Agrowastes (rice husks, rice straw and peanut husks) were treated by puff cooking at a pressure of 6 or 12kg/cm² prior to enzymatic hydrolysis. The puff cooking treatment resulted in a decrease in the relative content of hemicellulose and morphological changes. Components such as lignin and sugar were easily extracted with water or methanol from rice husks and rice straw treated at the pressure of 12kg/cm². The amount of lignin removed with NaClO₂ solution increased with increasing pressure applied for puff cooking. The degrees of enzymatic hydrolysis of rice straw and husks were also increased with increasing pressure applied for puff cooking, which simultaneously caused increases in the weight ratios of glucose as well as reducing sugar to the total sugar in the hydrolysates. However, in the case of peanut husks, little or no effect of puff cooking treatment on enzymatic hydrolysis or the degree of extraction of the components with water or methanol was observed.

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Changes to the conformation and functional properties of soy protein caused by mild acid treatment were investigated. When a 2% soy protein solution in 0.05 N HCl was heated for 30min at 95°C, the functional properties of the protein, including its solubility, emulsifying properties and foaming properties, were greatly improved. It was found that the soy protein can be deamidated preferentially without significant cleavage of the peptide bonds under these carefully controlled conditions in dilute acid (0.05 N HCl at 95°C for 30 min). The functional properties of the soy protein were increased greatly with an increase in surface hydrophobicity at an early stage of the mild acid treatment. These results suggest that the improvement in the functional properties of acidmodified soy protein may be mainly due to an increase in surface hydrophobicity induced by deamidation and acid-induced denaturation.

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A cellulolytic enzyme was extensively purified from a commercial crude cellulase preparation from Aspergillus niger by consecutive column chromatography. The purified enzyme was homogeneous on polyacrylamide gel as well as ampholine electrophoresis. The enzyme was an acidic protein with an isoelectric point at pH 3.67. The molecular weight of the enzyme was estimated to be 31, 000 by SDS-polyacrylamide gel electrophoresis. No carbohydrate moiety seemed to be associated with the enzyme protein.
The optimum pH was 4.0, and the optimum temperature of the enzyme was 45-50°C. The enzyme was completely stable over the range of pH 5.0-8.0 at 4°C for 24 hr, and retained about 50% of its original activity after heating at 70°C for 10min. The enzyme was partly inactivated by ImM Ag⁺, Hg²⁺, and Fe²⁺.
The enzyme was characterized as an endocellulase on the basis of its action on carboxymethyl cellulose and cellooligosaccharides. The enzyme split cellopentaose, retaining the β-configuration of the anomeric carbon atoms in the hydrolysis products. The Km value of the enzyme for carboxymethyl cellulose was 0.086%. It was active on carboxymethyl cellulose and cellooligosaccharides (cellotriose to cellohexaose), but not on either cellobiose or p-nitrophenyl β-D-glucoside under the assay conditions used.

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We tried to polymerize D-glucose to cellotriose, the smallest substrate for β-1, 4-glucan synthesis by the β-transglycosylase of Trichoderma longibrachiatum, without participation of high energy compounds such as nucleotide sugars. A commercial β-glucosidase (sweet almond) showed a typical condensation reaction of D-glucose, producing cellobiose when it was entrapped in a visking tube and incubated in 30% D-glucose solution. The reaction was done with immobilized enzyme covalently bound to polyacrylamide beads, and entrapped enzyme. Cellobiose (21.0mg) was obtained from 30 g of D-glucose in a 3-day reaction, where 0.29 unit of the entrapped enzyme preparation was incubated with 100ml of 30% D-glucose at pH 6.0 and 41°C. Gentiobiose was also produced in the mixture as a minor product. The immobilized β-glucosidase (Sumizyme C) preparation covalently bound to polyacrylamide beads could catalyze a transglucosylation reaction to produce cellotriose from cellobiose in a good yield without production of gentiobiose. The transfer reaction was optimal at pH 4.8 and 30°C. Cellotriose (11.2mg) was produced from the reaction mixture containing 68 mg of cellobiose and the enzyme preparation (0.1 unit) after 24-hr of incubation at the optimal conditions. Both immobilized β-glucosidases, sweet almond and Sumizyme C, may be used repeatedly without any loss of the initial activity.

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Three electrophoretically distinct β-glucosidases, β-glucosidase-1, -2 and -3, have been purified from a culture filtrate of Aspergillus aculeatus No. F-50 to homogeneity by ethanol fractionation, DEAE- and SP-Sephadex column chromatography, gel filtration with Sephacryl S-200 and isoelectric focusing. Both β-glucosidase-1 and -2 were potently active not only on salicin but also on insoluble cellooligosaccharide, of which the average degree of polymerization was 20. On the other hand, β-glucosidase-3 was only slightly active on the latter substrate. The molecular weights of β-glucosidase-1, -2 and -3 were estimated to be 133, 000, 132, 000 and 136, 000, respectively, and their isoelectric points to be 4.7, 4.3 and 3.5. All the β-glucosidases were rich in acidic amino acids and glycine, and were glycoproteins.

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The enzymic properties of three electrophoretically distinct β-glucosidases, β-glucosidase-1, -2 and -3, from Aspergillus aculeatus No. F-50 were investigated. β-Glucosidase-3 had a low optimum pH of 3.0, but the other two enzymes had optimum pHs of 4.0-4.5. Both β-glucosidase-1 and -2 were potently active not only on soluble cellooligosaccharides, such as cellotriose to cellohexaose, but also on insoluble cellooligosaccharide, of which the average degree of polymerization was 20. On the contrary, β-glucosidase-3 was only slightly active on the insoluble substrate. The combined use of either β-glucosidase-1 or -2 and endo-glucanase remarkably stimulated the hydrolysis of amorphous cellulose, yielding glucose only. But β-glucosidase-3 did not show such a synergistic effect, and the glucose content of the hydrolyzate was only about 60%.

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Large and small starch granules were isolated and characterized from grains of 6 cultivars of barley, one with waxy and 5 with nonwaxy endosperms, including types with both high protein and high lysine, and high-lysine types. Large granules of a given nonwaxy cultivar contained more amylose than small granules of the cultivar. Large granules of a cultivar contained more long B chains of amylopectin and had the lower ratio of Fr. Ill to Fr. II, which represent one of the structural characteristics of amylopectin, than those of small granules of the cultivar. Small granules had higher conclusion temperature and smaller heat of gelatinization than those of large granules of the cultivar by DSC. Small granules of a nonwaxy cultivar were digested about 4 times or more faster than large granules of the cultivar by Rh. glucoamylase.

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A simple and sensitive assay system was developed in the search for new herbicidal substances. The system is based on the following two phenomena observed in photosynthesis: (1) de novo starch synthesis, determined in excised leaf segments of barnyard millet (Panicum crus-galli), a C₄ plant, and Italian ryegrass (Lolium multiflorum LAM.), a C₃ plant; and (2) oxygen evolution in the cells of Scenedesmus obliquus, detected by using an oxygen electrode. The system can detect photosynthesis-inhibiting herbicides at a concentration as low as 0.1 ppm. After assaying 6, 500 microbial culture broths, 6 culture filtrates were selected for further study.

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Better producers of L-lysine were obtained by derivation of fluoropyruvate(FP)-sensitive mutants from Brevibacterium lactofermentum AJ3990. The coexistence of FP and excess biotin synergistically stimulated L-lysine formation by washed cells. FP inhibited 50% of growth and pyruvate dehydrogenase (PDH) activity of AJ3990 at 0.04 HIM and ImM, respectively. Therefore, the synergistic effect of FP and excess biotin seems to be due to the optimization of the PDH/pyruvate carboxylase activity ratio in L-lysine biosynthesis. This was confirmed by the derivation of FP-sensitive mutants which have the optimal level of PDH activity for L-lysine production. The best producer, AJ11204, had about 27% PDH activity as compared with the parental strain and accumulated 70 g of L-lysine per liter with a conversion yield of 50% from glucose in the presence of excess biotin.

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When brewing barley malt extracts were incubated with malt β-glucans, insoluble materials were formed in the reaction mixture. To investigate the cause of this, we studied various factors that may participate in the formation of these materials. The isolated malt β-glucans were similar to barley β-glucans with the β-(1 → 3) and (1 → 4)-linkages in a molar ratio of 1:2.38, and the molecular weight was 950, 000. Three enzymes were detected and purified from malt by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and isoelectric focusing. One of these enzymes was β-(1 → 4)-D-glucanase (I) with a molecular weight of 40, 000 and an optimum pH of 5.0. The other enzyme was β-(1 → 3), (1 → 4)-D-glucan 4-glucanohydrolase, with a molecular weight of 33, 000 and an optimum pH 5.0. The third enzyme was β-(1 → 4)-D-glucanase (II), with a molecular weight of 49, 000 and an optimum pH of 4.5. Among these three β-glucanases, β-(1 →4)-D-glucanases (I) and (II) had not been identified before in malt, and β-(1 →4)-D-glucanase (II) was most stable on heat treatment and formed most of the precipitates in the reaction mixture.

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In order to examine the relationship between tobacco aroma and the nitrogen-containing compounds in the tobacco headspace volatiles, the volatiles of flue-cured, Burley and Turkish tobaccos collected with Tenax GC were analyzed by a gas chromatograph equipped with a flame thermionic detector. Of the 21 compounds identified in the chromatogram, 2, 5-dimethylpyridine, 2, 4-dimethylpyridine, pyrrole and N-methylpyrrole have not been previously reported as present in the tobacco headspace volatiles. By principal component analysis and stepwise discriminant analysis, three principal components and discriminant functions with four peaks were obtained. The results of these statistical analyses classified the samples into each variety respectively.

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The antifungal activity of 2, 4-dihydroxyacylophenones and related compounds against Trichophyton spp and other fungi were investigated to determine their structure-activity relationships.
The activity of these compounds was found to be closely related to the length of the acyl and alkyl substituents attached to the 1, 3-dihydroxybenzene moiety In addition, differences m activity were observed depending on the position of the alkyl substituents and on the number of substituents attached to the 1, 3-dihydroxybenzene moiety. Some compounds tested showed potent antifungal activity against Trichophyton spp. and other fungi that was more active than amphotencm B.

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An attempt was made to use the disappearance of ovalbumin antigen as an index for the analysis of the process of digestion of ovalbumin in vitro and in vivo. The antigenic determinants of ovalbumin were highly sensitive to conformational changes in the protein. Mild heat-treatment and acid-denaturation abolished spontaneously all the reactivities of antigenic determinants. Experiments on protease digestion in vitro showed that native ovalbumin was hardly digested by pepsin at pH 2.7 or by pancreatin at pH 8.0, while acid- or heat-denatured ovalbumin was more easily digested by these proteases. The results suggested that the denaturation of ovalbumin was a rate-limiting step in its digestion, and the disappearance of ovalbumin antigen was a useful index of the initial process of digestion which preceded the proteolytic hydrolysis.
At 1 hr after the administration of an ovalbumin diet to rats, approximately 20% of the ovalbumin antigen administered was detected in the whole contents of the gastrointestinal tract. In the small intestine, the amount of intact ovalbumin comprised about half of the total protein of the intestinal contents. These results suggested that in the stomach and small intestine, denaturation of ovalbumin was a significant rate-limiting step in its digestion process. But after 2hr, little ovalbumin antigen was detected in the small intestine. This disappearance of the antigen from the small intestine cannot be well explained by the in vitro data.

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Tremerogen A-10 is an S-polyisoprenyl peptide mating pheromone secreted by the AB cells of the heterobasidiomycetous yeast Tremella mesenterica. We investigated the feasbility of the use of compactin (ML 236-B), a potent inhibitor of mevalonate synthesis in mammalian cells, for the study of the mating pheromone production. Compactin specifically inhibited mevalonate synthesis of the yeast cells without affecting protein synthesis. The secretion of tremerogen A-10 was effectively blocked by the drug. Accumulation of a large precursor polypeptide (M.W. 28, 000) for the mature pheromone (M.W. 1, 480) in the membrane fraction of compactin-treated cells was demonstrated by immunoprecipitation of ³⁵S-labeled proteins. The results suggested that the addition of the nonpolar residue to a polypeptide precursor is important for the production of tremerogen A-10, especially in the intracellular transport and processing of the precursor molecules.

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Eighty-seven strains of acetic acid bacteria were surveyed for plasmids by CsCl-ethidium bromide equilibrium centrifugation of cell lysates. Twenty-seven of the 33 strains of Acetobacter were found to harbor plasmid DNA and most strains contained multiple species of low-molecularweight plasmids. On the other hand, plasmid DNAs were detected in 23 of the 36 strains of Gluconobacter and most of them had molecular weights of more than 5 megadaltons. Of the 18 strains newly isolated from a vinegar factory, some of which were examined taxonomically, 17 contained plasmid DNA. The molecular weights of plasmids detected in this study were in the range of about one to over 17 megadaltons. The plasmids with molecular weights of less than 5 megadaltons were characterized with restriction endonucleases. The physical maps of two plasmids designated as pMVlOl and pMV102, which were found in the isolated strains, were constructed.

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β-Phenethyl alcohol (PEA) stimulated the inductive formation of inulin fructotransferase (IFT) in Arthrobacter ureafaciens at the transitional phase between the lag and exponential phases of cell growth. The stimulation by PEA was optimal at a concentration of 8.3 × 10^-3M. The formation of IFT was repressed by addition of actinomycin D, rifampicin and D-glucose in both the extracellular and cell-bound fractions. Cerulenin, procaine and tunicamycin inhibited the formation of IFT in the extracellular fraction. PEA overcame the repression by rifampicin, D-glucose and tunicamycin of IFT formation. On the basis of these results, it may be suggested that PEA acts directly or indirectly at the level of transcription process and in the glycosylation of protein(s) which may affect the IFT formation.

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FL-657B, which induced differentiation of Friend leukemia cells, was isolated from the culture fluid of Streptomyces sioyaensis and identified with trichostatic acid, a hydrolysis product of trichostatiir A and C. FL-657B induced hemoglobin biosynthesis of both dimethyl sulfoxidesensitive and -resistant Friend leukemia cells. FL-657B caused approximately 90% of the cells to be benzidine positive and reduced the growth to approximately 30-70% of the control at 2.42μg/ml. Hemoglobin newly biosynthesized by the induction of FL-657B showed a UV absorption pattern similar to that from the normal mouse.

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Interspecific recombinants have been produced between Streptococcus cremoris H-61 and S. lactis J-1 by polyethylene glycol-induced protoplast fusion. All of the fusants obtained showed mixed physiological properties of the two parents, and possessed plasmids derived from both parents at random. Physiological properties of primary colonies were stably maintained among the progenies after the single-colony isolation procedure. Similarly, in most of the fusants the plasmid profiles of the primary colonies were stably maintained, but one lost 2 out of the 7 plasmid bands. However, there was no indication that plasmids from either one of the parents were preferentially lost. These results showed that interspecific genetic transfer occurred on chromosomal and plasmid DNA on the protoplast fusion and that the fusants obtained were not heterokaryons, but true recombinants.

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The induction and mechanism of fatty liver in the rat by the synthetic carcinogen 2-acetylaminofluorene (AAF) were investigated.
The induction of this fatty liver was dose and time dependent, being gradually increased by the intake of a 0.05% AAF diet for 3 weeks. The AAF dosage was found to increase the activity of drug-metabolizing enzymes (p-nitroanisol demethylase and aniline hydroxylase) and to decrease the activity of pyruvate kinase and α-glycerophosphate dehydrogenase. The AAF dosage had no effect on the incorporation of [1-¹⁴C]acetate into the lipid fraction during in vitro incubation of liver slices. The supplement of adenine to the AAF diet had no effect on the accumulation of liver lipid.
It is suggested from the result of treatment with Triton WR-1339 that a block in the secretion of triglyceride from the liver is a major cause of the induction of fatty liver by AAF.

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Dextransucrases from Leuconostoc mesenteroides NRRL B-1416 and B-1375 strains were purified to electrophoretically homogeneous preparations. After successive column chromatographies, the enzyme fractions were treated with endodextranase, then subjected to preparative polyacrylamide gel electrophoresis. The purified dextransucrase from each strain had a dimeric structure of molecular weight 130, 000-133, 000. Alkaline treatment (pH 10.5) dissociated these dimer forms into the respective monomer forms having molecular weight of 64, 000-68, 000. The two enzymes were closely similar to each other in optimum conditions and thermal and pH stabilities. The purified B-1416 enzyme was activated 4.35-fold by the addition of exogenous dextran (0.5%), while the B-1375 enzyme was activated 2.76-fold. In the absence of exogenous dextran, both enzymes gave 5-10 min lag periods for reaction, which were abolished by the clinical dextran.

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Protoplasts of Pyricularia oryzae P₂ formed a cell wall and eventually reverted to a normal mycelial form in liquid medium. The process of the formation of two main cell-wall components, glucan and chitin, was studied from the onset of regeneration. Analyses using radioactive sugars suggested that chitin synthesis started after a short lag but glucan formation was delayed. Chemical analysis of regenerating cell walls using gas-liquid chromatography indicated clearly that chitin formation precedes glucan formation.

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Subcellular localization of muscle inorganic pyrophosphatase was examined using rabbit skeletal muscle homogenates. The pyrophosphatases were found to be contained in the microsomal, mitochondrial, and cytosol fractions. The microsomal and mitochondrial pyrophosphatases were most likely bound to the respective Subcellular fractions. The pyrophosphatases associated with microsome and mitochondria showed their optimal activities at about pH 5.5 and 7, respectively. They were not dissociated from the particles by washing with salt solution or by ten times freezing-thawing. The activity of microsomal acid pyrophosphatase was not affected by Mg, ²⁺ Ca, ²⁺ or EDTA, but that of the mitochondrial neutral pyrophosphatase was enhanced by the addition of Mg.²⁺ The microsomal acid pyrophosphatase was stable between pH values of 5.5 and 8.5 during storage at 4°C. The activity was inhibited by p-chloromercuribenzoate. The activity was irreversibly inhibited by sodium dodecyl sulfate, but reversibly inhibited by neutral salts and membrane solubilizing detergents such as Triton X-100, octaethylene glycol mono-n-dodecylether, and sodium cholate.

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Inhibitors of angiotensin I-converting enzyme were isolated from an enzymatic hydrolysate of bovine casein. The amino acid sequences of these inhibitors were Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys (CEI₁₂), Phe-Phe-Val-Ala-Pro (CEI₅), and Ala-Val-Pro-Tyr-Pro-Gln-Arg (CEI_β7). CEI₅ is a penta-peptide derived from the hydrolysate of CEI₁₂ with proline-specific endopeptidase, and CEI_β7 is a hepta-peptide derived from β-casein. These inhibitors potentiated bradykinin in the contraction of the uterus and the ileum of rats. The ileum was more sensitive to these inhibitors than the uterus. The bradykinin-potentiating activity of these inhibitors on the ileum lasted for more than 90 min even after washing the organ.

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A soil microorganism, identified as Acinetobacter calcoaceticus KB-2, was cultivated on palm oil as a carbon source for cell production. This organism grew with a specific growth rate of 1.10h^-1. The pH optimum for growth was between 6.5 and 7.0, and the temperature optimum was 39°C. Compared with other strains on water-insoluble substrates such as hydrocarbons and natural oils and fats so far reported, the cultivation time for this strain was short and the cell mass productivity was relatively high. More than 90% of the palm oil was assimilated by this strain, and the overall cell yield was 1.02 (g of cells/g of palm oil) after 8 hr cultivation with the concentration of 3% palm oil.

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Zein, an associate of two heterogeneous subunits, was fractionated into monomer, dimer and polymer (a mixture of the trimer and higher polymers) fractions. Sulfhydryl group analysis showed that almost all cysteine residues of the dimer and the polymer were involved in formation of intermolecular disulfide bonds. In the monomer, however, intramolecular disulfide bonds existed. To clarify in more detail the state of cysteine residues in the monomer, an experiment was carried out using a Thiopropyl-Sepharose 6B column. The possibility was shown that some of the cysteine residues were blocked or substituted. A model was presented to explain the state of cysteine residues in the monomer.

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We studied DNA breakage by phenyl compounds present in foodstuffs in vitro using λADNA and in cultured mammalian cells using RFL and HeLa cells. Strong in vitro activity was detected in o-and p-dihydroxyphenols, but the m-isomer had no activity. The same results were obtained with aminophenols and phenylenediamines. In flavonoids, the 3-OH group seemed to be active in the DNA breakage, in addition to the o-diphenol group. Cellular DNA breakage by the compounds was different from their in vitro activity and varied with the cell lines. RFL cells were preferable to HeLa cells for screening for DNA breaking substances, because of their greater sensitivity.

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The chloramphenicol-resistance (CP^r) plasmid pTZ12 (2.55kb) in Bacillus subtilis was genetically analyzed in detail, and the CP^r determinant and the functional unit of replication were mapped. The plasmids pTZ12 and pBR322 were digested with suitable restriction endonucleases and Hgated with T4 ligase. The ligated DNAs were introduced into E. coli by transformation and CP-resistant transformants were selected. In conclusion, the CP^r determinant was mapped between a TaqI site and a BclI site (about 900 base pairs) on pTZ12. A set of pTZ12-pBR322 recombinant plasmids isolated from E. coli was introduced into B. subtilis by transformation to test for ability to replicate in B. subtilis. From the results, the region of the functional unit of pTZ12 replication was mapped. It was also proved that the gene product of this CP^r determinant was chloramphenicol acetyltransferase (CAT) and the native CAT in the cells carrying pTZ12 was a dimeric protein with two identical subunits having a molecular weight of approximately 24, 000 (24 K).

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An efficient method for the Stereoselective synthesis of 2-amino-2-deoxy-D-arabinose and 2-deoxy-D-ribose is described.
The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2, 3-O-isopropylidene-D-glyceraldehyde with high erytro-selectivity (nearly 100%).
Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-D-arabinose and 2-deoxy-D-ribose.

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New 14-O-derivatives of (-)-indolactam V, which has the basic ring-structure of teleocidins without the monoterpenoid moiety, were prepared and their Epstein-Barr virus early antigen (EBV-EA) inducing activity was tested. A series of 14-O-alkyl derivatives was far less active than (-)-indolactam V, but the 14-O-acyl derivatives showed a high EBV-EA inducing activity. These results suggest that the free hydroxyl group at C-14 plays an important role in inducing the EBV-EA, and that the activity of the acyl derivatives arises through hydrolysis of the ester groups.

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Three non-reducing oligosaccharides were isolated from the fraction of cyclic (1→2)-β-Dglucan of Rhizobium meliloti J7017 by reversed-phase chromatography and paper chromatography. Methylation and ¹H-NMR analyses indicated that they were α-D-glucopyranosyl α-kojitrioside, α-D-glucopyranosyl α-kojitetraoside, and α-D-glucopyranosyl α-kojipentaoside.

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Seeds of grain amaranths contain a high amount (about 60% of total nitrogen) of albumin and globulin and a trace amount of prolamin. From salt-soluble extracts of A. hypochondriacus seeds, a globulin (440, 000 in apparent molecular weight and s⁰₂₀, w= 12-7) was purified by Sepharose 6B gel and DEAE-cellulose column chromatographies. The protein comprised at least four kinds of subunits whose molecular weights were 36, 000, 32, 000, 20, 000 and 18, 000, respectively. The amino acid composition of the globulin was almost similar to those of soybean and oat globulins.

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Cultured insect cells, TN-368 from the cabbage looper, swelled and burst upon treatment with the enzyme-digested delta-endotoxin of Bacillus thuringiensis var. aizawai. The cytotoxic sweelling was depended upon the amount of the delta-endotoxin added and the concentration of NaCl or KCl in the isotonic solution. The concentration of Na⁺ in the swollen cells approximately doubled in isotonic NaCl, while that of K⁺ decreased to 10% of the original cellular concentration. The cell swelling was inhibited by tetrodotoxin and also by ouabain in only KCl isotonic solution. On the other hand, 4-aminopyridine stimulated the swelling in the isotonic KCl solution, These results indicate that the delta-endotoxin induces the stimulation of Na⁺ influx and K⁺ efflux in the isotonic NaCl solution, and also stimulates the Na+, K+-ATPase in the isotonic KCl solution. The cytotoxic swelling was also blocked by cAMP, AMP, ATP, GTP, and NAD, but not by adenosine and GMP. These results suggest the participation of nucleotide derivatives in the action of delta-endotoxin.

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We cloned in E. coli the whole 17 nif genes (nifQ-J) of Klebsiella oxytoca NG13 using pBR322 as a vector, and constructed a recombinant plasmid, pNOW25 (nif⁺, Ap^r, 42.6kb). A non nif DNA fragment was deleted from the plasmid with Xhol, and a smaller plasmid, pNOK31 (nif⁺, Ap^r, 31.1 kb), was reconstructed.
We constructed the restriction map of the cloned nif genes. The map was the same as that of the K. pneumoniae M5al nif genes as to the EcoRI, HindIII, BamHI and Xhol sites, but differed considerably in the Pstl, SalI and BglII sites.
E. coli KO60 containing pNOW25 or pNOK31 can grow on a N-free medium. The acetylene reduction activities of KO60 (pNOW25) and KO60 (pNOK31) were 280nmol and 390nmol/48hr per 7ml of N-free liquid medium, whereas the activity of K. oxytoca NG13 was 3800 nmol. Thus, the expressed activity of the nif system of K. oxytoca is rather low in E. coli even if the nif genes are cloned on a multicopy plasmid.

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Two naturally occurring new sesquiterpene ketones, neoacolamone (1) and 7α-hydroxyneoacolamone (2), were isolated from the roots of Chloranthus serratus (Thunb.) Roem. et Schult. (Japanese name: Futari-shizuka, Chloranthaceae). The structures were elucidated on the basis of their physicochemical properties and chemical reactions. Three sesquiterpene ketones, acoragermacrone (3), acolamone (4) and zederone (5), and four additional sesquiterpenes including lindenanolides were also found in the same source. The relationships of these isolates to the constituents of C. japonicus and to the sesquiterpene biogenesis in the Chloranthus species are discussed.

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(±) Linalol is cyclized into cis and trans linalol oxides by various microorganisms. This reaction, assuming an intermediate epoxidation step, is analogous to the corresponding step proposed for the biosynthesis of ionophorous antibiotics.

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Pyrrolylmagnesium bromide reacted with thiol-and selenoesters in the presence of cuprous iodide to afford 2-acylpyrroles regioselectively.

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