Reactions of the p-nitrophenyl-cellobioside (G₂-pNP) hydrolyzing xylanase from Cellvibrio gilvus (XCEL) were investigated kinetically in detail. XCEL hydrolyzed only aglyconic bonds in various arylcellobiosides with close kinetic paramters together. Its kinetic parameters toward various p-nitrophenyl cellooligosaccharides were also close. Two more xylanases, from Streptomyces sp. E86 (XSTR) and from Aspergillus japonicus (XASP), were found to hydrolyze G₂-pNP at a lower rate compared with XCEL. The V_max of XSTR and XASP were comparable to that of XCEL, suggesting that the high G₂-pNP-hydrolyzing activity of XCEL was due to its small K_m. A xylanse from Robillarda sp. Y-20 (XROB) did not have any activity on G₂-pNP. p-Nitrophenyl-xylobioside (X₂-pNP) and p-nitrophenyl-glucosyl-xyloside (GX-pNP) were examined as substrates to the four xylanases. Three of the four xylanases hydrolyzed these substrates, only at their aglyconic bonds, rather faster than xylan, but XROB hydrolyzed them with a very small rate. Classification of xylanases based on their activity on the aryl-glycosides is discussed. The advantage of using X₂-pNP or GX-pNP for xylanase assay is also discussed.

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A kinetic analysis of splitting oligomeric substrates by poly(β-D-mannuronate)lyases (alginate lyases I, SP1 and SP2) from a marine mollusk was done. Monomer and oligomers of mannuronate and guluronate were prepared by hydrolyzing poly β-1, 4-D-mannuronate and poly α-1, 4-L-guluronate from alginate with H₂SO₄, respectively, and thereafter by gel filtration on a Bio-Gel P-2 column. Alginate lyases I apparently did not act on the trimer of mannuronate but did on the tetramer or those longer than that, indicating the increased k_cat/K_m with increasing polymerization degree. The kinetic analyses suggest that the size of the subsite structure of the enzymes is most likely to be able to bind the linear pentamer of mannuronate units.

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Emulsions of corn oil in solutions containing up to 1.5% mucilage from ruredzo (Dicerocaryum zanguebarium) in buffer solutions were prepared in a piston homogenizer. Emulsification was assessed by diluting samples of emulsions in sodium dodecyl sulfate and measuring absorbance at 500 nm. Viscosities of mucilage solutions were measured in a rotational viscometer. Emulsification capacity increased with mucilage concentration but was decreased in the presence of NaCl and CaCl₂. Except at 1.5% mucilage concentration, for which about 50% emulsifying ability was retained after 10 min, most of the emulsions were broken up after ten minutes. The optimum pH for the formation of emulsions was about 7.4 with poor emulsification at both higher and lower pHs. The changes in emulsifying capacity that were brought about by changes in pH, NaCl, and CaCl₂ concentration corresponded to changes in viscosity that were brought about by pH and salt concentration.

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The non-thermal effects of ceramics heater radiation on xanthine oxidase activity have been investigated using the enzyme, substrate, and competitive inhibitors which were irradiated on cooling. The K_m and V_max in the irradiated enzyme system were reduced to 51% and 85% of the non-irradiated control, respectively. The K_i for a competitive inhibitor, folic acid, in the irradiated enzyme system decreased to 22% of the non-irradiated control. A steady-state molecular kinetic analysis for the reaction estimates that the irradiated enzyme may be kept in a folding state, and the formation of a Michaelis complex has been accelerated, and the activated Michaelis complex has been stabilized, and that a solvation or an electrostriction of xanthine, folate, and an active center of the enzyme with water may be promoted by irradiating the components in an aqueous solution, by which modification of the enzyme activity has been regulated.

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Depolymerization of hyaluronic acid obtained from Streptococcus zooepidemicus by D-fructose 6-phosphate was investigated for characterization of reducing sugar-mediated degradation of biopolymers under physiological conditions. The extent of depolymerization was monitored by the decrease of viscosity of a reaction mixture containing 1.0% hyaluronic acid, D-fructose 6-phosphate, and 1.0×10^-2 mM of Cu²⁺ in phosphate buffer, pH 7.4. It was found that the depolymerization of hyaluronic acid was dependent on the concentration of the reducing sugar and was specifically accelerated by the presence of Cu²⁺. The reaction was found to be significantly inhibited by catalase, superoxide dismutase (SOD), 1, 2-dihydroxybenzene 3, 5-disulfonic acid (Tiron), and chelating agents such as EDTA and diethylene triamine penta-acetic acid (DETAPAC), although the inhibition by SOD was low. Almost the same depolymerization rates were observed in hyaluronic acid preparations of different molecular weight (1.1×10⁶, 8.8×10⁵, and 6.8×10⁵). The rates, however, were different for hyaluronic acids obtained from S. zooepidemicus, rooster comb, and umbilical cord. It was concluded that depolymerization of the polysaccharide was caused by active oxygen species generated by the autoxidation of D-fructose 6-phosphate in the presence of Cu²⁺, in a mechanism similar to that previously reported for the degradation of DNA and inactivation of virus in vitro.

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Sucrose phosphorylase from Leuconostoc mesenteroides was found to catalyze transglycosylation from sucrose to catechins. All catechins were efficient glycosyl acceptors and their transfer ratios were more than 40%. The acceptor specificity of the enzyme decreased in the following order: (-)-epicatechin gallate=(+)-catechin>(-)-epicatechin>(-)-epigallocatechin gallate>(-)-epigallocatechin. About 150 mg of the purified transfer product was obtained from 100 mg of (+)-catechin. Its structure was identified as (+)-catechin 3'-O-α-D-glucopyranoside (C-G) on the bases of the secondary ion mass spectrometry analysis, the component analyses of its enzymatic hydrolyzates, and the nulcear magnetic resonance analysis. The browning resistance of C-G to light irradiation was greatly increased compared to that of (+)-catechin. The solubility of C-G in water was 50-fold higher than that of (+)-catechin. The antioxidative activity of C-G in the aqueous system with riboflavin was almost equal to that of (+)-catechin. In addition, C-G strongly inhibited tyrosinase, in contrast with (+)-catechin, which is the substrate of tyrosinase. The inhibitory pattern of C-G was competitive using L-β-3, 4-dihydroxyphenylalanine as a substrate.

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Hydroxy and hydroperoxy fatty acids were labeled with 9-bromomethylacridine at room temperature. They were separated from the degradation products and less polar fatty acid derivatives on an octyl silicagel column, and put on an octadecyl silicagel column by on-line column switching. By this method, picomolar levels of the derivatives were measured with good reproducibility. The detection limit of 16-hydroxy-hexadecanoic acid as a representative was 0.9 pmol (S/N=3) and the relative standard deviation of its peak areas was 2.5% (18.5 pmol, n=7). The method was used for the measurement of hydroxy fatty acids derived from hydroperoxy fatty acids and phosphatidylcholine (PC) hydroperoxides spiked in human plasma. By incubation at 37°C for 4h with human plasma, the hydroperoxy fatty acid was reduced to the corresponding hydroxy fatty acid. In this condition, the PC hydroperoxides showed a considerable decrease, however, a small portion (2.5-3%) of PC hydroperoxides decomposed gave the corresponding hydroxy fatty acids.

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A DNA fragment responsible for the 4''-O-acylation of macrolide antibiotics was cloned from a mutant strain of the carbomycin producer Streptomyces thermotolerans. The gene encoding the macrolide 4''-O-acyltransferase was within a 2.7-kb region of the cloned fragment (15-kb). Streptomyces lividans carrying the region converted exogenously added tylosin to 4''-O-acyltylosins. Nucleotide sequencing of the region showed two open reading frames (ORFs). Expression assay using deleted plasmids showed that both ORFs were essential for optimal expression of the acyltransferase activity. One of them (acyB1) was identical with carE reported previously as a gene encoding 4''-mycarosyl isovaleryl-CoA transferase. The other (acyB2) was assumed to encode a novel regulatory protein that could activate acyB1 expression. acyB1 and acyB2 were highly conserved among streptomycetes with macrolide 4''-O-acyl transferase activity.

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In Eurycotis floridana, male calling behavior is associated with the sex pheromone released by the anterior part of tergites 2, 7, and 8. Glandular extracts of tergite 7 revealed a characteristic odor of caramel which is attractive at a distance for the females. We identified the two compounds responsible for this odor, 4-hydroxy-5-methyl-3(2H)-furanone and 4-hydroxy-2, 5-dimethyl-3(2H)-furanone. These compounds are known to be very important as flavor components of foods, but this is the first time that they have been identified as natural insect products. Various amounts of the identified compounds were tested, and their biological function is discussed.

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Evidence has been reported that bulb development in onion plants (Allium cepa L.) is controlled by endogenous bulbing and anti-bulbing hormones, and that gibberellin (GA) is a candidate for anti-bulbing hormone (ABH). In this study, we identified a series of C-13-H GAs (GA₁₂, GA₁₅, GA₂₄, GA₉, GA₄, GA₃₄, and 3-epi-GA₄) and a series of C-13-OH GAs (GA₄₄, GA₂₀, GA₁, and GA₈) from the leaf sheaths including the lower part of leaf blades of onion plants (cv. Senshu-Chuko). These results suggested that two independent GA biosynthetic pathways, the early-non-hydroxylation pathway to GA₄ (active GA) and early-13-hydroxylation pathway to GA₁ (active GA), exist in onion plants. It was also suggested that GA₄ and GA₁ have almost the same ability to inhibit bulb development in onion plants induced by treatment with an inhibitor of GA biosynthesis, uniconazole-P. The endogenous levels of GA₁ and GA₄, and their direct precursors, GA₂₀ and GA₉, in leaf blades, leaf sheaths, and roots of 4-week-old bulbing and non-bulbing onion plants were measured by gas chromatography/selected ion monitoring with the corresponding [²H]labeled GAs as internal standards. In most cases, the GA levels in long-day (LD)-grown bulbing onion plants were higher than those of short-day (SD)-grown non-bulbing onion plants, but the GA₁ level in leaf blades of SD-grown onion plants was rather higher than that of LD-grown onion plants. Relationship between the endogenous GAs and bulb development in onion plants is discussed.

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We have developed a versatile system that permits efficient manipulation of coryneform bacteria by gene disruption and replacement. Circumventing the methyl-specific restriction system in certain of these bacteria by using non-methylated plasmid DNA was necessary. Integrative plasmid DNA containing an in vitro disrupted copy of the Brevibacterium flavum thr B gene was introduced into B. flavum at an efficiency of 2.4×10² integrants/μg. Integration occurred by homologous recombination either via a Campbell-like mechanism or via a double crossover event. Integrants were stable for over 32 generations.

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A DNA segment encoding a signal peptide from yeast invertase was fused in frame to bglH gene encoding 87-kD-β-1, 3-glucanase from Bacillus circulans IAM1165 and was expressed in the yeast Saccharomyces cerevisiae under the control of the GAL1 gene promoter. Yeast cells containing this fused gene produced active β-1, 3-glucanase in the medium after a long period of incubation at low temperature. The enzyme produced by yeast was heterogeneous in size, and larger than the enzyme produced by Escherichia coli.

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Three Lactobacillus casei bacteriophages, LC-Nu, PL-1, and φFSW, were compared. Phage LC-Nu, which has not been previously characterized, originated from a local cheese plant in Finland. Phages PL-1 and φFSW (isolated in Japan) represent the most thoroughly studied L. casei phages so far. All three phages had similar morphotypes, but still had different patterns of structural proteins, as analyzed by SDS-PAGE. The phages differed also in types of genome organization: LC-Nu and PL-1 had cohesive ends in their DNAs, and the DNA of φFSW was circularly permuted. The initiation site and orientation of packaging of φFSW DNA were identified. The homologies between the phage genomes were analyzed by Southern hybridization. About one-third of each phage genome was highly homologous with other phages (homology over 85%), and two-thirds were slightly homologous (homology between 65% and 76%). DNAs from five industrial L. casei strains were also tested for homology with phage LC-Nu DNA. Phage LC-Nu related sequences were present in all the L. casei strains tested.

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A novel strain, MX-232, which produced much trehalulose (1-O-α-D-glucopyranosyl-D-fructose), was isolated from a soil sample in Udonthani, Thailand. The isolate was a Gram-negative aerobic rod, motile with peritrichous flagella, and had a high ability to convert sucrose into trehalulose and isomaltulose (palatinose, 6-O-α-D-glucopyranosyl-D-fructose) with α-glucosyltransferase. The results of physiological characterization, G+C content, and pathogenicity tests on several plants showed that MX-232 is a strain of Agrobacterium radiobacter. When free cells and 20% (w/v) sucrose solution were used in the reaction, the yield of trehalulose was 88-90% (w/w). And with immobilized cells, the yield was about 85% when 40-50% (w/w) sucrose solution was used as the substrate. Using immobilized cells in a column reaction was good for producing trehalulose. This report suggests MX-232 has the highest ability to produce trehalulose from sucrose.

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An extracellular enzyme that produces di-D-fructofuranose 2', 1;2, 1'-dianhydride (difructose anhydride I=DFA I) from inulin was purified from the culture broth of Streptomyces sp. MCI-2524. The purification enhanced the specific activity 7-fold with an overall yield of 17%. The purified enzyme, when electrophoresed on a SDS polyacrylamide gel, gave a single band corresponding to a molecular weight of 36 kDa. Gel filtration chromatography gave a single peak that eluted at a position corresponding to 70 kDa. The enzyme was active from pH 3.0 to pH 9.0, and at temperatures up to 65°C. Maximal activity was observed at pH 6.0, at 55°C. The enzyme was inhibited by Cu²⁺.

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Lymphatic absorption of [³H]cholesterol and [¹⁴C]sitosterol from a mixed micellar solution containing sodium taurocholate or taurochenodeoxycholate, and glycocholate or glycochenodeoxycholate infused into the duodenum was compared in rats with lymph and bile drained. Absorption of [³H]cholesterol into the thoracic duct lymph was clearly higher than that of [¹⁴C]sitosterol irrespective of the source of bile salts. Lymphatic absorption of cholesterol for 24h was reduced by 23% in the taurochenodeoxycholate micelles compared with the taurocholate micelles, and the reduction of sitosterol was 61%. A similar extent of reduction was also observed between the glycocholate and glycochenodeoxycholate micelles. The absorption rate of cholesterol in relation to sitosterol (cholesterol/sitosterol ratio) was significantly higher in the chenodeoxycholate micelles (around 12) than the cholate micelles (around 6 to 7). Thus, the difference in the absorption rate between cholesterol and sitosterol could not be explained by the differential affinity of these sterols for the different bile salt micelles, and was largely influenced by the source of bile acids.

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Endomitotic diploidization occurred as a result of heat treatment of spores during germination of the industrial yeast S. cerevisiae. Polyploid cells were obtained in both heterothallic and homothallic strains. In the heterothallic strains, the resultant diploid cells, with the ability to mate, were used to construct tetraploids. In the homothallic strains, tetraploid cells were obtained directly after a mating-type switch. In each strain, the highest frequency (approximately 10% of viable cells) of diploidization was obtained when spores were cultured for 30 min in liquid medium to initiate germination followed by subsequent heat treatment at, 55°C for 10 min.

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A new allosamidin analog (2), possessing an N, N'-diacetylchitobiosyl moiety as a constituent, was stereoselectively synthesized through the coupling reaction between the disaccharide thioglycoside derivative (5) and allosamizoline derivative (6). The inhibitory activity of 2 against chitinases derived from an insect, yeast and mold were tested and compared with that of allosamidin (1).

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The intracellular cadmium (Cd) content was measured with early stationary phase cells of a highly Cd-tolerant moderately halophilic bacterium Pseudomonas sp. No. 40 cultivated in 1 M and 3 M NaCl medium containing 0 to 2500μg of CdCl₂/ml. It was found that the Cd contents were greatly affected by the NaCl concentration of the medium. When the bacterium was cultivated in the 1, 2, 3, and 4 M NaCl medium containing 1500μg of CdCl₂/ml, the intracellular Cd content was 25.0, 4.1, 3.1, and 2.0 mg Cd per g of dry cells, respectively. The intracellular Cd content decreased with increases of NaCl concentration of the medium. The fact seems to reflect Cd-tolerance of the bacterium towards the growth in the medium of different NaCl concentration. It is worthwhile to note that the bacterium showed the highest Cd-tolerance (in 3 M NaCl) and the lowest Cd content among the bacteria so far known. The bacterial cells grown in the 1 M NaNO₃ and 1 M Na₂SO₄ medium accumulated 1.8-1.3 times as much Cd²⁺ as those in the 1 M NaCl medium in the presence of 50-200μg of CdCl₂/ml. It would also explain the difference in the Cd toxicity in the medium of NaNO₃, Na₂SO₄, or NaCl.

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Isoenzymes of aspartate aminotransferase (EC 2.6.1.1, AAT) were purified to homogeneity and crystallized from bran of rice (Oryza sativa cv. Koganemasari). The native molecular weights of AAT-1 and AAT-2 isoenzymes were 88, 000 and 94, 000 with the subunit molecular weights of 44, 000 and 47, 000, respectively, indicating that the holoenzymes of the isoencymes are dimers. The isoelectric points of AAT-1 and AAT-2 were pH 6.5 and 5.0, respectively: both isoenzymes have no subform. The isoenzymes showed similar K_ms for four natural substrates, with the exception that AAT-1 had a higher affinity for L-glutamate than AAT-2. Amino donor and acceptor specificities of the isoenzymes were almost identical and fairly high. Amino acid compositions of the isoenzymes were similar but not the same. The isoenzymes contained one mole of pyridoxal 5'-phosphate (PLP) per subunit and showed characteristic absorption spectra of PLP enzymes. Polyclonal antibodies raised against AAT-1 selectively cross-reacted with AAT-1 but not with AAT-2. Conversely, the antibody raised against AAT-2 selectively cross-reacted with AAT-2 but not with AAT-1.

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Aspartate aminotransferase (EC 2.6.1.1, AAT) occurs as two forms in rice bran (from Oryza sativa cv. Koganemasari), AAT-1 and AAT-2. Immunotitrations with antisera against the AAT isoenzymes indicated an immunological distinction between AAT-1 and AAT-2. Sequential titration of total AAT activity showed that AAT-1 and AAT-2 comparised 82% and 18% of the total activity in the crude extract of rice bran, respectively. AAT-1 and AAT-2 comprised 45% and 55% of the total AAT activity in the crude extract of top tissues of the rice plant. AAT-1 was mainly in the cytosolic fraction of the rice plant cells. On the other hand, AAT-2 was the major isoenzyme of AAT in mitochondria and peroxisomes. Minute activity of AAT-1 was found in the membrane fractions of the mitochondria and peroxisomes and was solubilized with Triton X-100.

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Such stress factors as mechanical (vibration), thermal (unfavorable temperature), optical (light), and starvation reduced the larval growth of the red flour beetle (Tribolium castaneum Herbst). Various biogenic amines, including octopamine (OA), dopamine (DA), serotonin (5-HT), epinephrine (E), norepinephrine (NE), their precursors, and metabolites, in whole-body T. castaneum were measured by high-performance liquid chromatography coupled with electrochemical detection (ECD). Tyrosine occurred in the highest concentration, followed by OA, tryptophan, and 3, 4-dihydroxymandelic acid. The amount of OA was much higher than that of tyramine (a precursor of OA in the biosynthetic pathway) and of synephrine (N-methyl OA). DA, 5-HT, E, NE, and their related substances occurred in extremely low quantities compared with OA. Insects were stressed by vibrating at 1, 10, 100, or 1000 Hz, optically under a 24-h light (15 W, 50 Hz) photoperiod, thermally by changing the incubation temperature from an initial value of 30°C, or by starvation, which resulted in dramatic changes of levels of biogenic amines, including OA.

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1-Aminocyclopropane-1-carboxylgte (ACC) deaminase, a pyridoxal phosphate enzyme that catalyzes cyclopropane ring-opening and deamination of ACC, formed a quinoid intermediate with D-alanine, as shown by the appearance of a 510-nm absorption band. The presence of D-alanine also stimulated the inactivation of ACC deaminase with iodoacetamide. The increase of absorbance at 510 nm and the stimulation of the enzyme inactivation were temperature-dependent with a critical point at around 20°C, indicating a conformational change of the enzyme. To identify a reactive thiol group, this stimulated inactivation and an iodoacetamide derivative, N-(iodoacetamidoethyl)-1-aminonaphthalene-5-sulfonic acid were used. The residue that was modified by the specific reagent was monitored by absorbance at 350 nm through the digestion by lysylendopeptidase and the fractionation of peptides, and it was located at Cys-162 near the midpoint of the whole peptide chain of the ACC deaminase.

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Alcohol acetyltransferase (AATase) catalyzes the esterification of isoamyl alcohol by acetyl coenzyme A. The enzyme was solubilized from the microsomal fraction of Saccharomyces cerevisiae Kyokai No. 7, using Triton X-100 and then purified by a series of chromatographic separations: Poly Buffer Exchanger 94 (PBE94), DEAE Toyopearl, Toyopearl HW60, hydroxyapatite, Octyl-Sepharose CL-4B, and hexanolaffinity column chromatography. When the solubilized enzyme was put on PBE94, two active fractions were obtained. The enzyme obtained after affinity column chromatography had a single band on an SDS polyacrylamide gel, and its molecular mass was estimated to be 60 kDa. The enzyme was most active at pH 8.0 and 25°C. It was stable between pH 7.5 and 8.5, but was unstable at tempratures above 10°C. The activity was markedly inhibited by heavy metal ions such as Cd²⁺, Cu²⁺, Zn²⁺, and Hg²⁺, and sulfhydryl reagents. The K_m for acetyl-CoA was 0.19 mM. The internal peptide sequences were also identified.

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In order to clarify the substrate specificity of the α-L-mannosidase activity of naringinase (Sigma), the following disaccharides and phenol glycosides were freshly prepared: methyl 2-O-(α-L-mannopyranosyl)-β-D-glucoside (1), methyl 3-O-(α-L-mannopyranosyl)-α-D-glucoside (2), methyl 4-O-(α-L-mannopyranosyl)-α-D-glucoside (3), methyl 5-O-(α-L-mannopyranosyl)-β-D-glucoside (4), methyl 6-O-(α-L-mannopyranosyl)-α-D-glucoside (5), 6-O-(α-L-mannpyranosyl)-D-galactose (6), p-nitrophenyl α-L-mannoside (7), and 4-methyl umbelliferone α-L-mannoside (8). These compounds, except for 3 and 5, were hydrolyzed with naringinase.

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A potato tuber disk assay of culture broths which had been confirmed to show neither antibacterial nor phytotoxic activity resulted in the discovery of a novel type of crown gall formation inhibitors, julimycin B-II and julichrome Q_1.3. These two bisanthraquinones, products of Streptomyces sp. TM-71, inhibited crown gall formation on potato tuber disks at a minimum inhibitory dose of 1.6μg/disk without affecting the growth of Agrobacterium tumefaciens or the germination of alfalfa seeds.

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A screening procedure for microorganisms which have an ability to produce a desired compound as their secondary metabolite is first proposed. In some cases, the target microorganisms can be expected to grow and be enriched in a medium containing the desired compound, namely one of the secondary metabolites of the microorganisms, as the sole source of carbon, degrading and assimilating the compound to the primary metabolites. This approach was applied to isolate alkano-δ-lactones producing fungi by using a medium containing alkano-δ-lactones as the sole source of carbon. We isolated Fusarium solani and Trichoderma viride that had the ability to biosynthesize 2-deceno-δ-lactone (massoialactone) and 2, 4-decadieno-δ-lactone(6-pentyl-α-pyrone), respectively, in a glucose medium.

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The degradative pathway of 5-alkanolides (alkano-δ-lactones) and 2-deceno-δ-lactone (massoialactone) by Fusarium solani PM-1, a massoialactone-producing fungus, was investigated. (±)-Alkano-δ-lactones were shown to be degraded first to a one-carbon-atom-less methyl ketone, 4-hydroxy-2-alkanones, after hydroxylation. The 4-hydroxy-2-alkanones were then converted to 2, 4-alkanediones or to 2, 4-alkanediols, and are suggested to be successively degraded by modified β-oxidation. (R)-Massoialactone, the main compound in the volatiles produced by the strain, was first saturated to (R)-decano-δ-lactone, and then this saturated lactone was degraded in the same way. These observations lead to a conceptional cycle of acetate moieties throughout the production and degradation of the secondary metabolites.

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An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological properties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50% decrease of growth yield are 27 and 16 g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512 mM) ethanol, 8.5 g/liter (94 mM) lactate, 2.9 g/liter (48 mM) acetate, and 0.9 g/liter (20 mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.

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Compounds that promoted the growth of pollen tubes were isolated from the style of Rhododendron mucronatum and were identified as azalein, (+)-catechin, and (-)epicatechin. Among these compounds, 50-100 ppm of (+)-catechin or (-)-epicatechin increased the growth of pollen tubes in Camellia japonica, R. mucronatum, Styrax japonica, and Pinus densflora by 16-20%. These compounds had no significant effects on the growth of pollen tubes in Lilium auratum and Narcissus pseudo-narcissus. Among the phenolic compounds with similar chemical structures as the promoters, catechol- and pyrogallol-type compounds had the activity and the latter types were more effective. As α-tocopherol also showed growth promoting activity, it was thought that the antioxidative activity of the phenolic compounds was responsible for the promotion of the pollen tube growth.

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The ability for such lymphocyte activation as growth of the spleen and Peyer's patch (PP) cells from unprimed BALB/c mice, and of lymph node (LN) cells from mice immunized with hen's egg ovomucoid (OM) was assayed by using sonicated microorganisms. The occurrence of immunopotentiating activity was strikingly dependent on the properties of individual strains, rather than on the species or microbial culture conditions. All the tested Bifidobacterium strains, especially B. adolescentis M101-4, showed strong mitogenic activity in those assays, and also enhanced the production of the anti-OM antibody by LN cells, while they did not induce the growth of thymocytes. Similar results were obtained from experiments using germ-free (GF) mice. These results suggest that the activation with B. adolescentis M101-4 was not due to any secondary stimulation of lymphocytes primed with bacteria in the gut or environment, but to direct or indirect activity toward B cells that was intrinsic to the strain.

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A non-secretory ribonuclease (RNase PK₃) was isolated from porcine kidney, and its primary structure was analyzed. RNase PK₃ consisted of 126 amino acid residues. The amino acid sequence of RNase PK₃ has high sequence homology with non-secretory RNases from human urine and bovine kidney.

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A plant endonuclease with 3'-nucleotidase activity was purified from scallion bulbs to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 38, 000 by SDS-PAGE and 40, 000 by Sephadex G-100 gel filtration. The enzyme rapidly hydrolyzed yeast RNA and denatured calf thymus DNA to acid-soluble substances, and hydrolyzed the plasmid pBR322 to yield small DNA fragments at low enzyme concentrations. These four activities were eliminated by treatments with EDTA and tetraethylenepentamine. The enzyme had maximum activity at pH 8.5-9.0 for 3'-AMP, 3'-GMP, and 3'-UMP, at pH 6.5 for 3'-CMP and yeast RNA, and at pH 6.0 for denatured calf thymus DNA and pBR322. During digestion of yeast RNA by the enzyme at pH 6.5, 5'-GMP was released most rapidly, followed by 5'-UMP, 5'-AMP, and 5'-CMP. These properties were different from those of endonucleases isolated from other sources such as mung bean sprouts and wheat seedlings.

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The sex pheromone of Bombyx mori, bombykol [(10E, 12Z)-10, 12-hexadecadien-1-ol], can be biosynthesized in four steps: construction of a hexadecanoic moiety from acetyl CoA, Δ-11-desaturation, Δ-10, 12-desaturation, and reduction of the acyl group. This biosynthesis is regulated by a hormone named the pheromone biosynthesis activating neuropeptide (PBAN). To examine the steps that are accelerated by this neurohormone, pheromone glands excised from decapitated females were incubated in vitro with either ¹⁴C-labeled sodium acetate or one of three fatty acids [hexadecanoic acid, (Z)-11-hexadecenoic acid, or (10E, 12Z)-10, 12-hexadecadienoic acid]. After analyzing the radioactivity that was incorporated from each precursor into bombykol and the biosynthetic precursors, it was observed that the first three steps proceeded in glands both treated and untreated with synthetic PBAN of B. mori; however, the last step proceeded only in the treated glands. From this in vitro experiment, it can be concluded that the main regulatory role of PBAN is in the reduction of the acyl group in B. mori, as was shown by our previous in vivo experiment.

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Poly(γ-glutamic acid) (PGA) hydrolase was purified from the culture filtrate of a filamentous fungus, Myrothecium sp. TM-4222 and its general properties, especially the mode of hydrolytic action on the γ-glutamyl bond of PGA, were investigated. The purified preparation demonstrated a homogeneous band on an acidic slab gel of pH 4.3 with polyacrylamide gel electrophoresis. The enzyme showed its maximum activity at 37°C and at pH 5.0, being stable up to 40°C. The molecular mass was estimated to be 68 kDa by gel filtration. The hydrolytic action of the enzyme was specific for PGA, but not for other γ-glutamyl peptides or amides. The enzyme converted 38% of the original PGA with an average molecular mass of 500 kDa to smaller peptides, and then depolymerized these fragments to a mixture of γ-oligopeptides which consisted of only L-glutamic acid. L-Glutamic acid monomer was negligible in the reaction mixture. The remaining 62% of PGA was resistant to the enzyme action, in which D-glutamic acid was mainly detected. This study demonstrated a novel endo-type specificity of hydrolysis on PGA by the enzyme.

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Two-dimensional analysis of [Ca²⁺]_i in the intact body of a free-living soil nematode, Caenorhabditis elegans, was done. The effects of DOPA on the level of [Ca²⁺]_i were measured and compared with the data from microspectrofluorometry. DOPA caused a temporal elevation of [Ca²⁺]_i in the intestinal tract. The pattern of [Ca²⁺]_i transient from image analysis accords well with that from microspectrofluorometry. The subcellular distribution of fura-2 in C. elegans was also examined and it was shown that the fluorescence indicator was mainly in the cytosol fraction.

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Chloroflexus aurantiacus J-10-fl was found to contain two types (protease I and protease II) of thermostable proteases which were separated by Butyl-Toyopearl 650 M chromatography. Protease I was purified to electrophoretic homogeneity from the culture broth of C. aurantiacus J-10-fl. The molecular mass of protease I was estimated to be approximately 66 kDa by SDS-PAGE, and the value of approximately 66 kDa was also obtained by the Hedrick-Smith method, indicating that protease I was a monomer. The isoelectric point was 6.2. Protease I activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, and o-phenanthroline. The optimum pH for the activity of protease I was around 8.0. Addition of Ca²⁺ increased the pH and heat stabilities of protease I. The activity was stable between pH 4.0-11.0 and up to 75°C, and the maximum activity was observed at 70°C in the presence of 2 mM CaCl₂. Protease I was resistant to the treatment by denaturing reagents (8 M urea or 1% SDS) at pH 8.0 and 20°C for 24 h. The sites of cleavage. in oxidized insulin B chain by protease I were similar to those by other microbial neutral metalloproteases. Elastase activity of protease I was not detected.

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A system (pair) of bacteriophage pA1 and the host bacterium Vibrio sp. A1 was isolated from seawater. The lysate of host cells infected with the phage showed a significant becteriolytic activity with wider lytic action spectra, more susceptibility at alkaline pH(=9) and higher stability of lytic activity in the lysate compared with other phage-induced lysins reported so far.

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In shaking culture, nitrate reductase activity in the cell-free extracts of Bacillus licheniformis increased with the addition of NH₄Cl to the medium containing NaNO₃ as a single nitrogen source, where amounts of nitrogen sources were sufficient for cell growth. This increase of nitrate reductase activity therefore suggests that the activity is not for nitrate assimilation but for other physiological functions containing a dissimilatory nitrate reduction.

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A β-glucosidase was intensively purified with high recovery from a commercial preparation of Aspergillus niger by consecutive column chromatography. The enzyme was an acidic protein with a pI of 3.8, and split cellotriose to produce specifically β-D-glucose. Substrate specificity studies demonstrated that the purified enzyme required absolutely the C-4 configuration of the terminal, nonreducing β-D-glucose residues in the substrate molecules.

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Given the importance of transketolase, TK (EC 2.2.1.1) for both pharmacological studies and synthetic purposes, the need for a simple and inexpensive assay is patent. We describe here a simple and inexpensive TK assay using the unphosphorylated ketoses L-erythrulose, 4-deoxy-L-erythrulose, and 4-deoxy-D, L-erythrulose as donor substrates instead of D-xylulose-5-phosphate, with D-ribose-5-phosphate as the acceptor substrate.

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Collageriase liberated DNA from HAF-induced aggregate of hepatoma AH109A cells with dispersion of the aggregate. The DNA was isolated from the aggregate by electrophoresis, and the effects of the DNA on the aggregation activity of HAF were examined. The DNA did not aggregate hepatoma AH109A cells, but it increased the cell number of the aggregate induced by HAF. These results suggest that the DNA from hepatoma AH109A cells is essential for formation of the HAF-induced aggregate and it provides adhesion among the cells of the aggregate.

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Recently we reported increased serum levels of copper and of ceruloplasmin activity in rats fed a diet with excess tyrosine. In this study, we found increases in the apparent absorption of copper from the intestines and in the concentrations of kidney copper and metallothionein in rats on the same diet.

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A comparison was made of the fatty acid composition in the liver for various sizes of squid (Illex argentinus). The proportion of 14:0, 16:0, 16:1 n-7, and 18:1 n-9 increased, while that of 18:0, 20:4 n-6, and 22:6 n-3 decreased with increasing liver weight. The proportion of 20:5 n-3 was found to be almost constant for any size of liver.

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A compound was isolated in crystal form from an extract of Gymnema sylvestre dried leaves. The compound was identified to be conduritol A by chemical methods, and the physicochemical characteristics also coincided with the data in the literature. Conduritol A was tested for its effect on intestinal glucose absorption or glucose tolerance in rats. The results show that the absorption of glucose in vitro was completely inhibited in the presence of 0.2 mg/ml of conduritol A and that the blood sugar level was effectively depressed by administering conduritol A at 10 mg/kg of rat body weight.

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An autolysate of bonito bowels was treated with ultrafiltration, loose RO concentration, ion-exchange chromatography, and reverse phase chromatography to increase its potency to inhibit angiotensin I-converting enzyme (ACE) activity by 16-fold. Oral administration of the partially purified autolysate decreased the systolic blood pressure of spontaneously hypertensive rats (SHR) in a dose-dependent manner at the doses of 1 g peptides/kg or higher. The relationship between the antihypertensive activity (in vivo) of the partially purified preparation and its ACE inhibitory activity (in vitro) in comparison with previously reported ACE inhibitory peptides is discussed.

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Three strains of purple nonsulfur photosynthetic bacteria were isolated from seawater. They showed the characteristic properties of Rhodobacter species. Their biochemical, physiological, and chemotaxonomical properties were different from those of previously known species.

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Capsaicin was converted into the corresponding glucoside when administered to cell suspension cultures of Coffea arabica cultured in a modified Murashige and Skoog's medium with 5μM 2, 4-dichlorophenoxyacetic acid and 0.5μM kinetin. The glucoside was identified as capsaicin-β-D-glucopyranoside by FAB-MS, ¹H-NMR, and hydrolysis with α- and β-glucosidases. The pungency of the glucoside was approximately 1/100 of that of capsaicin.

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The minimum inhibitory concentrations of ten fatty acids and their methyl esters on Staphylococcus aureus and MRSA #10 were investigated. The highest antibiotic activity was observed for γ-linolenic acid (C_18:3), and when the concentration was 500μg/ml in the medium, the MRSA cells were all killed.

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