The NaCl concentration of the growth medium affected hydrogen production by Lyngbya sp. (No. 108) strain. Cells grown in medium containing 3% NaCl produced the most hydrogen. The carbohydrate content of this strain also increased with increasing NaCl concentration of the growth medium up to 720 μg/mg cells at 5% NaCl. In the presence of 20 μmol/ml MFA (monofluoroacetic acid), inhibition of hydrogen production was observed. We extracted the glycogen from this nonheterocystous filamentous cyanobacterium, Lyngbya sp. (No. 108), and observed that glycogen and carbohydrate consumption of this strain is coincident with hydrogen production.
These results led us to the conclusion that the reserve glycogen or other carbohydrate were used as sources of electron donors for hydrogen production, and that the NaCl concentration of the medium affected the hydrogen production by this strain.

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The uranium-adsorbing abilities of 60 species of commercially available crude drugs were investigated. The abilities to adsorb uranium from non-saline water differ with different species of crude drugs. High uranium-adsorbing abilities were observed in Corni fructus, Geranii herba and Chaenomelis fructus. With the aim of improving handling and adsorbing characteristics, several crude drugs were immobilized with formaldehyde. Most of immobilized crude drugs tested adsorbed large amounts of uranium from non-saline water. The selective adsorption of heavy metal ions (Cd²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, Zn²⁺, and UO²₂⁺), differs extremely with different species of crude drugs. Most of the crude drugs adsorbed uranyl and copper ions far more readily than other metal ions. Thus, crude drugs of plant origin, such as Corni fmctus, Geranii herba, and Chaenomelis fructus, could be used for the recovery and removal of uranium from seawater, and waste and mine water containing uranium.

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Three additional structural gene loci (Acp, EstB-1 and Pgm, encoding the enzymes acid phosphatase, esterase B and phosphoglucomutase, respectively) were identified in commercial and research-maintained lines (155) of Agancus bispoms. The addition of these new marker loci effectively increases the total number of biochemical loci now available in A. bispoms by 60%. Allozyme variability at all eight structural gene loci (Aat, Acp, Adh, EstB-1, Gpt, Pep-1, Pep-2 and Pgm) was used to classify the lines into only 37 genotypic classes out of more than 3 million possible. A recalculated UPGM (unweighted pair-group method) cluster analysis, based on coefficients of similarity for all lines examined, is presented. The limited genetic diversity in this germplasm collection, as evidenced by linkage disequilibrium between loci and a lack of fit to Hardy-Weinberg expectations, commercially remains untapped.

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It was found that TRG promoted the growth of duckweed Lemna paucicostata 151 in a wide range of concentrations and the effect was the strongest at 20 μM, at which the growth rate was 1.8 times that of the control. The biosynthesis and metabolism of TRG in L. paucicostata 151 was investigated in relation to the plant growth-promoting and flower-inducing activities of the compound to obtain fundamental information on plant growth regulation. Properties of the TRG synthesizing enzyme, nicotinate methyltransferase, were elucidated. Various potent inhibitors to the enzyme were found: TRG (product inhibition), benzoic acid, salicylic acid, picolinic acid, isonicotinic acid, and pyrazinamide. It should be noted that all these compounds except TRG are flowering inducers of the plant. Some relationship between the metabolism of TRG and plant growth regulation is postulated.

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The protoplast fusion technique has been applied successfully to Aspergillus usamii mut. shirousamii and Aspergillus niger, leading to the recovery of recombinant progeny. A strain obtained from a heterokaryon using d-camphor showed a 2-fold DNA content per conidium and an about 1.2-fold conidial diameter compared to those of the original strains, and was regarded as a heterozygous diploids. The diploid strain was treated with benomyl as a haploidizing agent, many segregants being obtained. The segregants were phenotypically single and double auxotrophs, and prototrophs. The prototrophic segregants were selected and their haploidy was confirmed by their conidial sizes and DNA contents per conidium. A prototrophic segregant strain derived from these haploids was considered phenotypically to be a recombinant with some excellent characteristics, that is, higher amylase and citric acid productivities than those of the original strains. The results indicate that the segregants of diploid strains induced by benomyl cause genetic changes affecting enzyme and citric acid production.

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Neurospora sp. ATCC 46892 grew on pregelatinized polished rice, even as a sole carbon source, and simultaneously produced a strong fruity odor. The main components of the aroma were ethyl caproate, ethanol and isoamyl alcohol, as judged on gas chromatography. The effects of carbon or nitrogen sources and impregnating nutritive solutions on ethyl caproate production were examined, and the optimum conditions for the ester production were determined, under which more ethyl caproate, which is responsible for the fruity odor, and less isoamyl alcohol were produced than in a liquid culture. The cell-free extract of the solid state culture (which we call N-koji) contained a strong alcohol acyltransferase activity, which caused abundant ethyl caproate production in the culture. The odoriferous N-koji was applied to the making of sake, a Japanese alcoholic beverage, and as a result a sake rich in ethyl caproate was obtained. It was suggested that the solid-state culture was one of the useful methods for fruity odor formation.

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A therapeutically) important fibrinolytic enzyme, urokinase, has been modified with fragmented human serum albumin (mol wt., 1000-7000) by reaction with glutaraldehyde as a coupling agent. The chemically modified enzyme was separated by gel filtration with Sephadex G-150 into products which underwent conjugation with albumin fragments to various extents. The major modification product, with a molecular weight of about 70, 000, and retaining 40 % of the original urokinase activity, was significantly more resistant to inactivation by protease inhibitors in rat plasma than the unmodified enzyme. It also showed an in vivo half-life ten times longer than the native one during circulation in blood. The rabbit antiserum raised against the modified urokinase reacted not only with the antigen but also with both the unmodified enzyme and the human serum albumin. The modified enzyme did not elicit specific antibodies in an immunized rabbit, and the enzyme that had been modified excessively with albumin fragments lost the antigenicity of urokinase. These results indicate that the chemical modification with albumin fragments is useful for improvement of in vitro and in vivo stabilities of chemotherapeutic enzymes.

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Gluconobacter suboxydam contains a large amount of cytochrome c in addition of cytochrome o in the respiratory chain of the membrane. The content of cytochrome c was found to be influenced by the extracellular pH in growth which is lowered by oxidation products of several sugars or sugar alcohols in the growth medium. Lowering of the external pH caused an increase in cytochrome c content concomitantly with an increase in alcohol dehydrogenase activity, while neither cytochrome o content nor glucose dehydrogenase activity is increased. The increase in cytochrome c content was also accompanied by an increase in KCN-insensitivity for the respiratory activity. The change in cytochrome c content appeared to be mainly due to the change of a cytochrome c corresponding to the second subunit of alcohol dehydrogenase. Furthermore, the extracellular pH of G. suboxydans also affected a respiration-driven proton translocation; the H⁺ /O ratio of the cells grown at lower pH was much lower than the value, about 2, of the cells grown at higher pH. In the presence of KCN, however, both the cells grown at high and low pHs could not generate a membrane potential. Thus, this study indicates that G. suboxydans produces a KCN-insensitive and non-energy-generating respiratory bypass, which may be related to a cytochrome c associated with alcohol dehydrogenase, concomitant with a decrease of the extracellular pH during growth.

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An aerobic, methane-utilizing bacterium (methanotroph, strain M) was isolated that degraded trichloroethylene (TCE) in a pure culture at a high concentration, 10ppm. Strain M used methane or methanol as the sole carbon and energy source for growth, and no growth occurred on nutrient broth or glucose. The strain exhibited intracv toplasmic membranes along the cell periphery, and the taxonomical properties indicated that strain M was a new methanotroph belonging to obligate type II. In addition, strain M was able to degrade not only TCE, but also halogenated alkenes (i.e., 1, 1-dichloroethylene, both cis- and trans-1, 2-dichloroethylene, and 1, 2-dibromoethylene) and alkanes (i.e., 1, 1, 2-trichloroethane, 1, 2-dichloroethane and chloroform), but not tetrachloroethylene, 1, 1, 1-trichloroethane, carbon tetrachloride or aromatic compounds.

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Yeast alcohol dehydrogenase (ADH), diaphorase (DI) and NAD were co-immobilized on Sepharose that had been repeatedly modified with hexamethylenediamine and glutaraldehyde. The activity and re-usability of the gel were investigated with changing the immobilization conditions of the enzymes and the reaction conditions of the glutaraldehyde used in the modification. The results suggested that the immobilization temperature and the immobilization time of the enzymes mainly had an effect on the stability and activity, respectively. The degree of polymerization of the glutaraldehyde affected both the activity and the re-usability, and A₂₃₅/A₂₈₀ was used as an index of the degree of polymerization. The optimum conditions were as follows: a temperature and reaction time in the immobilization of the enzymes of 20°C and 7 hr, respectively, and a degree of polymerization of the glutaraldehyde used in the modification of A₂₃₅/A₂₈₀ = 20. The gel prepared under these optimum conditions was applied to a flow injection analytical system for ethanol. A good linear relationship between the concentration and the response was observed in the range of 20-80 HIM, suggesting that the gel would be applicable to ethanol analysis.

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The cDNA sequence coding for tuna growth hormone (tGH) was placed under the control of the repressible acid phosphatase (PHO5) promoter of a yeast, Saccharomyces cerevisiae, in an expression plasmid, pAM82. The yeast cells transformed with the plasmid synthesized tGH only when the cDNA was attached to the vector through a synthetic oligonucleotide linker having a similar sequence to the 5'-flanking region of the PHO5 structural region. The amount of tGH produced in yeast cells accounted for more than 3% of the total cellular protein and the product was immunologically identified as tGH by Western blotting using polyclonal antibodies specific to tGH.

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Rubusoside derivatives by transgalactosylation of various β-galactosidases were isolated and their structures were analyzed. Escherichia coli β-galactosidase produced mainly 13-O-β-D-glucosyl-19-O-[β-D-galactosyl-(1→6)-β-D-glucosyl]-steviol (RGal-2). Bacillus circulans β-galactosidase produced mainly 13-O-β-D-glucosyl-19-O-[β-D-galactosyl-(1→4)-β-D-glucosyl]-steviol (RGal-la) in the early stage of the reaction and then produced 13-O-[β-D-galactosyl-(1→4)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol (RGal-1b). With decreasing the amount of these products (RGal-la and RGal-1b), RGal-2 was produced.

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Incubation of rubusoside with raffinose in the presence of various α-galactosidases yielded three galactosylated rubusosides. Mortierella vinacea α-galactosidase transferred a galactosyl residue preferentially to the C6-hydroxyl group of the glucosyl residue at the 13-hydroxyl group of rubusoside to produce mainly 13-O-α-D-galactosyl-(1→6)-β-D-glucosyl-19-O-β-D-glucosyl-steviol. α-Galactosidases from Absidia veflexa, Escherichia coli, and coffee beans transferred a galactosyl residue to the C6-hydroxyl group of both glucosyl residues at the 13-hydroxyl and 19-carboxyl groups of rubusoside, yielding the above galactosylated rubusoside and 13-O-β-D-glucosyl-19-O-α-D-galactosyl-(1→6)-β-D-glucosyl-steviol.

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High hydrostatic pressure of 4000 to 10, 000 kg/cm² was applied to fresh egg white and yolk and the resulting gels were analyzed with their texture, protease susceptibility, and nutrients.
Egg white and yolk set to a stiff gel at or above 6000 and 4000 kg/cm², respectively. Both gels were softer and more elastic than heat-induced gels. Taste and flavor of the pressure-induced gels were natural without a cooked taste and flavor. Subtilisin digestibility of the pressure-induced gels were compatible or superior to the heat-induced gels. In the former gels, no destruction of vitamins or amino acid residues and no formation of unusual compounds such as lysinoalanine were detected.
Based on these results, it is proposed that high pressure is useful in food processing and preservation to avoid adverse effects on natural foods.

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The tryptophan synthase genes, trpA and trpB, of Bacillus stearothermophilus IFO13737 were cloned by transformation of tryptophan auxotrophic mutations of the trp genes into Escherichia coli. The genes are located in the order of trpB and trpA, according to their coding orientation, in a 2.5 kb EcoRV-HindIII DNA fragment. The complete nucleotide sequence of this DNA was determined. The trpA and trpB genes consist of 810bp (269 amino acid residues) and 1215bp (404 amino acid residues), respectively. The 5'-proximal portion of the trpB gene was found to overlap 20 nucleotides of the upstream coding region of the trpA gene. The homology of the amino acid sequences of the trp gene products of trpA and trpB of B. stearothermophilus is 35 and 50%, respectively, to those of E. coli, and 55 and 70%, respectively, to those of B. subtilis.

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Oryzacystatin, a proteinaceous cysteine proteinase inhibitor (cystatin) from rice seeds and probably the first well-defined cystatin superfamily member of plant origin, was immunologically investigated for its occurrence in rice seeds during maturation and germination. The enzyme-linked immunosorbent assay (ELISA) using anti-oryzacystatin Immunoglobulin G showed that all the investigated 23 cultivars of rice, Oryza sativa L. japonica, contained Oryzacystatin at 1-4 mg% in their seeds. Particularly, Oryzacystatin levels were high in precocious cultivars and low in sticky rice cultivars. The use of the ELISA method for the representative rice cultivar, Nipponbare, gave the result that in the seed maturation process, Oryzacystatin was synthesized in precedence to total seed protein. In the germination process, Oryzacystatin tended to decrease in accordance with degradation of total seed protein.

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The amino acid sequence of Indian peafowl egg-white lysozyme has been identified. The reduced and carboxy methylated lysozyme was digested with trypsin followed by purification of the resulting peptides by reverse-phase HPLC. The tryptic peptides obtained were sequenced using the DABITC/PITC double coupling manual sequencing method. The alignment of the tryptic peptides were deduced by comparison with corresponding peptides of hen egg-white lysozyme. This protein proved to consist of 129 amino acid residues, and a relative molecular mass of 14423 Da was calculated. Amino acid sequence comparison of peafowl lysozyme and other phasianoid bird lysozymes revealed a maximum homology ratio of 98% with turkey lysozyme.

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An alkalopsychrotrophic strain, Micrococcus sp. 207, inducihly and extracellularly produced amylase and pullulanase. The main hydrolysis product from amylose, with a crude enzyme preparation, was maltotetraose. The optimum temperature for activity of the amylase was 60°C and that for pullulanase 55°C. The activities at 0 to 30°C exhibited similar activation energy values. In an optimized production medium at pH 9.7, the highest yields of these enzymes were obtained after cell growth at 18°C for 4 days. At pH 8.5, the yields of amylase and pullulanase became maximum after 3 days cultivation. With more prolonged cultivation, the yield of amylase but not that of pullulanase activity decreased. These enzymes were not produced at temperatures above 30°C. Sucrose was not effective as an inducer, but it stimulated cell growth and enhanced the enzyme productivities with soluble starch.

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We have isolated four independent genomic DNA fragments encoding a rice storage protein, glutelin, by using its cDNA as a probe. Restriction mapping and sequencing analyses showed that all four clones encode type II mRNA of glutelin and have very similar nucleotide sequences. However, several differences were found among the four clones with respect to the nucleotide sequences of their coding and S'-upstream regions, some of which confirmed corresponding changes in the amino acid sequences of the encoded proteins. These results indicate that the genes encoding type II mRNA species of glutelin constitute a multi-gene family of closely related members.

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A new main tea constituent, 2-O-(β-arabinopyranosyl)-myo-inositol (1, ca. 0.8 % of a Japanese green tea) was characterized by ¹³C-NMR spectroscopy. The inositol glycoside (1) has been found not only in all the tea extracts so far examined but also in the homogenate of Camellia sinensis var. sinensis ("Yabukita cultivar"), suggesting its physiological importance in carbohydrate metabolism in the tea plant.

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Chemical modification of histidine residues in ricin E was studied with regard to saccharide binding. The analytical data indicate that 6 out of 7 histidine residues in ricin E are eventually modified with diethylpyrocarbonate (DEP) at pH 6.0 and 25°C in the absence of specific saccharides. Modification of histidine residues greatly decreased the cytoagglutinating activity of ricin E, and only 10% of the residual activity was found after modification of 6 histidine residues/mol. The data of affinity chromatography using lactamyl- and galactosamine-cellulofine columns suggest that modification of histidine residues does not have much effect on the binding ability at the low affinity saccharide-binding site of ricin E but abolishes the binding ability at the high affinity saccharidebinding site. In the presence of lactose, one histidine residue/mol was protected from the DEP modification with retention of a fairly high cytoagglutinating activity. Such a protective effect was also observed for specific saccharides such as galactose and N-acetylgalactosamine, but not for glucose, a non-specific saccharide. On treatment with hydroxylamine, the modified ricin E restored 67% of the cytoagglutinating activity. Based on these findings, it is suggested that in the high affinity saccharidebinding site of ricin E there exists one histidine residue responsible for saccharide binding.

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We screened the immunoglobulin production stimulating factor (IPSF) in foodstuffs, using human-human hybridoma HB4C5 cells cultured in serum-free ITES-ERDF medium, and found that egg yolk lipoprotein (YLP), lactoferrin, Block Ace, and casein had IPSF activity. The maximum IPSF activity was obtained at concentrations over 100μg/ml in YLP, 10μg/ml in lactoferrin, and 25μg/ml in Block Ace and casein. These IPSFs stimulated the IgM production of human-human and mouse-mouse hybridomas, but their effect on IgG producers was very small. This suggests that IgG production of hybridomas is regulated differently from their IgM production.

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The eleven strains of conidium-forming yeasts classified in the basidiomycetous anamorphic genera Sterigmatomyces, Kurtzmanomyces, Tsuchiyaea and Fellomyces, and the teleomorphic genus Sterigmatosporidium were examined as to the partial sequences of 18S rRNA and 26S rRNA. The positions determined (in Saccharomyces cerevisiae) were 1451 through 1618 of 18S rRNA, and 1618 through 1835 and 470 through 626 of 26S rRNA. The partial sequence determination of positions 470 through 626 of 26S rRNA indicated that T. wingfieldii should be included in the group comprised of Fellomyces and Sterigmatosporidium species. However, the genera Sterigmatomyces, Kurtzmanomyces, Tsuchiyaea and Fellomyces constituted their own separate clusters as to the partial sequences of positions 1451 through 1618 of 18S rRNA and 1618 through 1835 of 26S rRNA. The classification of the conidium-forming yeasts in the four basidiomycetous anamorphic genera mentioned above was proved to be reasonable from the phylogenetic point of view.

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Pseudomonas putida ATCC 12633 and some other fluorescent Pseudomonas strains utilized 1, 4-diguanidinobutane (arcaine) and its homologues with 3-7 methylene groups as sole nitrogen sources. The P. putida strain produced a novel enzyme which hydrolyzed 1, 4-diguanidinobutane to agmatine and urea; diguanidinobutane amidinohydrolase (EC class 3.5.3.) was proposed as the name for this enzyme. This enzyme hydrolyzed diguanidinoalkanes with 3-10 methylene groups; higher reaction rates were observed with 1, 4-diguanidinobutane, 1, 5-diguanidinopentane, and 1, 6-diguanidinohexane. The enzyme was also active toward agmatine, although the rate was below 1% of that toward 1, 4-diguanidinobutane, and it was suggested to catalyze the hydrolysis of the higher homologues of agmatine at higher rates.
The enzyme was induced by α, ω-diguanidinoalkanes with 3-7 methylene groups. The substrate specificities of the enzymes individually induced by the diguanidinoalkanes with 3-7 methylene groups were very close to each other. It was then concluded that only one enzyme was produced to catalyze the initial step of the degradation of the diguanidinoalkanes commonly.

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Two crystalline minor products (14 and 15) were newly obtained by the oxidation of β-caryophyllene (1) with lead tetraacetate. The structures were determined as 4, 4-dimethyltricyclo-[6.3.2.0^{2, 5}]tridec-8-en-1-ol (14) and 4, 4-dimethyl-9-oxatetracyclo[6.4.2.0^{2, 5}.0^{8, 10}]tridecan-1-ol (15) by HR-MS, LR-MS, IR, ¹H-NMR, ¹³C-NMR, and single crystal X-ray analysis data. Products 14 and 15 are novel sesquiterpenes.

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Sulfonamide-resistant mutant strains were derived from a mutant strain, K₃-15, of Flavobacterium sp. 238-7 to improve the menaquinone (MK)-4 productivity. A mutant strain, SP0736, with resistance to 50 μg/l of sulfapyridine in a chemically-defined medium, had improved productivity of MK-4. By optimization of the culture conditions, the mutant strain SP0736 produced 280 mg/l of the culture broth (240 mg/l of MK-4 and 40 mg/l of MK-6) at 6 days of cultivation. The productivity of strain SP0736 was 50% higher than that of the parent strain, K₃-15.

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The biological activities of eckol, a novel phlorotannin with a dibenzo-p-dioxine skeleton, were examined. Eckol inhibited the antiplasmin activity of α₂-plasmin inhibitor very efficiently (IC₅₀; 1.6μg/ml) as well as those of α₂-macroglobulin and α₁-antitrypsin. However, its inhibitory effect on the antithrombin III-heparin complex was very weak. Eckol also showed inhibitory activity on thrombin (IC₅₀; 12μg/ml), but not on plasmin. Its inhibitory activity was reduced in whole human plasma, but at concentrations of above 200μg/ml it enhanced urokinase-induced fibrinoly sis in human plasma. Studies on the inhibitory spectra of several derivatives of eckol showed that the dibenzo-1, 4-dioxane skeleton was necessary for inhibition of plasmin inhibitor. These observations suggest that eckol or its derivatives may be useful clinically for potentiating thrombolytic activity.

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