Previously, we reported that induction of peroxidation of synthetic phosphatidylcholines (PCs) containing a polyunsaturated fatty acid by Fe²⁺-EDTA in the presence of ascorbate resulted in the formation of four types of PCs with an sn-2-oxidatively fragmented acyl group, which had platelet-aggregating activity due to interaction with platelet-activating factor (PAF) receptors. These PCs were compounds with a short-chain monocarboxylate, ω-hydroxymonocarboxylate, dicarboxylate, and dicarboxylate semialdehyde residue, respectively. In this study, we investigated the PAF-like lipids formed during peroxidation of PCs from hen egg yolk, salmon roe, sea urchin eggs, and krill in an FeSO₄/EDTA/ascorbate system. The platelet-aggregating activities of these oxidized PCs were all inhibited by FR-900452, an antagonist of PAF. The activity of oxidized krill PC, which was equivalent of 89.8±8.8 pmol 16:0-PAF/μmol of starting PC, was about 5 times those of oxidized PCs from salmon roe and sea urchin eggs, and about 50 times that of oxidized hen egg yolk PC. The PAF-like phospholipids that had different combinations of long-chain alkyl or acyl groups with one of the above four types of short-chain acyl groups were identified by gas chromatography-mass spectrometry. The results indicated that foodstuffs that are rich in 1-O-alkyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine are potential sources of compounds with high PAF-like activity formed by deleterious lipid peroxidation.

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The biological activity of KM-01, a fungal metabolite isolated from Drechslera avenae, was tested with various bioassays of known plant hormones. It inhibited the action of brassinosteroid dose dependently, whereas no activity was observed with auxin and cytokinin bioassays. Although a synergistic effect with GA₃ at low concentrations and an inhibitory effect on ABA were observed, we conclude that KM-01 is the first selective brassinosteroid inhibitor found in natural sources.

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When angiotensin fragments, Val-Tyr and Angiotensin III (ANG III), with potent ACE inhibitory activity were intravenously administered to spontaneously hypertensive rat (SHR), a significant reduction of diastolic blood pressure was observed. After incubation of ANG III with SHR plasma, four fragments with ACE inhibitory activity, Val-Tyr (ANG (3-4)) (IC₅₀=26.0μM), Ile-His-Pro-Phe (ANG (5-8)) (11.6μM), Tyr-Ile-His-Pro-Phe (ANG (4-8)) (457.5μM), and Val-Tyr-Ile-His-Pro-Phe (ANG (3-8)) (6.55μM), were confirmed to generate in SHR plasma. Compared the metabolic behavior of ANG II in SHR plasma with that in normotensive Wistar plasma, the initial degradation rate (3.07nmol/ml/min) in Wistar plasma was about 2-fold higher than that in the SHR one (1.75nmol/ml/min).

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Japonica-type rice grains stored at 40°C for 2 months were treated with a freeze-dried powder of acetic acid bacteria and then cooked. It was found that the originally existing n-hexanal were decreased by more than 35%. Moreover, the stale flavor had been signiticantly reduced compared to that of non-treated rice grains. Simultaneous treatment with freeze-dried cells of the acetic acid bacteria and a surfactant was 1.3-1.9 times more effective for reducing the amount of n-hexanal than with the cells of acetic acid bacteria alone. The optimum conditions for reducing the stale flavor were investigated by using a combination of the acetic acid bacterial cells and the surfactant. The optimum conditions were as follows : soaking temperature, 50°C ; soaking time, 30-180 min ; pH 4.5-7.0, optimally at pH 6.0. Treating Indica-type rice grains with the freeze-dried cells of acetic acid bacteria was also effective for reducing the specific off-flavor of the rice.

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The major compounds formed from the N^α-benzoylarginine amide (BzArgNH₂) and 3-deoxyglucosone (3DG) reaction system were purified by reversed-phase HPLC. The major product was identified as R-4-imidazolone (I) ; R=2-(4-benzoylamino-5-pentamide)amino-5-(2, 3, 4-trihydroxybutyl) as described in a previous paper (ref. 10). Intermediate products to the formation of compound I were identified as R-4(5H)-imidazolone (II), R-4(5-hydroxy)-imidazolone (III), and R-4-dihydroxyl-2-imidazoline (IV). Periodical changes in the amounts of compounds I, II, III, and IV suggest the formation of compound I via compound II by dehydration and oxidation from the BzArgNH₂-3DG reaction system to be the major pathway, the formation of compound I via compound III being the other pathway.

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The inhibitory effect of phosphorylated oligosaccharides, which were prepared from potato starch, on the formation of calcium phosphate in vitro were investigated. Phosphorylated oligosaccharides from potato were fractionated by ion-exchange chromatography into two fractions, PO-1 and PO-2. Fraction PO-1 was composed of maltotriose, maltotetraose, and maltopentaose to which one phosphate group was attached. Fraction PO-2 was predominantly composed of maltopentaose and maltohexaose to which at least two phosphate groups were attached. The average degree of polymerization of dephosphorylated PO-1 and PO-2 was evaluated to be 4.02 and 5.82, respectively. Fraction PO-2 was the main component having an inhibitory effect on calcium phosphate formation. In addition, among the phosphorylated monosaccharides, glucose-1, 6-diphosphate and fructose-1, 6-diphosphate were more effective inhibitors of the formation of calcium phosphate than glucose-6-phosphate and fructose-6-phosphate. These results suggest that the strength of the inhibitory effect might depend on the number of phosphate groups attached to each sugar molecule.

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The effects of prodigiosin 25-C (PrG) which preferentially suppresses cytotoxic T cells (CTL), was examined in comparison with concanamycin B (CMB), a specific inhibitor of vacuolar type H⁺-ATPase (V-ATPase). PrG and CMB directly inhibited the cytotoxic function of CTL and neutralized acidic organelles of CTL in vitro. In addition, PrG or CMB was injected in C57BL/6 mice after immunization with an allogeneic mastocytoma, P815. PrG and CMB inhibited the killing activity of CTL against the tumor and reduced the population of CD8⁺ cells without affecting CD4⁺ and B220⁺ populations in the spleen. PrG and CMB had only a negligible effect on antibody production induced by sheep red blood cells (SRBC) and mitogenic responses of lymphocytes. These results suggest that PrG and CMB have similar immunosuppressive properties at least through their inhibitory effects on acidification of intracellular organelles required for the effective function of CTL.

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The pH-sensitive swelling of a natural-polyelectrolyte complex gel, prepared from xanthan and chitosan, was investigated using a model based on the Donnan equilibrium theory with special attention to the dissociation behavior of the polyelectrolytes. First, the pH dependence of the degree of ionization for the xanthan-chitosan complex was evaluated by the potentiometric titration method, and the value of the dissociation constant for analyzing the swelling behavior was obtained. Second, the validity of the Donnan equilibrium was confirmed by measuring the concentration of sodium ion in the gels. By analyzing the swelling behavior of the gel using the model, it was suggested that the network properties of the gel altered with changes in the ambient pH. These results indicate that analysis using the parameters evaluated by potentiometric titration is useful for investigating the swelling behavior of ionic gels.

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Linoleic acid and oleic acid markedly increased the influx of ⁴⁵Ca into isolated intestinal epithelial cells, and this increase reflected a rise in the intracellular calcium level. Methyl linoleate had no effect, while glutamic acid and somatostatin both inhibited the linoleic acid-induced influx of ⁴⁵Ca. In addition, methyl linoleate had no effect, while glutamic acid inhibited linoleic acid-induced hormone-responsive pancreatic exocrine secretion.

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Plasmid DNAs in the range from 2.9 to 12.6 kbp were transferred into Bacillus subtilis ISW1214 intact cells by the use of electroporation. The transformation efficiency (transformants per μg plasmid DNA) decreased with increases of size of the DNA. However that of 2.0×10³ transformants per μg of DNA were done routinely, by using a plasmid with a large molecular size of 12.6 kbp. The size of plasmid DNA in the range of 3.7 to 12.6 kbp did not affect the molecular efficiency (transformants per molecule input DNA). The transformation efficiency as high as 9.3×10⁴ transformants per μg of purified plasmid pUB110 was obtained, using a cell concentration of 7.6×10¹⁰ cells/ml and DNA concentration of 4 μg/ml in buffer containing 0.3 M sucrose, 1 mM CaCl₂, and 1 mM sodium citrate (pH 6.0) under optimal pulse conditions of an electric field strength of 7 kV/cm and a duration of 500 μs with a single squared pulse at 0°C. The gene expression for antibiotic resistance after electroporation was completed within 1.5 h. The transformants were confirmed to harbor the same intact plasmid by agarose gel electrophoretic analysis.

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The thermophilic protease aqualysin I (AQI) gene (aqul), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacl^q gene. Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F' episome was used as a host cell for aqul expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.

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A cDNA and genomic gene encoding calmodulin were isolated from Aspergillus oryzae using a part of the calmodulin gene from A. nidulans as a hybridization probe. The gene was in a 3.4-kb SphI fragment and Southern-blot analysis of genomic DNA suggested the existence of a single copy of the calmodulin gene in A. oryzae. The nucleotide sequence analysis showed that the gene consists of five introns and six exons. Although the nucleotide sequence homology with that of A. nidulans was not so high (68%), the deduced amino acid sequence was 100% and 84% identical with calmodulin of A. nidulans and chicken, respectively. The cDNA encoding A. oryzae calmodulin was expressed under the control of the GAL1 promoter in the calmodulin null mutant (cmd1) of yeast, Saccharomyces cerevisiae, and could function as a calmodulin gene.

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The main problem with dried-salted fish (DSF) products is lipid oxidation. PUFA of fish oil is very easily oxidized, and sodium chloride is known to be a pro-oxidant. Many researchers have found that the products of lipid oxidation had negative effects on a variety of species, so we evaluated the effect of a desalting and defatting treatment on the lipid oxidation of Indonesian DSF. The dietary effect of untreated DSF, defatted DSF and desalted DSF on diarrhea, on the internal organs, on hepatic, serum, and urinary lipid peroxidation, and on hepatic and serum alpha-tocopherol were evaluated by using rats. The defatting treatment had a significant effect (p<0.01) on reducing the lipid oxidation variables of the DSF sample and on protecting the rats from diarrhea. Compared with the rats in the casein group, these in the untreated DSF group had significantly higher values (p<0.05) for hepatic, serum and urinary lipid peroxidation, but significantly lower values for hepatic and serum alpha-tocopherol. No significant differences were observed between the rats fed with casein and defatted DSF.

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Inactivation of Saccharomyces cerevisiae by high pressure treatment from 120 to 300 MPa in the range of -20 to 50°C followed pseudo first order reaction kinetics. The regression analysis of 43 inactivation rates showed that pressurization at sub-zero temperatures (-20 and -10°C) enhanced the effects of pressure as pressurization at higher temperatures : i.e., pressurization at 190 MPa and -20°C gave the same effect as pressurization at 320 MPa and room temperature. The results imply that high pressure treatment applied to food sterilization at lower temperatures has a greater effect with smaller pressure without destroying the original taste and flavor. Additional effects of sugars and slats on inactivation of yeast are also described.

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The incipient timing of cerebral strokes in the stroke-prone spontaneously hypertensive rats (SHRSP) was biochemically determined by investigating the relationship between the glutathione peroxidase (GSH-Px) activity in erythrocytes and the extent of stroke lesions. When the blood pressure of SHRSPs was maintained at over 240 mmHg, the GSH-Px activity fell, and the body weight also decreased. In SHRSP whose GSH-Px activity in erythrocytes had dropped below 23 units/ml of blood, the incidence of cerebral strokes was 98% (n=88/90). The hematocrit level did not change even after the GSH-Px activity had dropped to 23 units/ml of blood. The reduced GSH-Px activity in erythrocytes observed during continued hypertension was found to be due to a decrease in GSH-Px protein, and not to any inactivation of the enzyme, as evident from immunochemical titration. At the moment when the GSH-Px activity had dropped to 23 units/ml of blood, and the control diet was changed to one based on fish or a hydralazine treatment given, the activity recovered, and an increase in body weight and prolongation of the life-span were observed. It was deduced from these findings that tracing the GSH-Px activity in erythrocytes in SHRSP would serve as an indicator for predicting and prognosing stroke lesions.

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Esterase activity of Brevibacterium linens 62 and Brevibacterium sp. R312 was detected. Each strain had esterase activities that hydrolyzed p-nitrophenyl acetate and α-naphthyl acetate. Biosynthesis and optimum pH and temperature of the two esterase activities showed that the latter were caused by different esterases. The influence of the culture medium and the growth substrate on biosynthesis of the esterase systems were studied. Hydrolysis of methylthioacetate and phenethyl acetate by cell extracts of the two strains was done. No enzymatic ester synthesis reaction was observed. However, transfer reactions by cell extracts of the two strains were done.

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The expression and induction of polyphenol oxidase (PPO) in apple cell culture were examined. PPO activities were measured using chlorogenic acid or L-Dopa as the substrate. The isozymes of PPO were analyzed by electrophoresis and Western blotting. The isozymes expressed in cell culture were different from those in fruit and leaf. Chlorogenic acid oxidase activity in cell culture was raised 10 times by adding macerozyme, and a new isozyme of L-Dopa oxidase was induced by adding it to the culture.

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The presence of granule-bound starch synthase and ribulose bisphosphate carboxylase/oxygenase in precursors to plastids in apexes of developing stolons of potatoes and in plumules of winter wheat was analyzed by immunogold staining and electron microscopy. From the labeling with gold particles of sections, the heretofore designated "proplastid" could be separated into at least two types ; one type devoid of granule-bound starch synthase and ribulose bisphosphate carboxylase/oxygenase and the other already containing these enzymes. The immunogold staining and electron microscopy also showed that these two types of organelles contained DNA and that these two groups seemed to be developmentally related to the formation of plastids. The former type was indistinguishable from the plastid initial of wintering perennials in terms of ultrastructure and can be classified as a plastid initial. The latter type is homogeneous in terms of ultrastructure and the stage of development relative to mature plastids, and it should be referred to as a genuine proplastid.

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Glycinin acidic subunit A_1a and its trypsinized product (A_1a/Try) were found to potentiate insulin-mediated antilipolysis antagonized to isoproterenol in 3T3-L1 preadipocytes and adipocytes. A_1a and A_1a/Try protected 3T3-L1 preadipocytes from the cytotoxic action of trypan blue. Furthermore, the oxygen consumption in rat liver mitochondria that was accelerated during incubation, which implies an increase in the permeability of the inner membrane, was suppressed by the addition of A_1a and A_1a/Try. On the other hand, the chymotryptic product of A_1a did not show such effects. These results suggest that specific amino acid alignment is required for the functions of A_1a and that the insulin-stimulating action of A_1a and A_1a/Try is closely correlated with their stabilizing effect on cell membranes.

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N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp. 5f-1 was a zinc-metalloenzyme which contained 2.06-2.61 g·atom of Zn per mole of enzyme. The zinc atom was required for the catalytic activity and stability of the enzyme. The N-terminal amino acid sequence of Pseudomonas sp. 5f-1 D-AGase showed 32% identity to that of Alcaligenes xylosoxydans subsp. xylosoxydans A-6.

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An injection of Escherichia coli lipopolysaccharide (LPS) increased the activity of histidine decarboxylase (HDC) in bone marrow (BM) cells of C3H/HeN mice much more than in C3H/HeJ mice, which are resistant to various effects of LPS. In WBB6/F1 (W/W^v) mice, which are genetically deficient in mast cells, HDC activity increased more than in C3H/HeN mice. Cultured BM cells of W/W^v mice spontaneously synthesized histamine in a HDC-dependent way. LPS caused a slight increase in HDC-associated histamine synthesis by these cells. Treatment of the BM cells with murine recombinant granulocyte-macrophage colony stimulating factor (mrGM-CSF) increased the histamine synthesis. In addition, treatment with mrGM-CSF made the cells respond to LPS by a dose-dependent increase in HDC activity and histamine synthesis. Most dish-adherent BM cells that had been treated with both mrGM-CSF and LPS for 48h were stained for nonspecific esterase and not for chloroacetate esterase, and had twice as much HDC activity as the nonadherent cells had. Immunocytochemical analysis of the BM cells of W/W^v mice treated with both mrGM-CSF and LPS showed that HDC was in the cytoplasm of cells having Mac-1, a macrophage-differentiation antigen. These results suggest that cells of the macrophage lineage in the BM of mice synthesize histamine.

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Extensive screening for cellulose-producing Acetobacter strains suitable for agitated culture was done by developing the screening conditions. A total of 2096 strains were isolated ; isolation from fruits was particularly efficient. The cellulose productivities of 412 isolates were estimated by culturing in two different media under both shaken and static conditions. No correlation between the amounts of cellulose accumulated in shaken and static cultures was observed. Higher cellulose accumulation was obtained in the shaken cultures using a corn steep liquor/fructose-based medium than a conventional yeast extract/peptone/glucose-based one. Many isolates showed higher cellulose accumulation than well-known cellulose-producing strains. The producer that yielded the highest cellulose accumulation in shaken culture was selected and named Acetobacter sp. BPR 2001. Using this strain, cellulose was produced in a jar fermentor.

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Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells. Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP-1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3 ; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191-1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-1 into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu₂cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-1. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu₂cAMP, and triiodothyronine (T₃), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-1 was increased by Bu₂cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu₂cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations. These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver.

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We have isolated an α-glucosidase(AGL)-encoding gene (agdA) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus niger gene as a probe. Southern hybridization analysis showed that the agdA gene is on a 5.0-kb ScaI fragment and there is a single copy in the A. oryzae chromosome. Comparison with the A. niger agdA gene indicated that the agdA gene contains three putative introns from 52 to 59 nucleotides long, and that it encodes 985 amino acid residues. The deduced amino acid sequence of A. oryzae AGL is 78% homologous with the A. niger AGL. The high degree of homology with the amino acid sequence bordering the putative catalytic residue of a number of AGL enzymes, and this enzyme suggests that Asp492 is a catalytic residue of A. oryzae AGL. The cloned gene was functional. Transformants of A. oryzae containing multiple copies of the cloned agdA gene showed a 6-16 fold increase in AGL activity. Like the Taka-amylase A and glucoamylase genes of A. oryzae, expression of the agdA gene was induced when maltose was provided as a carbon source, but expression was not induced by glucose. This result suggested that cis-element(s) involved in maltose induction may be also present in the agdA promoter region.

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A bacterium isolated from soil was found to accumulate abundant trehalose in its medium. The bacterium was identified as a strain of Micrococcus varians from its taxonomical characteristics and was designated M. varians strain No. 39. Several other strains of Micrococcus and Deinococcus also accumulated extracellular trehalose but M. varians strain No. 39 produced the largest amount. Addition of manganese ions, and excess thiamine and nicotinamide, stimulated trehalose accumulation. Addition of 20 g/liter CSL led to the maximum conversion yield of trehalose in the production phase. Keeping the pH of the culture broth at 6.0 facilitated the maximum trehalose production rate. Cultivation of strain No. 39 at 32°C during the growth phase and 34°C during the production phase resulted in maximal trehalose production. Trehalose non-assimilating mutants (strain No. 7 and 9) derived from strain No. 39 accumulated about 15% more trehalose than the parent strain. Strain No. 7 produced 40 mg/ml trehalose from 100 mg/ml glucose under optimized conditions in a 5-liter jar fermentor.

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A novel ice-nucleating bacterium, KUIN-4 was isolated from a cherry leaf, which was unsusceptible to frost injury. Strain KUIN-4 was identified as Pseudomonas sp. from its characteristics and taxonomics ; the optimum temperature and pH for its growth were 18°C and 4.5, respectively. When strain KUIN-4 was cultured aerobically in CYE medium (pH 4.5) for 48h at 18°C, the ice-nucleating activities of strain KUIN-4 cells and culture broth (extracellular ice-nucleating matter) after removal of the cell were obtained. The ice-nucleating temperature, T₅₀(°C), was indicated to be -2.8°C in cell suspensions (4.25×10⁷ cell/ml) of strain KUIN-4. Also, it had become apparent that the ice-nucleating activity involved class A, B, and C structures as judged by its freezing difference spectrum in D₂O versus H₂O. The ice-nucleating activity of this strain as well as other ice-nucleating bacteria was significantly decreased by heat treatment (40°C, 30min). The ice-nucleating activity from this strain had unique features, which was stable under acidic conditions (pH 3.5-5.0) and was weakly inhibited by denaturants and protein-modifying reagents.

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Abscisic acid (ABA) and indole-3-acetic acid (IAA) were identified by full-scan GC-MS in phloem exudates collected from cotyledons of Pharbitis nil Chois., strain Violet. The occurrence of gibberellin A₁ (GA₁), GA₁₉, and GA₂₀ was also demonstrated by gas chromatography-selected ion monitoring (GC-SIM). The levels of these plant hormones were analyzed by HPLC or GC-SIM in the phloem exudates, and in the cotyledons from seedlings grown under conditions that were inductive and non-inductive for flowering. The amounts of plant hormones in the phloem exudates collected for 6h were 3-50% of those originally present in the cotyledons. The levels of plant hormones were higher in the phloem exudates collected from cotyledons grown under the inductive conditions. These results suggest that the transport of plant hormones from the cotyledon via the phloem is significant, and that endogenous plant hormones are involved in regulating the flowering of P. nil.

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The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by ATP or fructose 1, 6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.

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The occurrence of a new brassinosteroid of (22S, 24R)-3β, 22-dihydroxy-5α-ergostan-6-one, named cathasterone, was demonstrated by a GC-MS analysis in cultured cells of Catharanthus roseus. Its endogenous level was in the range of 2-4ng/g fw, similar to those of brassinolide and castasterone. A feeding experiment with a deuterium-labeled substrate revealed that cathasterone was converted to teasterone and typhasterol. This is the first report of the natural occurrence of cathasterone as a brassinosteroid being the biosynthetic precursor of teasterone.

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Acinetobacter calcoaceticus LMD 79.41 is a unique bacterium containing a soluble quinoprotein D-glucose dehydrogenase (sGDH) in addition to the membrane-bound quinoprotein D-glucose dehydrogenase (mGDH) which is distributed extensively in Gram-negative bacteria. sGDH has been shown to be a distinct enzyme from mGDH, though both enzymes contain a tightly bound pyrroloquinoline quinone (PQQ) as their prosthetic group. In this study, sGDH was detectable in all strains tested of A. calcoaceticus but not in other Gram-negative bacteria tested, indicating that sGDH can be useful as a taxonomic marker for A. calcoaceticus. The binding process of PQQ with both enzymes was examined by using the apoenzymes purified from a PQQ-deficient mutant strain of A. calcoaceticus. sGDH was able to bind two moles of PQQ in one mole of the homodimer with a fairly high affinity. The binding reaction was much faster at alkaline pH than at acidic pH, and required the presence of some divalent cations such as Cd²⁺, Ca²⁺, Sr²⁺, or Mn²⁺. On the other hand, mGDH bound one mol of PQQ in the monomeric enzyme with a relatively slow reacting process, which was optimum at acidic pH and in the presence of different types of divalent cations such as Mg²⁺, Ca²⁺, Zn²⁺, or Sr²⁺ . Thus, it is suggested that sGDH and mGDH have distinct structures around their PQQ binding site. Furthermore, binding of PQQ affects the conformation of both enzymes, which can be shown from the diminishing intrinsic fluorescence of the enzymes and the increase in resistance against proteolysis upon PQQ binding. Data also suggest that the conformational changes caused by PQQ-binding are more dramatic in sGDH than in mGDH. Based on the results obtained, the differences in PQQ-binding mode between the enzymes and the physiological meanings of sGDH are discussed.

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DDMP saponin, which is widely distributed in leguminous seeds, scavenged oxygen radicals when assayed using the xanthine oxidase-NH₂OH method, electron spin resonance (ESR), and chemiluminescence. One mg of DDMP saponin/ml scavenged superoxide at a degree equivalent to 17.1 units of superoxide dismutase/ml by the ESR spin trapping method. This scavenging activity of DDMP saponin is caused by the DDMP moiety attached to the triterpene aglycone since soybean saponin Bb, which is derived from soyasaponin βg, but which lacks this group, did not show the scavenging activity.

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The absorption of (-)-epigallocatechin gallate (EGCg) into the circulation system was studied in rats. EGCg was detected in rat plasma after an oral administration of 50mg, the concentration of EGCg in the plasma reaching the highest level about an hour after dosing, and then decreasing quickly.

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A simple method for preparation of poly-mannuronate from alginate has been developed. By making the best use of the substrate specificity of a poly-guluronate lyase, we prepared a poly-mannuronate of which the properties were almost identical to those of the poly-mannuronate produced by Haug's acid hydrolysis method. Our method is very useful in terms of time and labor saving.

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The inhibitory activity of (9R, 10S, 12Z)-9, 10-dihydroxy-8-oxo-octadecenoic acid and its diacetate, acetonide and methyl ester toward tea pollen tube growth were different, the inhibition by the diacetate being the strongest. Each compound of the fatty acid and its derivatives exhibited more inhibition than its C-9 epimer. The fatty acid and its C-9 epimer showed the same toxicity against HeLa cells.

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We found that Acetobacter xylinum KU-1 produced cellulose from D-arabitol. The maximum cellulose production was obtained when it was grown in a medium containing 2.0% D-arabitol, 1.0% tryptone, and 1.0% yeast extract (pH 5) at 30°C for 96h statically. The productivity was more than 6 times as much as that of D-glucose [productivity (mg/ml-medium): from D-arabitol, 12.4; from D-glucose, 2.0].

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We screened extracts of edible plants for inhibitors of phagocytosis by peritoneal exudate macrophages. 1'-Acetoxychavicol acetate was isolated from the ethyl acetate extract of Languas galanga, and this compound strongly inhibited phagocytosis at an IC₅0 value of 1.2μM with negligible effects on pinocytosis and cell viability. Target(s) of 1'-acetoxychavicol acetate was suggested to be downstream of the signal transduction pathway that is mediated by protein kinase C.

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Glutathione reductase was purified from iron-grown Thiobacillus ferrooxidas AP19-3 to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 100, 000 and was composed of two identical subunits of molecular weight (M_rs, 52, 000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A purified enzyme reduced one mole of the oxidized form of glutathione (GSSG) with one mole of NADPH to produce two moles of the reduced form of glutathione (GSH) and one mole of NADP⁺. The glutathione reductase was most active at pH 6.5 and 40°C, and had an isoelectric point at 5.1. The Michaelis constants of glutathione reductase for GSSG, NADPH, and NADH were 300, 26, and 125μM, respectively.

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We contemplated the formation of γ-dodecalactone through microbial conversion consisting of two steps, the first for oxidizing oleic acid into 10-hydroxystearic acid and the second for the formation of γ-dodecalactone from the hydroxy acid. Microorganisms were screened for production of 10-hydroxystearic acid from oleic acid. A bacterium which could produce the hydroxy acid with a transformation yield of 61.5% at a concentration of 5% oleic acid was isolated. The hydroxy acid was found to be biotransformed to γ-dodecalactone by baker's yeast. The enantiomeric composition of the biosynthetic γ-dodecalactone was estimated to be the (R)-configuration and the optical purity of the lactone was estimated to be 87.6% e.e. The biotransformation yield from oleic acid to γ-dodecalactone was 22.5%.

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fic gene expression at the stationary phase is dependent on RpoS (σ³⁸). For this paper, the fic promoter (P fic) mutants were isolated; C at the position -34 was changed into T. They did not show the stationary phase-specific expression dependent on RpoS in vivo. Furthermore, it was confirmed that they were recognized by Eσ⁷⁰ as efficiently as Eσ³⁸ in vitro.

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Heat treatment was done to improve the substrate specificity for sulfite in Thiobacillus thiooxidans JCM7814 cells. Cell suspension in 0.1M sodium citrate-NaOH buffer (pH 5.5) was heated at 60°C for 30min. Sodium sulfite, sodium sulfide, and sodium dithionite at the concentration of 0.01M protected the cells from thermal inactivation of sulfite oxidation. Sodium sulfite was the most effective among the three additives. Intact cells can oxidize various inorganic sulfur compounds, but the heat-treated cells oxidized only sulfite.

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The dynamical properties of subsite C of hen egg-white lysozyme were investigated using Kyn62-lysozyme as an active analogue. Time-resolved fluorescence depolarization studies showed that the segmental motion of kynurenine which was important in subsite C was described with two components of which the rotational correlation times were φ₁=150ps and φ₂=1.4ns, respectively. Although these two segmental motions retained 90% of motional freedom, the slower motion was completely restricted and the degree of freedom was lost to 40% during the interaction with oligomers of N-acetyl-D-glucosamine.

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Pyruvate decarboxylase from brewer's yeast was modified with the N-hydroxysuccinimide ester of an amylose glycylglycine adduct and some properties of the resulting conjugate were studied with regard to thermostability. By this conjugation the optimum temperature of the activity of pyruvate decarboxylase shifted from 35°C to 40°C. The conjugate showed a greater resistance than the enzyme to inactivating by heat treatment. It is suggested that AG-ONSu can be advantageously used for stabilization of the thermolabile enzyme.

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Previous study has shown that a crude manganese peroxidase (MnP) preparation from the fungus could bleach oxygen-alkaline treated hardwood kraft pulp (OKP) with manganese, glucose, and glucose oxidase. Using purified MnP instead of the crude one also did OKP bleaching with Tween 20. We conclude that MnP is important in this fungal bleaching system.

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Two chitinases (Chi-A and Chi-B) purified from Streptomyces sp. J-13-3 had the same molecular weights (31, 000) and enzymatic properties (optimum pH and temperature of pH 6.0 and 45°C) but had significantly different isoelectric points (3.9 for Chi-A, 3.5 for Chi-B). Chi-A and -B had identical N-terminal amino acid sequences (ADXAAAWNASSVYTGGGSASYNGHN), similar amino acid compositions, and immunological cross-reactivities. A concomitant decrease of Chi-A and increase of Chi-B was observed in their productions during cultivation.

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The structure of precarthamin (1), one of the yellow pigments of safflower petals and a precursor of carthamin (2), was determined on the basis of NMR, mass, and UV/VIS spectra.

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The medium composition of alkalophilic Bacillus sp. Ya-B strain, which produces a new alkaline elastase with very high elastolytic activity, was examined to study its effect on elastase production. The total activity increased 3-fold by using an acid hydrolysate of soybean and the yield after the precipitation with ammonium sulfate was markedly improved.

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Proglycinins containing multiple modifications, that is, with two kinds of single modifications, designed previously to improve food functions, were assessed for ability to form proper conformations. This was done to discover the modification tolerability of proglycinin. Based on our results, three new criteria are proposed for judging formation of proper conformations. Our results suggest that proglycinin molecules tolerate different combinations of single modifications without misfolding.

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An allergenic peptide was isolated from the chymotryptic hydrolysate of gluten by gel filtration and HPLC. The primary structure of the peptide was determined as Ser-Gln-Gln-Gln-Gln-Pro-Pro-Phe-Ser-Gln-Gln-Gln-Pro-Pro-Phe-Ser-Gln-Gln-Gln-Gln-Pro-Pro-Phe-Ser-Gln-Gln-Gln-Pro-Pro-Phe. The amino acid sequence similarity shows that the peptide originated from a low-molecular-weight glutenin chain.

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Ketone bodies were assayed in the urine of ten-week-old vitamin B₁₂-deficient rats by a high-performance liquid chromatography. The urinary excretion of ketone bodies (352.5±68.3μmol/day) as well as of methylmalonic acid was increased significantly by dietary vitamin B₁₂ deficiency.

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Three mutants with lower levels of tryptophan uptake were derived from a tryptophan-producing strain of Corynebacterium glutamicum. They all showed improved production kinetics at the latter half of fermentation and accumulated 10 to 20% more tryptophan than their parent. These results indicate that prevention of amino acid uptake can be a useful strategy to bypass remaining regulatory steps and thereby to increase the yield and productivity in amino acid high-producing strains.

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