The plasma level of mucosal mast-cell protease was examined to find whether such measurements could be an indicator of allergic response to β-lactoglobulin (β-LG) challenged orally by rats. Brown Norway rats, which had been raised on a bovine milk-free diet, were systemically sensitized on day 0 with a low dose of β-LG, and then by an oral administration of β-LG for 3h on day 14. The oral challenge with β-LG in saline, when compared to saline alone, resulted in a systemic elevation of rat mast-cell protease II (RMCPII), one of the specific markers for gut mucosal mast-cell secretion. The challenge with β-LG in a fat emulsion further increased the level of plasma RMCPII. This manipulation, however, was not successful for detecting any significant difference in mucosal leucotriene C₄, another allergic mediator. An oral challenge with polymerized β-LG did not induce any elevation of the protease, but resulted in a lower plasma level of β-LG-specific IgG. This animal model is thus relevant to investigate the events regulating the mucosal hypersensitivity and humoral immunity to food proteins.

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Purified dextransucrases [EC 2. 4. 1. 5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively. DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V₀) on Sepharose 6B, while the DSM-G was eluted at 1.2 × V₀ volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.

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Several chitosan preparations, either with a comparable degree of deacetylation but differing viscosity or with comparable viscosity but a differing degree of deacetylation, were examined for their effect on the fecal fat excreted from rats fed on a high-fat diet. As the viscosity or deacetylation degree of a chitosan preparation increased, the more its effect on the apparent fat digestibility by rats became conspicuous. A supplement of ascorbic acid to each chitosan diets resulted in a significant depression of fat digestion and absorption in the lumen. The chitosan intake caused a higher level of fat to be excreted in the feces of the corn oil-receiving rats than the lard-receiving ones, although the effect was strong with both diet groups.

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We investigated the mechanism for the inhibition of fat digestion by chitosan, and the synergistic effect of ascorbate. The important inhibition characteristics of fat digestion by chitosan from observations of the ileal contents were that it dissolved in the stomach and then changed to a gelled form, entrapping fat in the intestine. The synergistic effect of ascorbate (AsA) on the inhibition of fat digestion by chitosan is thought not to be acid-dependent but due to the specificity of AsA itself, according to the data resulting from using preparations supplemented with sodium ascorbate (AsN). The mechanism for the synergistic effect is considered to be 1) viscosity reduction in the stomach, which implies that chitosan mixed with a lipid is better than chitosan alone, 2) an increase in the oil-holding capacity of the chitosan gel, and 3) the chitosan-fat gel being more flexible and less likely to leak entrapped fat in the intestinal tract.

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Synthetic α-tocopherol (α-Toc) contains equal amounts of eight different stereoisomers arising from three chiral centers in the phytyl tail. Of these, the four stereoisomers with the 2R configuration are generally more active than their corresponding 2S-isomers. We investigated the change in distribution of α-Toc stereoisomers in the plasma and tissues after intravenously administering all-rac- and SRR-α-Toc acetate. The concentration of 2R-isomers in the rat plasma after administering all-roc-α-Toc acetate was higher than that of the 2S-isomers. On the other hand, the concentration of 2R-isomers in the liver was lower than that of 2S-isomers up to 6h. In the rat plasma after administering only SRR-α-Toc acetate, no SRR-α-Toc was detected after 6h, although SRR-α-Toc in the liver was retained at a higher level than in the other tissues. We presume that the intravenously administered 2R- and 2S-isomers were easily transported into the liver from the plasma, the 2R-isomers being preferentially released from the liver into the blood, whereas the 2S-isomers remained in the liver for up to 6h.

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The binding constant (K) and number of binding sites (N) of atrazine to isolated photosystem (PS) II membranes were measured with an apparent correlation between N and the activity of oxygen evolution. Upon the addition of an electron acceptor, N became equal to the total number of the population of PS II reaction centers irrespective of having oxygen-evolving activity, about 4 mmol per mole of chlorophyll, with a concomitant decline of K from 1.32 (±0. 34)x10⁷M^-1 to 4.09 (±0.40)x10⁶M^-1. NH₂OH and NaCl treatments, which inactivate oxygen evolution, affected neither the binding to PS II membranes of the extrinsic 33-kDa protein or of atrazine. The atrazine binding sites that are latent in CaCl₂-treated PS II membranes was partially restored by the reconstitution of the membranes with isolated extrinsic 33-kDa protein. An oxidizing system involving the 33-kDa protein may provide a suitable structure of PS II reaction center complex for atrazine binding. The level of inhibition of oxygen-evolving activity by atrazine under the saturating intensity of light parallels the fraction of the photosystem (PS) II reaction center with the quinone-binding site blocked by atrazine. In contrast, under a rate-limiting intensity of light, percents of remaining oxygen-evolving activity after the addition of atrazine correlated with the 1.33th power of the fraction of atrazine-free binding sites. Inhibition of PS II complexes more than one that bound with atrazine suggests a cooperation between PS II complexes to evolve oxygen under weak light intensity.

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The purpose of this study was to find whether the regulation of urea synthesis was mediated through the activation of N-acetylglutamate synthesis by ornithine when the thyroid status was manipulated. Experiments were done on three groups of rats, given 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) without triiodothyronine (T₃) treatment, treated with PTU + T₃, or neither PTU nor T₃ (control). Urinary excretion of urea and the liver concentrations of ornithine and N-acetylglutamate in rats given PTU + T₃ were significantly lower than in rats given PTU alone. The liver concentration of N-acetylglutamate was correlated with the liver concentration of ornithine (r=0.920, p<0.001). Ornithine administration (0.5mmol/100g body wt) elevated the liver concentration of N-acetylglutamate in all three groups. The results suggest that the greater concentration of ornithine in the hypothyroid (PTU alone) rats is likely to increase the N-acetylglutamate concentration in liver and stimulate urea synthesis.

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Cellulose production by Acetobacter strains is enhanced by the addition of a small amount of cellulase to the production culture. The effect of an endo-β-1, 4-glucanase from Bacillus subtilis on the cellulose production by Acetobacter xylinum BPR2001 was examined by adding various amounts of the purified glucanase to the culture. The addition of a small amount of this glucanase enhanced cellulose production. Furthermore, it reduced the amount of a polysaccharide called acetan produced. However, an active-site mutant enzyme of the glucanase, which showed no enzyme activity but still had cellulose-binding ability, had no effect on cellulose production. It was concluded, therefore, that the endoglucanase activity itself, but not the cellulose-binding ability, was essential for the enhancement of cellulose production. The structural properties of the cellulose produced in the presence of the endoglucanase were found to be almost identical to those of native bacterial cellulose.

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Lip was modified with various hydrophobic groups, such as Z, Boc, lauroyl, and palmitoyl, using water-soluble acylating reagents. The chemical modification enhanced the dispersibility of Lip in organic solvents, and changed the substrate selectivity, which depends on the length of the carbon chain of triglycerides in hydrolytic reactions. As for the synthesis of triglycerides, Z-modified Lip catalyzed this reaction faster than unmodified Lip. Z-, Boc-, and fatty acid-modified Lips also catalyzed diverse esterification reactions including that of ethyl eicosapentaenoate, lactose oleate, and cholesterol oleate, and the yields of these reactions increased depending on the carbon number of the introducing group.

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A multi-channel continuous-flow analyzer equipped with biosensing devices was developed for multicomponent measurement and its use in automating routine analysis was evaluated. Biosensing was achieved by the aid of an immobilized enzyme reactor installed in the channel, and the channel switching process for the sensing of a different compound was made by using a column-switching rotary valve. Another rotary valve was used for auto-sampling. Both of the two rotary valves were interfaced to a system controller and work conjugatively in a programmed manner. Signal subtraction between different channels was found to be more precise compared with the multi-channel flow-injection analysis method, which is of merit for an analysis utilizing enzyme relay reaction (as for sucrose analysis) or for background signal subtraction. Glucose, lactate, and sucrose content in real samples were measured automatically with high reproducibility, and the results agree well with the kit method.

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The thiol-dependent gelation of bovine serum albumin (BSA) was compared between the intact form (molecular mass of 66kDa) and the domain I-truncated fragment (molecular mass of 45kDa). The hardness of the gel that was induced by incubating with 70mM 2-mercaptoethanol (2-ME) was the maximum at pH 7.5 for intact BSA, but the maximum was at pH 6.5 for the fragment. The gel hardness was increased with increasing protein concentration in a similar manner for intact BSA and for the BSA fragment, although the BSA fragment had a maximum gel hardness at a lower 2-ME concentration than that for intact BSA. Non-reducing SDS-PAGE revealed that the fomation of intermolecular disulfide bonds was involved in the gelation of both the protein forms. Both the disulfide-reduced form of the fragment and intact BSA were found by CD spectra to assume a molten globule-like state. The β-strand content in this state appears to have been closely correlated with the difference in pH optimum for gelation of the two forms of BSA.

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The superoxide dismutase (SOD)-like activity of natural antioxidants was evaluated by measuring the inhibition of pyrogallol autoxidation that is catalyzed by the superoxide radical. Among 22 water-soluble antioxidants tested, L-ascrobic acid, L-ascorbic acid 6-palmitate, glutathione (reduced form), (+)-catechin, and (-)-epicatechin showed effective SOD-like activity. To analyze lipophilic antioxidants, an optically clear organic system composed of diethyl ether, surfactant (dioctyl sulfosuccinate, AOT) and water, called reverse micelles, was developed. The optimum concentrations of AOT, water and pyrogallol for determining SOD-like activity were found to be 50mM, 1.3M, and 40mM, respectively. After proving that pyrogallol autoxidation was mediated by the superoxide anion in that system, the SOD-like activity of 24 lipophilic antioxidants was measured. Cinnamon oil, γ-oryzanol, extract of rosemary leaf, L-α-lecithin, and L-α-cephalin exhibited activity, although the activity of some antioxidants could not be measured because of the intense color or low solubility.

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Urea and citrulline are important constituents in some fermented foods and beverages that control their fermentation processes. An HPLC method using reversed phase mode with sodium dodecyl sulfate and a ureido group-specitic postcolumn reaction with diacetyl monoxime and antipyrine was used for the simultaneous determination of urea and citrulline in soy sauce, wine, and Japanese rice wine. The method was simple and proved to be reliable for the analysis of fermented foods and beverages.

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One of the major allergenic proteins in the soybean 7S-globulin fraction, which was recognized by sera of about 25% soybean-sensitive patients with atopic dermatitis, was identified as α-subunit of β-conglycinin. The IgE antibodies recognizing the α-subunit did not show a cross-reactivity against both α'-and β-subunits known to be highly homologous to α-subunit, and also against other allergenic components identified in soybeans [Ogawa et al., J. Nutr. Sci. Vitaminol., 35, 555-565 (1993)]. The IgE-binding site(s) was estimated to be located on the peptide 232-383 of α-subunit.

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"Process whey protein" was prepared by heating bovine milk whey protein isolate solution at neutral pH under salt-free conditions. The process whey protein solution, being clear, was heated at various pHs (2.0 to 11.0) and NaCl concentrations (0 to 200 mM), and the turbidity and gel properties of the products were then examined. For comparison, the properties of the whey protein isolate treated under the same conditions were measured. The whey protein isolate formed a transparent gel or sol below pH 3 and above pH 7 at low NaCl concentration after heating, but the process whey protein formed transparent gels and sols over a wider range of pH and NaCl concentrations than those of the whey protein isolate. More elastic, firmer, and denser gels were obtained from the process whey protein than from the whey protein isolate. The process whey protein provides a novel food material with useful properties.

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The complete amino acid sequence of pokeweed leaf chitinase-B (PLC-B) has been determined by first sequencing all 19 tryptic peptides derived from the reduced and S-carboxymethylated (RCm-) PLC-B and then connecting them by analyzing the chymotryptic peptides from three fragments produced by cyanogen bromide cleavage of RCm-PLC-B. PLC-B consists of 274 amino acid residues and has a molecular mass of 29, 473 Da. Six cysteine residues are linked by disulfide bonds between Cys20 and Cys67, Cys50 and Cys57, and Cys159 and Cys188. From 58-68% sequence homology of PLC-B with five class III chitinases, it was concluded that PLC-B is a basic class III chitinase.

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The isolation and partial characterization of PAS-4 glycoprotein (78kDa) from bovine milk fat globule membrane (MFGM) is described. PAS-4 was selectively extracted with Triton X-114 nonionic detergent and then fractionated on DEAE-Sepharose at pH 7. 5. The PAS-4 fraction that was not bound on DEAE-Sepharose gave a single band by SDS-PAGE. The recovery of PAS-4 was 57.4% from MFGM. An amino acid analysis found a high percentage of nonpolar residues. Approximately 7.2% of carbohydrate from PAS-4 was composed of mannose, galactose (Gal), N-acetylglucosamine, N-acetylgalactosamine (GalNAc), and sialic acid, most of the Gal and GalNAc in PAS-4 being released after mild alkaline hydrolysis. This indicated that PAS-4 contained both N- and O-linked sugar chains in concordance with the results of lectin affinity. PAS-4 had apparent isoelectric points of 7.45, 7.41, and 7.32, but these were shifted to pI 7.47 by a neuraminidase treatment. The apparent molecular weight of PAS-4 after de-glycosylation with N-glycanase was approximately 57, 000 by SDS-PAGE.

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Alkene monooxygenase from the propene utilizer Nocardia corallina B-276 was separated into three components, and all components were purified to homogeneity and their properties were examined. The epoxidase, with a molecular mass of 95kDa, was considered to catalyze the oxidation of the substrate propene to propylene oxide. It consisted of 53- and 35-kDa subunits, which contained approximately 2-mol of non-heme iron per mole of protein. The reductase, molecular mass 40 kDa, was found to contain an FAD and an Fe₂ S₂ cluster. A third protein, which we have called the coupling protein, with a mass of 14 kDa, appears to function as a regulator of activity. The purified AMO system required NADH as an electron donor, and catalyzed alkene epoxidation only. Acetylene, a specific inhibitor for methane monooxygenase, did not inhibit the AMO activity.

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The effects of the flower lipid-saccharide complex and unsaponifiable matter (1 g/kg of diet) from the sunflower on liver lipid metabolism and intestinal flora was studied in rats given cholesterol-enriched diets. After six weeks of feeding, the microsomal cholesterol concentration in the liver had been significantly reduced with the sunflower diet. The ratio of cholesterol/phospholipid was also reduced by the sunflower diet. The 3-hydroxy-3-methylglutaryl coenzyme A reductase activity of the sunflower groups was significantly lower than that of the control group. There was no significant difference in the cholesterol 7α-hydroxylase activity, although this tended to increase with dietary sunflower consumption. The number of Bacillus in the cecum flora was significantly higher in the lipid-saccharide complex group than in the other groups, while Bifidobacterium and Eubacterium in the cecum flora was significantly higher in the un-saponifiable matter group when compared to the control group. These results suggest that the lipidsaccharide complex and unsaponifiable matter in the sunflower are related to liver cholesterol synthesis and intestinal flora.

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A fluorescent reagent, o-phthalaldehyde (OPA), competitively inhibited porcine pancreatic α-amylase (PPA) with K_i values of 0.7-0.9mM, while α-amylase from Bacillus subtilis (BS) was uncompetitively inhibited, with K_i values of 5.8-7.6mM. In both cases, OPA gave a time-dependent irreversible inactivation, where the amylase activity was lost faster than the maltosidase activity. Zymograms of the course of OPA modification showed that PPA was converted into at least six, faster moving components and BS gave two components. The OPA modification was retarded by the addition of the substrate analog, cyclodextrins, and the OPA modified enzymes decreased in affinity for the substrate soluble starch. Stoichiometric measurement showed that both PPA and BS was inactivated by the incorporation of 1 mol of OPA per mol of enzyme. The role of OPA modification of α-amylases was discussed in relation to the regulation of catalytic activity of enzymes.

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Candida boidinii is a methylotrophic yeast that grows well on oleic acid, D-alanine, or methanol as sole carbon sources. All of these growth substrates induce peroxisomal proliferation ; we refer to them as PICs (Peroxisome Inducing Carbon sources). While the activity of catalase, an important peroxisomal enzyme, was induced by all the PICs, peroxisomal oxidases were induced in a PIC-specific manner. To better understand peroxisomal proliferation, two mutant strains that completely lost the ability to grow on all of the PICs were isolated and analyzed. Peroxisome matrix enzymes (catalase, D-amino acid oxidase, alcohol oxidase, etc. ) in both mutant strains were mislocalized to the cytoso1, and they were active there. Electron microscopy revealed the lack of normal large methanol-induced peroxisomes. We conclude that these mutants are impaired in peroxisomal assembly. Furthermore, the two mutants differed in the catabolite inactivation response to glucose. Finally, although both mutant strains could not grow on D-alanine as a carbon source, they could use D-alanine as a single nitrogen source. Thus, peroxisome assembly seemed to be important for the growth of C. boidinii on all of the PICs.

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Our continued screening to find tipA promoter-inducing substances resulted in the isolation of promoinducin from a mycelial extract of Streptomyces sp. SF2741. Based on various 1D- and 2D-NMR studies, including field gradient (FG)-COSY, HSQC, FG-HMBC, phase-sensitive ¹³C-decoupled HMBC and NOESY experiments, promoinducin's structure was established to be a thiopeptide composed of threonine, some unusual amino acids masked at their carboxyl groups by thiazole or methyloxazole rings, sulfomycinamate and five dehydroalanine residues. Promoinducin induced the tipA promoter at 40ng/ml, and also exhibited strong antibacterial activity against some Gram-positive bacteria.

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The denaturation and renaturation of a poly (α-L-guluronate) lyase from a marine bacterium with sodium dodecyl sulfate, guanidine hydrochloride (GHCI), and urea were investigated mainly by reflection of changes in the enzyme activity, far- and near-ultraviolet circular dichroism, and fluorescence. Amino acid analysis of the enzyme was also done to characterize the protein. The enzyme activity was lost with 6M GHCI and 3M urea in the assay mixture, but not 3% sodium dodecyl sulfate with the high activity. However, the far-ultraviolet circular dichroic spectra indicated that the secondary structure was hardly affected by treatment with 6M GHCI, but was influenced by 3M urea and 3% sodium dodecyl sulfate. By contrast, the near-ultraviolet circular dichroic spectra were attenuated by treatment with 6M GHCI and 3M urea, suggesting changes in the environments of tryptophan residues. Removal of the denaturants by dialysis almost restored all of the original protein conformation. The enzyme was more susceptible to denaturation by urea than by GHCI.

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3'-O-Caffeylerigeroside (pyromeconic acid 3-O-β-D-glucoside 3'-O-caffeyl ester) was obtained from the leaves of Erigeron annuus as a new pyromeconic acid derivative, and its structure was elucidated. Together with the γ-pyrone derivative, pyromeconic acid (3-hydroxy-4H-pyran-4-one) and its β-glucoside (erigeroside) were also isolated from the aerial parts of E. annuus. The siderophile activity of pyromeconic acid was also studied.

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Iso- and anteiso-fatty acids are detected in more than trace amounts in rat skin surface lipid. The terminal portion of even carbon number iso- and anteiso-fatty acids are synthesized respectively from valine (Val) and isoleucine (Ile) by essentially the same reaction sequences established for straight chain fatty acids. This paper describes the stereospecific biosynthesis of these branched chain fatty acids (BCFAs) and alcohols (BCFALs) in rat skin. Dependence of the concentration of these BCFAs on dietary L-Val and L-Ile was studied. Concentrations of even carbon number iso- and anteiso-fatty acid increased respectively with dietary L-Val and L-Ile. The saturation dose appeared to be 2% for L-Val and 1% for L-Ile. Supplementation of the diet with 2% D-Val, however, did not affect the concentration of even carbon number iso-fatty acid in rat skin surface lipid despite a comparable serum Val level to that of the 2% L-Val group. A similar experiment using 1% DL-Ile found that L-isomer, but not D-isomer, in the circulation was used for the biosynthesis of anteiso-fatty acids. This view was applicable to the incorporation of D-Val and DL-Ile into related BCFALs.

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μ-Calpain quickly split the α-connectin in myofibrils into β-connectin, and then produced a 1700-kDa component. Cathepsin D also split α-connectin into β-connectin, further degrading it to fragments smaller than the 1700-kDa component with increasing incubation time. The action of cathepsin D on the connectin molecule was distinctly different from that of μ-calpain in terms of the splitting rate and manner. When freshly excised muscle was exposed to a temperature of 37°C, complete disappearance of connectin (α, βand 1700-kDa component) was observed within 36h. In contrast, at 2°C, about 75% of connectin was retained as β-form even after 3 weeks. The present data suggest that the degradation of connectin in muscle might be caused by μ-calpain in the early stage of aging, and then with time, this action is replaced by m-calpain or cathepsin D. However, the possibility of other intrinsic proteases participating in the degradation of connectin still remains.

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The homoconjugate addition of various organocopper reagents to doubly activated cyclopropane (1)was investigated. The otpimized yield (81%) was obtained when using higher-order cyano cuprate in the presence of trimethylsilyl cyanide, and the reaction was applied to synthesize (±)-juvabione intermediate 9.

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During the decay of wood by the typical white rot fungus Coriolus versicolor, Laccase III was the most abundantly secreted phenol oxidase. In this study, we proposed a possibility of the intermediate degradation steps of polymeric lignin by a purified Laccase III using synthetic [β-¹³C] and [β-¹⁴C] lignin (DHP). When the [β-¹⁴C] DHP was incubated with Laccase III, the water-soluble degradation product formed was about 8%, of the applied [β-¹⁴C] DHP. The enzymic attack of Laccase III catalyzed the cleavage of the intermonomer linkages in the side chain structure of the polymeric lignin. In polymeric lignin metabolism by this fungus, laccase activity was closely related to the accumulation of water-soluble degradation Products.

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Kerase, a serine protease from Streptomyces fradiae, was immobilized on porous glass by covalent attachment of the enzyme through its amino groups. Chemical modification studies have shown that the amino groups can be used to establish covalent attachment of this protease to porous glass, modification of four lysine residues (44.4% of the accessible amino groups) resulting in a 6.5% loss of enzymatic activity. After immobilization, the optimal reaction pH range changed from 7.5-8.5 to 9-10, the protease being stable over a broad pH range of 6-12, while the soluble protease was irreversibly denatured in the alkaline pH range (pH > 8). Moreover, the optimal reaction temperature was changed from 55 to 65°C, showing higher thermal stability. Kerase immobilized onto porous glass was stable for at least 28 days when working in a repeated-batch process of three cycles per day, with an activity loss of 22.1±3.1%.

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Constituent sugars of the polysaccharide bioabsorbent from Alcaligenes latus B-16, which can hold water at more than 1000-fold (maximum 2000-fold) its own weight, were identified by three methods, gas chromatography, high-pressure liquid chromatography, and gas mass spectroscopy. This polysaccharide bioabsorbent is composed of four different sugars : glucose, rhamnose, fucose, and glucuronic acid. The molar ratio of the constituent sugars was : glucose : rhamnose : glucuronic acid : fucose = 1.8 : 1.1 : 1 : 1.1

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To get immortalized endothelial cells, human endothelial cells were transfected with a temperature-sensitive simian virus 40 DNA. The majority of transfected cells showed a prolonged life span and temperature-dependent cell growth. One of the clones, #F5-1, grew either at 33 or 37°C and could be cultured more than 110 generations. The cells expressed several endothelial cell specific characteristics.

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Bacillus subtilis No. 344 is a cytidine-producing mutant strain derived from wild type strain No. 122. When 3-deazauracil-resistant mutants were derived from strain No. 344, some of the mutants had higher productivities of cytidine. Among them, strain No. 428 accumulated 14.2mg/ml cytidine in the culture. Cytidine 5'-triphosphate (CTP) synthetase from strain No. 428 changed to be free from feedback inhibition by CTP, compared with the enzyme from strain No.344.

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A membrane-bound hydrogenase was purified aerobically by one step using a hydroxyapatite column after solubilization by acetone treatment from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1. The enzyme consists of two polypeptides of 63 and 31 kDa, respectively. The amino-terminal amino acid sequences of both subunits were homologous to membrane-bound type [Ni-Fe] hydrogenases from other origins. The thermostability under a hydrogen gas atmosphere is highly stable at 50°C, which is the optimum temperature for the cell growth.

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To analyze the functional contribution to the ssiA function of subsections of the ssiA-determinant sequence based on their dimensions, we constructed ssiA mutants carrying insertions and deletions. Results of the examination of the ssiA mutants told us that, in addition to the base sequence, the dimensions were crucial factors for the functional contribution of the subsections of ssiA.

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Seeds of soybean [Glycine max (L. ) Merr. var. Bay] were subjected to X-ray irradiation (21.4kR), and the M₂ generation was evaluated for the stearic acid content in the seed oil. Treatment with X-ray irradiation significantly increased genetic variability in the stearic acid content of the oil from Bay variety in comparison with the control plants. Among the 2513 M₂ plants tested, one mutant named M25 was selected for its stearic acid content of 20. 8%, about seven-fold higher than that of the original variety. An inverse relationship of stearic acid with oleic and linoleic acids was observed. Mutant M25 always had higher stearic acid content under different environmental conditions in the M₃ generation.

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USF-19A, a soybean lipoxygenase (SBL) and human 5-lipoxy-genase (5-LO) inhibitor, was isolated from Streptomyces sp. USF-19 strain. Its chemical structure was determined by spectroscopic evidence to be a new member of the antimycin antibiotic family. The IC₅0 value of USF-19A against 5-LO was 28. 0μM.

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The adsorption of benzoic acid and its derivatives to swollen chitosan beads increased with decreasing pK_a value, except for those compounds having a hydroxyl group in the aromatic ring. The hydroxyl group had the ability to stimulate adsorption, this effect being stronger than that of pK_as. The presence of glucose and methanol also stimulated the adsorption of benzoic acid.

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An antisense gene for Aralia cordata cinnamyl alcohol dehydrogenase (CAD) was introduced into tobacco plants. Two transgenic plants showed 55 and 20% reduction of the CAD activity compared to that of the control plant. Lignin content measured by the acetyl bromide method showed no significant differences between these plants and the control plant. However, the content of p-hydroxycinnamaldehyde groups in lignin was higher in the transgenic than in the control plants. The increase of p-hydroxycinnamaldehyde groups correlated inversely with the levels of CAD activity. Phloroglucinol staining of transverse sections of stem internode indicated that the fiber secondary wall reacted more strongly with the reagent in the transgenic plants. These results suggested that suppression of CAD activity in tobacco plants results in an increase of cinnamaldehyde groups in lignin, but no significant change in lignin content.

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Microorganisms degrading carbazole (CA), a model substrate of heterocyclic nitrogen compounds in crude petroleum oil, were screened under microaerobic conditions, i. e. nitrogen gas substituted conditions. Eight bacteria were isolated and identified. For example, Bacillus sp. KUKK-4 degraded 31% of CA when cultivated for 28 days in a medium initially containing CA at 1000mg/l with shaking under the microaerobic conditions.

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Under optimized conditions using Candida rugosa IFO 1364 grown on 1% D-sorbitol, 17. 4 mg/ml of [(2-hydroxyethyl)thio]acetic acid (HETA) was produced from 20 mg/ml of thiodiglycol (TDG) after 2 days of incubation at 30°C in the reaction mixture. The molar conversion ratio of TDG to HETA was 78%.

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Isolation of 2R, 5R-dihydroxymethyl-3R, 4R-dihydroxypyrrolidine (DMDP, 1) was isolated from the fermentation broth of Streptomyces sp. KSC-5791 by chromatographic techniques including preparative HPLC in a Shodex Asahipak NH2P-50 column. Compound 1 inhibited trehalases of Corynebacterium sp. ¹⁾ and diamondback moth (Plutella xylostella), but did not inhibit porcine kidney trehalase.

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Racemic 2-(6-benzyloxy-2, 5, 7, 8-tetramethyl-2-chromanyl) ethanol (5) was successfully resolved by employing asymmetric benzoylation and lipoprotein lipase (LPL). (S)-5 was obtained with more than 99% e. e.

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There was significant binding in autoclaved cells of Enterococcus faecalis FK-23 toward Trp-P1 and Trp-P2. Although the binding ability towards aflatoxins B₁, B₂, G₁, and G₂ was less compared with the significant binding toward Trp-P1 and Trp-P2, higher percentage binding toward these aflatoxins was observed when the amount of the cell preparation was increased.

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The activity of biotin synthesis from dethiobiotin was found in cell-free extracts of an Escherichia coli bio B transformant. Among the sulfur compounds tested, only S-adenosyl-L-methionine (Ado-Met) had a significant effect, while methionine and cysteine were inert. The activity was linearly stimulated by increasing protein concentration. When the dialyzed cell-free extracts were used for the reaction, NADP⁺, NADPH, and FAD among the well-known cofactors tested promoted the activity. Furthermore, in the presence of AdoMet, cysteine was apparently effective for biotin synthetic activity.

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Williopsis salicorniae IFO 10733 (type strain), which is characterized by the formation of saturn-shaped ascospores, by the incapability of assimilating nitrate, and by a lower DNA base composition (36.7 mol% G + C), was examined for its partial base sequences of 18S and 26S rRNAs. In the 18S rRNA partial base sequencings, it had an identical base sequence with the type strain of Ogataea glucozyma ( ≡ Pichia glucozyma, ≡ Hansenula glucozyma), which produces hat-shaped ascospores and has the ability to assimilate nitrate and methanol and a higher DNA base composition (45.1 mol% G + C). In the 26S rRNA partial base sequencings, the base differences were four, and the percent similarity was 87 between the type strains of the two species. The data obtained are discussed phylogenetically and taxonomically.

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Polypeptides were solubilized from a fresh egg yolk suspension, and the reaction mixture was clarified by using an enzyme preparation. Among the various enzyme preparations, only Newlase F could clarify the yolk suspension when used alone. Since Newlase F has both protease and lipase activities, it was suggested that several mixed enzyme preparations which had resulting protease and lipase activities could be available for use. When a 1O% yolk suspension was treated with Papain W-40/Lipase F, for example, the proteins of egg yolk were clarified within 4h, and the egg yolk oil rose to the surface of the liquid. About 80% of the polypeptides were liberated to the transparent liquid.

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Four gibberellins (GAs), GA₁₉, GA₂₀, GA₂₄, and GA₅₃, were identified by gas chromatography-mass spectrometry (GC-MS) from the dormant bulbils of Dioscorea japonica Thunb. ex Murray (Japanese yam), suggesting that two biosynthetic pathways of GAs, early 13- and non-13-hydroxylation pathways, are operating in the bulbils. Abscisic acid was also identified by GC-MS.

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A cDNA for proctase B from Aspergillus niger var. macrosporus was isolated and sequenced. The deduced amino acid sequence (394 residues) of the preproform of the enzyme was highly homologous (98% identify) with those of aspergillopepsins I from A. awamori and A. saitoi, and moderately homologous (68% identity) with that of A. oryzae. Most of the sequence differences were found in the carboxyl-terminal domain.

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Two new congeners (22R, 23R, 24S)-22, 23-dihydroxy-24-methyl-5α-cholestan-3α-ol 2 and (22R, 23R, 24S)-22, 23-dihydroxy-24-methyl-5α-cholestan-3-one 4 that are termed 6-deoxotyphasterol and 3-dehydro-6-deoxoteasterone, respectively, occur in relatively large amounts in the mature pollen of Cupressus arizonica. GC-MS, NMR spectroscopy, the reduction of 4 to 2, and the independent formation of 2 by the reduction of typhasterol were used to identify the new compounds. In the rice lamina bioassay, 2 showed weak activity. 6-Deoxocastasterone, castasterone, typhasterol, an epicastasterone-like compound, teasterone, 28-homocastasterone, 3-dehydroteasterone, brassinolide, and dolichosterone (or 24-epibrassinolide) were also present. These brassinosteroids were identified by co-chromatography with standards after being converted for an HPLC analysis of bioactive fractions. Six other peaks have not yet been assigned. 6-Deoxotyphasterol and 3-dehydro-6-deoxoteasterone should prove useful for exploring the early stages of the biosynthetic pathway(s) to brassinosteroids.

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DNA intercalation of 9-aminoacridine and its derivatives was evaluated by the UV-VIS absorption-Scatchard plot technique, and it was suggested that two molecules of 9-amino-2, 7-dibromoacridine were located between neighboring base pairs of DNA.

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