This paper describes the molecular and cellular mechanisms of food allergy and oral tolerance including immunomodulation. Food allergy is triggered by an aberrant immune response elicited by oral administration of dietary antigens. Oral tolerance is a state of immunological unresponsiveness induced by oral administration of dietary antigens and is thought to serve to suppress food allergy. This review first describes the characteristic properties of T and B cells relating to milk allergy and also the location of binding sites to T and B cells on allergen molecules. The immunogenicity of allergens is shown to be reduced by the modulations of the T cell binding site, using sophisticated methods such as site-specific mutagenesis. Furthermore, this review focuses on oral tolerance with special reference to the identification of lymphocyte compartment subsets and the immunological mechanism relating to oral tolerance. Finally, the application of oral tolerance for the treatment of allergy is speculated on.

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The structure of aggregates formed by heating dilute BSA solution was analyzed with the fractal concept using light scattering methods. BSA was dissolved in HEPES buffer of pH 7.0 and acetate buffer of pH 5.1 to 0.1% and 0.001% solutions, respectively, and heated at 95°C, varying the heating time t_a. The fractal dimension D_f of the aggregate in the solution was evaluated from static light scattering experiments. The polydispersity exponent τ and the average hydrodynamic radius <R_h> of the aggregates were calculated from dynamic light scattering experiments using master curves obtained by Klein et al. The values of D_f and τ of heat-induced aggregates of BSA at pH 7.0 were about 2.1 and 1.5, respectively, the values of which agreed with those predicted by the reaction-limited cluster-cluster aggregation (RLCCA) model. On the other hand, D_f of heat-induced aggregates at pH 5.1 was about 1.8, which agreed with that predicted by the diffusion-limited cluster-cluster aggregation (DLCCA) model. The dependence of <R_h> for the sample of pH 7.0 on t_a was similar to that of the polystyrene colloids reported previously.

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An NADP-specific glutamate dehydrogenase [L-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp. KSM-635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, with an isoelectric point of pH 4.87, had a molecular mass of approximately 315 kDa consisting of six identical subunits each with a molecular mass of 52 kDa. The pH optima for the aminating and deaminating reactions were 7.5 and 8.5, respectively. The optimum temperature was around 60°C for both. The purified enzyme had a specific activity of 416 units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH optima and at 30°C. The enzyme was specific for NADPH (K_m 44 μM), 2-oxoglutarate (K_m 3.13 mM), NADP⁺ (K_m 29 μM), and L-glutamate (K_m 6.06 mM). The K_m for NH₄Cl was 5.96 mM. The enzyme could be stored without appreciable loss of enzyme activity at 5°C for half a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethanol, although the enzyme activity was abolished within 20 h by freezing at -20°C.

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Theanine, L-N-ethylglutamine, is one of the major components of amino acids in Japanese green tea. To characterize the mode for intestinal absorption of theanine, the ionic dependency and kinetic properties of the theanine- and glutamine-evoked transmural electrical potential difference changes (ΔPD) were investigated in vitro by using everted sacs prepared from the guinea pig ileum. Both theanine and glutamine applied to the luminal side induced dose-dependent increases in ΔPD (increase in serosal positive value). The theanine- and glutamine-evoked ΔPD values conformed to the Michaelis-Menten relationship, with ΔPD_max not being different, whereas the half-saturation concentration was lower for glutamine (3.1±0.2 mM) than for theanine (21.4±0.6 mM). The theanine-evoked ΔPD value was much smaller when theanine was applied in the presence of glutamine than when applied alone. The theanine- and glutamine-evoked ΔPD values were both inhibited by removing Na⁺ from the luminal solution. These results suggest that the intestinal absorption of theanine and glutamine is mediated by a common Na⁺ -coupled co-transporter in the brush-border membrane, the affinity of which is lower for theanine than for glutamine.

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Lipid hydroperoxides in oils and foods were measured by a flow injection analysis system with high sensitivity and selectivity. After sample injection, lipid hydroperoxides were reacted with diphenyl-1-pyrenylphosphine (DPPP) in a stainless steel coil, then the fluorescence intensity of DPPP oxide, that was produced by the reaction, was monitored. By this method, trilinolein hydroperoxide showed good linearity between 0.4 and 79 pmol and their detection limits were 0.2 pmol (signal-to-noise ratio=3). The method made it possible to inject samples at 2-min intervals. There was a good agreement of the amounts of lipid hydroperoxides in oils and foods between by the batch method with DPPP and by the proposed method (coefficient of correlation: r=0.999; n=21; peroxide value=0.09-167 meq/g). With this method, the calibration graph of trilinolein hydroperoxide was useful for all samples tested.

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The N-terminal part of the CreA protein encompassing two zinc fingers was expressed in Escherichia coli as a fusion protein with the maltose binding protein (MalE) of E. coli. Our results show that CreA binds to the promoter of the Taa-G2 gene encoding Taka-amylase A of Aspergillus oryzae. DNase I footprinting experiments showed that CreA bound to three sites with high affinity and to one site with low affinity within the first 401-bp region upstream of the transcription initiation site. All of the sites contained sequences related to the CreA consensus binding site (5'-SYGGRG-3'), and are suggested to participate in repression of the Taa-G2 gene in response to glucose.

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A cDNA and a gene encoding endo-inulinase from Penicillium purpurogenum were isolated, and were cloned for the first time. Two oligonucleotide probes, which were synthesized based on the partial amino acid sequences of the purified endo-inulinase, were used to screen a cDNA library. A 1.7-kb DNA fragment encoding endo-inulinase was isolated and analyzed. A single open reading frame, consisting of 1548-bp, was found to encode a polypeptide that comprised a 25-amino acid signal peptide and 490-amino acid mature protein. All the partial amino acid sequences of the purified enzyme were discovered in the deduced ones. The deduced amino acid sequences of endo-inulinase had similar sequences to those of fructan hydrolases. A 3.5-kb chromosomal DNA fragment encoding endo-inulinase was also isolated and analyzed. The same ORF with the cDNA clone was identified. There were no introns in the endo-inulinase gene.

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The accumulation of a mixture of 5-alk(en)ylresorcinol homologs (ARs) composed of C^13:0, C^15:1, C^15:0, C^17:1, and C^17:0 carbon chains in rice seedlings (Oryza sativa, cv. RD-25) that exhibited high antifungal activity against the rice blast fungus was specific to etiolated plants. ARs accumulated specifically in the etiolated plants, this accumulation being much more in the underground sections, and decreased rapidly when the etiolated seedlings were exposed to light. Indica- and Africa-type cultivars and weedy- and wild-type rice accumulated the ARs, but Japonica-type cultivars did not. Interestingly, the ratio of ARs in the wild rice varieties distantly related to the cultivars was significantly different from that of the wild rice closely related to the cultivars and weedy types.

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We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys²⁷², His³³¹, and Asp³⁰¹) were found in the highly conserved region, but the region surrounding Tyr⁵¹¹, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These findings suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.

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Cell-bound isopullulanase (pullulan 4-glucanohydrolase: EC 3.2.1.57, IPU) from Aspergillus niger ATCC 9642 [Y. Sakano et al., Denpun Kagaku, 37, 39-41 (1990)] was separated into two active components, IPU F1 (pI=5.0) and IPU F2 (pI=4.9), using a Mono-P HR 5/20 column. The substrate specificity on pullulan and panose, specific activity, optimum pH, pH stability, and susceptibility to certain chemical reagents were similar between IPU F1 and IPU F2. IPU F1 and F2 had an identical N-terminal amino acid sequence, A-V-T-A-D-N-S-Q-L-L-. However, IPU F1 contained more total carbohydrate (15.3%) than IPU F2 (12.4%). SDS-polyacrylamide gel electrophoresis showed that the molecular weight of IPU F1 (71, 000) was greater than that of IPU F2 (69, 000). After deglycosylation of IPU F1 and F2 with peptide-N-glycosidase F, the molecular weights of IPU F1 and F2 became 59, 000.

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A gene of alanine racemase, a typical prokaryotic enzyme, was evaluated as a new index for detecting Escherichia coli in foods. An alanine racemase gene fragment containing a non-conserved sequence of the gene was amplified from genomic DNA of E. coli by a polymerase chain reaction, and then labeled with digoxigenin as a probe for detecting E. coli. Food samples and bacteria were each treated as at 25°C for 10 min in 0.1 N NaOH containing 0.5% SDS, before being directly spotted on to nylon membranes for DNA hybridization. The probe was specific for E. coli; all 48 strains of E. coli examined, including such pathogenic strains as E. coli O157:H7, showed positive signals, whereas all 59 strains of non-E. coli species, except for one strain (Shigella sonnei), did not show a signal. Various foods inoculated with E. coli K-12 showed positive signals, whereas no uninoculated foods showed any signal. Quantification of E. coli cells in the death phase by the hybridization method showed good correlation with that by the plate culture method. The alanine racemase gene could prove useful as an index for detecting E. coli in foods.

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LiP of Phanerochaete chrysosporium was chemically modified with MPSS, and acetic and benzoic acid N-hydroxysuccinimide esters to increase the activity in organic solvents. IEF demonstrated the LiP was chemically modified with these modifers. The modification of LiP was also confirmed by GFC and anion-exchange chromatography analyses. In addition, the modified LiP retained its original activity in aqueous solution. Furthermore, these modified LiPs had higher 3, 3'-dimethoxybenzidine oxidation activity than the native LiP in aqueous 70% water-miscible organic solutions including ethylene glycol and diethylene glycol. However, the activities of these modified enzymes were found to be negligibly low in water-immiscible organic solvents such as benzene and toluene. Furthermore, the effects of these introducing groups and the properties of organic solvents on the reaction of LiP in water-miscible organic solvents were investigated. The reaction of LiP modified with aliphatic acid was found to depend on the viscosity of the reaction system containing 70% water-miscible organic solvents. This suggests that the viscosity of the reaction system containing water-miscible organic solvents is important for the reaction of LiP modified with aliphatic acids.

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A β-primeverosidase concerned with alcoholic aroma formation during tea processing was purified from cv. Yabukita (Camellia sinensis var. sinensis). The acetone powder was subjected to ammonium sulfate precipitation and then to chromatographic purification with CM-Toyopearl 650M to give three active fractions, Glucosidases (Gls)-I, II, and III fractions. The main Gl-II fraction was characterized as a β-primeverosidase, which hydrolyzed a β-primeveroside into an aglycone and a primeverose (6-O-β-D-xylopyranosyl-β-D-glucopyranose) and was mainly concerned with alcoholic tea aroma formation. The Gl-II fraction was further seperated into Gls-IIa (main) and IIb (minor) by ion-exchange Mono S column chromatography. Gl-IIa was determined to be a monomeric protein of 61 kDa with pI 9.4. Gl-IIa was stable at temperature lower than 45°C and at between pH 5 and 7, and showed its highest activity at 50°C and at pH 5.

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Two new alcoholic aroma precursors, cis- and trans-linalool 3, 7-oxides 6-O-β-D-apiofuranosyl-β-D-glucopyranosides (1 and 2), as well as two already known compounds, (Z)-3-hexenyl β-D-glucopyranoside (3) and methyl salicylate 6-O-β-D-xylopyranosyl-β-D-glucopyranoside (β-primeveroside: 4), and another new monoterpendiol glycoside, 8-hydroxygeranyl β-primeveroside (5) have recently been isolated as aroma precursors in tea leaves (Camellia sinensis var. sinensis cv. Maoxie) ready for oolong tea processing.

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The generation of fluorescence and 3-deoxyglucosone (3DG), browning, polymerization, and impairment of the amino acid residues of lysozyme incubated with glucose were investigated at 37°C and 50°C at pH 7.4 in a phosphate or TAPSO buffer under aerobic and non-aerobic conditions with or without DETAPAC as a chelating reagent. Browning, the generation of fluorescence, and polymerization were accelerated under the non-aerobic, compared to aerobic, conditions. Moreover, the formation of 3DG was also significantly increased under non-aerobic conditions. The incubation of both reaction systems resulted in noticeable losses of arginine and lysine residues. DETAPAC significantly inhibited the advanced Maillard reaction under both aerobic and non-aerobic conditions. However, DETAPAC had no effect on the impairment of lysine and arginine residues. The generation of fluorescence, browning and polymerization of lysozyme in the TAPSO buffer were markedly inhibited under both aerobic and non-aerobic conditions. These observations suggest that transition metals in the phosphate buffer may have accelerated the formation of Amadori compounds via Schiff's base. In addition, under non-aerobic conditions, the formation of advanced glycation end products from 3DG via Amadori compounds is presumed to be the major pathway, because the formation of N^ε-(carboxymethyl)lysine, glyoxal, and glucosone was accelerated by an oxidative reaction catalyzed with transition metal ions. These presumptions are supported by the results from a lysozyme-3DG reaction system.

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By supplying styrene in the gas phase as the sole carbon and energy source, styrene-degrading aerobic microorganisms were readily isolated from soil samples. They were identified as Corynebacterium pseudodiphtheriticum and similar species, or Pseudomonas sp. Growth experiments on some aromatic compounds, resting-cell reactions with them, and the measurement of degrading enzyme activities suggest that Corynebacterium sp. AC-5 and ST-5 strains metabolize styrene through styrene oxidation into styrene oxide, then convert it into phenylacetaldehyde by a reaction using styrene oxide isomerase, and phenylacetaldehyde is reduced to 2-phenylethanol. The Corynebacterium sp. ST-10 strain did not have styrene oxide isomerase, and metabolized styrene oxide by an unknown enzymatic reaction. Possible metabolism of styrene was proposed for the Corynebacterium strains.

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P-1 was partially hydrolyzed with 0.01, 0.05, and 0.1 M trifluoroacetic acid (TFA), successively, and the dialyzable (E-1, E-2, and E-3) and non-dialyzable (I-1, I-2, and I-3) fractions were prepared and analyzed chemically and immunochemically. Either I-1 or E-1 reacted with anti P-1 serum as strongly as P-1 and were mitogenic. The cross-reactivity of I-2 and I-3 was less than I-1 with anti P-1 serum. However, they were as mitogenic as I-1. The cross-reactivity of E-2 and E-3 to anti P-1 serum was also very weak, and they were not mitogenic. The E-1 fraction had a similar sugar composition to I-1 and P-1. E-2 was a monosaccharide, all of Ara, and would be from the linkage of furanosyl residues in P-1. The composition of E-3 was free from Ara and the structure of E-3 was similar to that of I-3. E-3 would be considered to be deleted arabinofuranose from E-1. These results suggest that the mitogenic activity measured by the alkaline phosphatase assay is a property of the core part, I-3, but that P-1 contains several epitopes other than the core part by the immunochemical analysis.

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A method of obtaining mutants defective in the regulatory function of Ig μ gene expression was developed. Such mutants are useful for discovering the functions of transcription factors and isolating their genes, especially those of DNA-unbinding factors. Cells expressing Ig μ and Eco-GPT simultaneously under control of each μ enhancer were prepared as follows: myeloma X63Ag8.653 cells were transfected with pSV-VμMeΔCH1 encoding a μ gene modified for cell surface μ expression without light chains, selected by neomycin resistance, transfected with the Eco-GPT gene connected to the μ enhancer, and selected with mycophenolic acid in a medium containing xanthine. The μ_m/Eco-GPT cells were mutated with ethane methyl sulfonate (EMS), and selected with toxin-conjugated anti-μ antibody, and then with 6-thioguanine. The μ_m^-/Eco-GPT^- mutants obtained were fused with X63Ag8.653 cells. Fusion caused the mutants to recover μ_m expression, suggesting that some trans-acting transcription factor other than the μ-encoding gene itself was probably mutated.

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The structures of N-linked sugar chains of the storage glycoproteins in mature pea seeds have been estimated. Nine pyridylaminated (PA-) N-linked sugar chains were derived and purified from the hydrazinolysate of the storage glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping, considering the data of sugar composition analysis or sequential exoglycosidase digestions. The deduced structures were further analyzed by IS-MS/MS analysis. Every relevant fragment ion derived from all PA-sugar chains could be assigned on the basis of deduced structures. The estimated nine structures fell into two categories; the first was a typical oligomannose type (Man_8-3GlcNAc₂; 77.7%) which can be hydrolyzed by endo-β-N-acetylglucosaminidase PS [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 228-232 (1996)], the second was a xylose-containing type (Man_4-3Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 22.3%). Among these structures, Man₈GlcNAc₂ (19.7%), Man₆GlcNAc₂ (24.7%), and Man₃Fuc₁Xyl₁GlcNAc₂ (18.8%) were the dominant structures.

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Flames from town gas-oxygen, hydrogen-oxygen, and ethylene-oxygen mixtures, when blown against the surface of an aqueous solution of amines and amino acids, induced an oxidation reaction in the aqueous phase, while an acetylene-oxygen flame failed to oxidize the compounds in solution. The hydrogen flame caused direct hydroxylation of the aromatic rings of phenylglycine homologs. The isomeric ratio of o-, m-, and p-hydroxyphenyl-amino acids produced was in accordance with that obtained by using the reaction systems of Fe²⁺-H₂O₂-EDTA and Fe²⁺-ascorbic acid-H₂O₂-EDTA, which are known to involve a hydroxyl radical as the agent for hydroxylating the aromatic rings. These results strongly suggest that the active species of flame-induced oxidation in an aqueous solution was the hydroxyl radical which was produced in the flames and extracted into the aqueous phase.

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Brown Norway rats were primed intraperitoneally with β-lactoglobulin for 3 wk to induce reaginic antibody, during which time they were fed diets containing 10% each of coconut oil (CO), high oleic safflower oil, safflower oil (SO), or fish oil, then they were challenged for 3 h orally with the antigen. The dietary SO, compared to other dietary fats, resulted in lower circulatory release of rat chymaseII (RChyII), an indicator of degranulation of mucosal mast cells in the intestine, in response to the antigen. Addition of 0.5% curcumin to the CO or SO diet lowered the release. The SO diet, compared to CO diet, tended to increase the concentration of reaginic antibody, but the influence of curcumin addition was not prominent. These results indicate that dietary ingredients differently influence the synthesis of immunoglobulin E and degranulation of mast cells.

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Poly(vinyl alcohol) dehydrogenase (PVADH) purified from Pseudomonas sp. 113P3 catalyzed an oxidation of poly(vinyl alcohol) (PVA) in the presence of pyrroloquinoline quinone (PQQ) to give a β-diketone structure on PVA. Although PVADH oxidized not only enzymatically oxidized PVA but also chemically oxidized PVA, PVA-degrading microorganisms, Pseudomonas sp. 113P3 and Arthrobacter tumescens sp. 52-1 grew on the enzymatically oxidized PVA, but not on the chemically oxidized PVA. This suggests that the growth of PVA-degrading microorganisms is affected by the structure of oxidized PVA.

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Photosynthetic pigments in cultured green cells and in leaves of tobacco were investigated. Total contents of pigments related to the xanthophyll cycle in cultured cells were almost three times greater than those in leaves, and they increased with an increase in the intensity of growth light, suggesting cultured cells are much more susceptible to light than leaves and avoid light stress by the xanthophyll cycle.

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The spin-lattice relaxation time was measured by proton NMR of hydrated tobacco cut-fillers. The relaxation decays of adsorbed water were expressed by a single phase system below 70% relative humidity, while a two-phase system was applicable to water adsorbed at more than 80% relative humidity. From the two-phase model, it was considered that 0.12-0.13 kg water/kg dry tobacco is bound water.

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The thermal stability of three individual molecular species of soy-bean β-conglycinin, α₃, β₃, and modified α₃ (amino terminal Val1-Arg126 amino acid groups deleted), was studied by the change in far- and near-ultraviolet CD spectra and in surface hydrophobicity. The α₃ molecule was less thermostable than β₃, its lower stability being derived from the core of the molecule rather than the extra polypeptide chain on the amino terminal side.

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A total of 1752 strains of osmophilic yeasts were isolated from honey and pollens. Forty-three strains of osmophilic yeasts produced polyols, among which 6 strains produced erythritol in good yields. On the other hand, 52 osmophilic yeasts converted sucrose to fructooligosaccharides, among which 8 strains produced both extra and intracellular β-fructofuranosidase, which converted sucrose to fructooligosaccharides. This investigation concluded that osmophilic yeasts converted sucrose not only to polyols, but also to fructooligosaccharides in good yields.

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The lipid composition of some commercial bakers' yeasts having different freeze-sensitivity in frozen dough was investigated to clarify the correlation between their lipid composition and freeze-tolerance. The total lipid content including neutral lipid, free fatty acid, sterol, and phospholipid ranged between 23.0 to 32.2 mg/100 mg protein of the yeasts tested. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were the main phospholipids found in all yeast strains, but no distinct difference in these components between freeze-tolerant and freeze-sensitive strains was observed. Palmitoleic (C16:1), oleic (C18:1), palmitic (16:0), and stearic (C18:0) acids were the major fatty acids present in total lipid and phospholipid, and unsaturation indices of fatty acid in these lipid components were almost equal by the strains. The molar ratios of sterol to phospholipid of freeze-sensitive strains were higher than those of freeze-tolerant strains. The difference in the sterol-phospholipid ratio that influences the fluidity of plasma membranes in yeast cells was supposed to reflect the difference in freeze-sensitivity of bakers' yeast.

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Polar orange pigments were extracted from the cultured cells of a marine bacterium, strain PC-6, which was temporarily identified as Flavobacterium sp., and purified by chromatographic methods. The structures of two new carotenoid sulfates, (2R, 3S, 2'R, 3'R)-4-ketonostoxanthin 3'-sulfate (1) and (2R, 3R, 2'R, 3'R)-nostoxanthin 3-sulfate (2) were determined by spectroscopic methods.

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We introduced chromosome-mediated genes into mouse thymidine kinase-deficient FM3A (FM3Atk^-) cells, by electroporation. The effects of some parameters on the electric shock-mediated transfection of FM3Atk^- cells were investigated. Gene transfer of mouse L929 metaphase chromosome DNA into FM3Atk^- resulted in a maximum frequency of (3.0±0.3)×10^-5 at a cell density of 2.0×10⁸/ml and chromosome dosage of 5.0×10⁷ cell equivalents/ml in a buffer containing 0.25 M mannitol, 0.5 mM MgCl₂, 0.1 mM CaCl₂, and 1 mM Tris-HCl (pH 7.1). The highest yield of the transformants was obtained at an electric field strength of 1 kV/cm and a capacitance of 35 μF, with a single exponentially decaying pulse at 0°C. The incubation conditions of 60 min at 0°C was optimal for post-shock incubation after electroporation. The tk gene was detected in the transformants by in situ hybridization analysis.

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The genes encoding for trehalose-producing enzymes, a glycosyl-trehalose-producing enzyme (glycosyltransferase) and a glycosyl-trehalose-hydrolyzing enzyme (α-amylase), from Sulfolobus solfataricus KM1 were cloned and expressed in E. coli. The nucleotide sequence of the glycosyltransferase gene and the α-amylase gene indicated proteins with lengths of 728 and 558 amino acids and molecular masses of 86-kDa and 65-kDa, respectively. Regions highly conserved in the α-amylase family exist in the amino acid sequences of these enzymes.

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Kinetic experiments were done on a chitinase from Bacillus sp. with four kinds of 6-O-hydroxyethylchitin (HE-chitin, glycolchitin) with different degrees of substitution (ds) for the 6-O-hydroxyethyl group as substrates. The K_m increased markedly by increase in the ds, and the value for HE-chitin with the ds of 1.0 was 4.5-fold higher than that for a substrate with the ds of 0.33. In contrast, the V_max decreased in response to an increase in the ds, and the value for the former substrate was about a half of that for the latter one.

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Many microorganisms isolated from fish viscera formed trans fatty acids. One of them was identified as Cladosporium sphaerospermum. This is the first report of a fungus forming trans fatty acids. Several bacteria, identified as Pseudomonas sp., Pseudomonas putida, Marinomonas sp., and Schewanella putrefaciens, also formed trans-octadecenoic acids, which increased on growth at high temperature or with phenol. The trans-octadecenoic acids comprised a mixture of various double bond-positional isomers, such as Δ8, Δ9, Δ10, Δ11, and Δ12.

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We established an avidin-biotin method for the sensitive determination of rat IgE and found that a non-specific signal was generated depending on the reaction temperature. When the sera of rats immunized or not with ovalbumin (OVA) were fractionated in a hydroxyapatite column and OVA-specific IgE was determined by the avidin-biotin method at 4°C or 37°C, OVA-specific IgE peaks were detected at 37°C, even with nonimmunized rats, but not at 4°C.

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Arphamenine A, an Arg-Phe analog without a peptide bond, has been reported to be a possible inhibitor of the peptide transporter [H. Daniel and S. A. Adibi, FASEB J., 8, 753 (1994)]. The present study demonstrates that arphamenine A is not a real inhibitor, but is a substrate for the transporter, being transported transepithelially under a proton gradient. The substrate specificity of the peptide transporter was wider than expected.

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The first example of stereoselective reduction with aerobic thermophiles is reported. Various α- and β-keto esters were reduced stereoselectively to the corresponding alcohols by the aerobic thermophiles, Bacillus strains. In particular, the reduction of ethyl 3-methyl-2-oxobutanoate with B. stearothermophilus DSM 297 gave the corresponding (R)-alcohol with high yield in excellent enantioselectively (>99% e.e.). The conversions of keto esters to the corresponding hydroxy esters with Bacillus strains were increased by introduction of glycerol in the reaction mixture as an additive.

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A fluorescent derivative of D-glucose, 2-NBDG, which was previously developed for the evaluation of glucose uptake activity by living cells, was used on Escherichia coli cells and its fate after incorporation in the cells was investigated. 2-NBDG was converted to another fluorescent derivative (2-NBDG metabolite) immediately after it was taken by E. coli cells. This 2-NBDG metabolite was then decomposed to non-fluorescent forms. 2-NBDG metabolite was decomposed into the original 2-NBDG by G6Pase with concurrent liberation of inorganic phosphate. Furthermore, FAB/MS analysis showed that its molecular weight was 420, the same value as that of 2-NBDG 6-phosphate. These indicate 2-NBDG metabolite should be 2-NBDG 6-phosphate. Based on these results, the feasibility of 2-NBDG as a fluorescent non-toxic probe for glucose uptake activity and its application to viability assessment of various living systems are discussed.

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Light irradiation of the culture broth of three odoriferous Neurospora strains resulted in a decrease in the amount of ethyl caproate in the broth. Light reduced the activities of an acyl CoA: alcohol acyltransferase (AACTase) but it increased the esterase activity. Another volatile, 1-octen-3-ol, was also tested but no clear effect was found.

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A freshwater filamentous cyanobacterium, Nostoc spongiaeforme TISTR 8169, produced and excreted a violet pigment, named nostocine A, in the culture medium. Nostocine A inhibited the growth of some typical strains of microorganisms, algae, cultured plants, and established animal cell lines.

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We have isolated a chalcone synthase gene 2 (ST-CHS2) from potato by rapid amplification of cDNA ends by PCR. CHS2 cDNA had high homology to tomato LET-CHS2 (98%), petunia PHCHSJ (94%), potato ST-CHS1B (92%), petunia PHCHSA (92%), and LET-CHS1 (90%) at the overall 389-amino acid level. Genomic hybridization analysis indicated that CHS genes of potato comprise a family of at least six individual members.

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Substantially complete removal (99.78%) of the allergenic soybean protein, Gly m Bd 30 K, was attained by applying simple centrifugation at 10, 000×g and at pH 4.5 in the presence of Na₂SO₄ to defatted soy milk from a genetically mutated soybean cultivar (Tohoku 124) which lacked the α- and α'-subunits of conglycinin. The removal rate was ten times higher than that (97.44%) from a normal soybean cultivar of I.O.M. grade.

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l-Menthol was glucosylated by the α-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as glucosyl donor. When 50 mg of l-menthol and 1 M maltose in 10 mM citrate-phosphate buffer (pH 7.0) were incubated for 24 h at 30°C, a menthylglucoside was selectively obtained as a product. The molar conversion yield based on supplied menthol was 4.5%. The product was identified as l-menthyl α-D-glucopyranoside (α-MenG) by ¹³C-NMR analysis.

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A new route for the synthesis of phosphatidylinositol (PI) having a polyunsaturated fatty acyl group was developed by using lipase and phospholipase C as biocatalysts to supplement the normal chemical reactions.

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