor produced the known carcinogen sterigmatocystin. The amount of sterigmatocystin produced on rice was 410 and 280fig for the strains Ml3 and C132, respectively, while it was 730 μg for the typical strain MYA-0056.Pure sterigmatocystin was stable to irradiation in the dry state. There was a linear relationship between the destruction of sterigmatocystin and the irradiation dose, and 52 Mrad irradiation was necessary for complete destruction. Consequently, A. versicolor in animal feeds must be sterilized prior to its producing sterigmatocystin.

View full abstract

Both the enantiomers of 3-hydroxy-4, 5-dimethyl-2(5H)-furanone (sotolon) were synthesized from the enantiomers of tartaric acid. Both of them were shownto exhibit the same sugary flavor and insect attractancy as those of (±)-sotolon.

View full abstract

Two new sesquiterpene alcohols, 1 and 2, were isolated from a culture filtrate of Aspergillus oryzae. The structures of these compoundswere deduced on the basis of their spectral evidence and synthesis of one of them.

View full abstract

The gene (cya) responsible for adenylate cyclase activity of E. coli was cloned onto pBR322 and the hybrid plasmid carrying the cya gene was designated as PCAK-1 1. The hybrid plasmid was 6.0 Mdin size and contained a 3.3Md HindIII fragment derived from E. coli strain CA7902R chromosomal DNA. In various E. coli hosts, the hybrid plasmid coded for high adenylate cyclase activity. E. coli cells dosed with PCAK-11 accumulated a large amount of cAMPin both cells and broth. Especially marked accumulation of CAMPwas found in a strain deficient in CAMP-receptor protein (CRP).

View full abstract

A mutant blocked in L-proline hydroxylation was derived from a neoviridogriseins producing organism, Streptomyces griseoviridus P8648. This mutant, G-89, did not produce trans-4-hydroxy-L-proline which is the direct precursor of cis-4-hydroxy-D-proline in viridogrisein. Consequently, the organism selectively produced neoviridogrisein II in which the imino acid position is occupied by D-proline.

View full abstract

The incorporation reaction of [³H-methyl]deoxyribothymidine triphosphate (³H-dTTP) into the TCA-insoluble fraction by isolated nuclei of Saccharomyces cerevisiae was characterized as follows : (i) addition of three deoxyribonucleotides (dATP, dGTP and dCTP) is required for maximal incorporation, (ii) the buoyant density of the incorporated products is approximately 1.70, (iii) the incorporation reaction is strongly inhibited by inhibitors of DNA synthesis, (iv) 98% of radio-activities in the products are recovered in the DNA fraction, (v) the products are DNasesensitive and appear as long fibers at a high frequency, and (vi) the increase in accumulation of small-fragment DNA is time-dependent during the first 3-min incubation, followed by a rapid increase in large-fragment DNAs.Thus, it was concluded that the isolated yeast nuclei have an ability to synthesize or replicate nuclear DNA in vitro. Theability of isolated nuclei was strongly stimulated by cytoplasmic factor(s) in the cellular-soluble fraction prepared from mid-log cells of S. cerevisiae.

View full abstract

A photo-bromination of l, 6-anhydro-2, 3, 4-tri-O-benzoyl-β-D-glucopyranose and its [6, 6-²H₂]-derivative gave the corresponding (6S) 6-bromocompounds stereospecifically. They were transformed into (6S)-D-glucose-6-²H and (6R)-D-glucose-6-²H respectively.

View full abstract

Selenium-independent glutathione peroxidase was purified from a cell-free extract of Mucor hiemalis by ammoniumsulfate fractionation, column chromatographies on DEAE-Sephadex and hydroxylapatite, and gel filtration on Bio-Gel P-100. The purified enzyme was homogeneous on ultracentrifugation. The enzyme had a molecular weight of 45, 000 and an isoelectric point of 5.2. The enzyme could reduce cumenehydroperoxide and r-butyl hydroperoxide, but could not reduce hydrogen peroxide. The enzymewas highly specific for glutathione as a hydrogen donor. Mucor glutathione peroxidase was proved to be different from mammalian selenium-dependent glutathione peroxidase I and selenium-independent glutathione peroxidase II in some physicochemical and enzymatic properties.

View full abstract

From the myceliumof Guignardia laricina, four pigments and three other compoundswere isolated and their structures were determined to be 6-ethyl-5-hydroxy-2, 7-dimethoxy-l, 4-naphthoquinone (I), 6-(l-ethoxyethyl)-5-hydroxy-2, 7-dimethoxy-l, 4-naphthoquinone (II), 6-(l-hydroxyethyl)-5-hydroxy-2, 7-dimethoxy-1, 4-naphthoquinone (III), 6-ethyl-1-acetonyl-1, 5-dihydroxy-2, 7-dimethoxy-4-naphthalenone (IV), ergosterol peroxide (V), 9, 1 1-dehydroergosterol peroxide (VI) and isoevernin aldehyde (VII). II, III and IV were new compounds and VII was found for the first time in nature. In the lettuce seedling test, II and III showed remarkable phytotoxicity. V, VI and VII were less active and I and IV were inactive at the concentration tested. Furthermore, III showed strong activity not only in a larch leaf spot test, but also in the antimicrobial assay.

View full abstract