This article covers molecular designs to develop several new fluorometric reagents and their applications to increase the sensitivities up to the picomole level using HPLC for the measurement of biomolecules. The methods were designed to demonstrate the physiological activities, for example (1) N-(9-acridinyl)maleimide (NAM) for the measurement of SH, -S-S-, and sulfite such as cysteine, (2) diphenyl-1-pyrenylphosphine (DPPP) for the hydroperoxides in lipids, serum, tissues, and foodstuffs, (3) 9-bromomethylacridine (9-BrMA), (4) 2-(anthracene-2, 3-dicarboxylimide)ethyltrifluoromethane sulfonate (AE-OTf) for carboxylic acids, and (5) The chiral fluorometric labelling reagent (S)-(+)-2-tert-butyl-2-methyl-1, 3-benzodioxole-4-carboxylic acid (TBMB) to identify the chiralities of amino acids, sugars, and mono- and diacylglycerols.

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In pheromone science, molecular asymmetry is important both chemically and biologically. Techniques for the determination of the absolute configuration of pheromones are discussed first. Enantioselective synthesis of pheromones is the central part of the research on molecular asymmetry in pheromone science. Synthesis of sordidin, the banana weevil pheromone, is given as an example in that area. Molecular asymmetry governs the biodiversity of pheromone perception as illustrated by the detailed discussion on the relationships between absolute configuration and pheromone activity.

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The effects were compared of the addition of graded levels of L-cystine and of L-cysteine (0.3, 3, or 5%) to a 10% casein diet on several metabolic parameters in rats. The growth-promoting effect of cystine was equivalent to that of cysteine. Supplementation of these two amino acids elevated serum cholesterol, liver ascorbic acid, liver nonprotein sulfhydryl (SH) and kidney metallothionein, and reduced the activity of serum ceruloplasmin. The responses of serum cholesterol, liver nonprotein SH, and serum ceruloplasmin to cystine were greater than of those to cysteine. When the basal diet was supplemented with 0.3% of these amino acids, the elevation of liver ascorbic acid by cystine supplementation was less than that by cysteine supplementation. However, when supplemented with 5% of these amino acids, the elevation of liver ascorbic acid by cystine was greater than that by cysteine. There was no difference in the influence of cystine and cysteine on kidney metallothionein. This study demonstrates that dietary cystine and cysteine had the same influence on growth, but had a differential influence on such metabolic parameters as liver nonprotein SH, serum ceruloplasmin, serum cholesterol, and tissue ascorbic acid.

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κ-Casein gene promoter was localized within a 570-bp fragment ( -552/+18) of a 5'-flanking region by the gene transfection assay. Deletion mutation analysis in mammary epithelial cell line, HC11, suggested that there are regulatory element in a region from -439 through -125. Some nuclear proteins from lactating rate mammary gland bind to this region specifically. One of them expressed preferentially during pregnancy bound to a 132-bp fragment (-439/-308) and another expressed preferentially during lactation bound to a 183-bp fragment (-307/-125).

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Mitochondrial serine hydroxymethyltransferase, L-serine: tetrahydrofolate 5, 10-hydroxymethyltransferase (EC 2.1.2.1), (m-SHMT) was extracted and highly purified from Euglena gracilis z. The specific activity increased from the crude extract with 10% yield up to 580-fold through the following steps: ammonium sulfate fractionation, DEAE-cellulose column chromatography and rechromatography, and affinity chromatography with L-lysine-Sepharose 4B. The molecular weight of the purified m-SHMT was 88, 000 by gel filtration through Sephadex G-200, and 44, 000 by SDS-PAGE. One mol of the purified enzyme contained two mol of pyridoxal 5'-phosphate (PLP), indicating that the enzyme is a dimer. Characteristics of the enzyme were examined and compared with SHMTs of other origins. The m-SHMT of Euglena gracilis z had L-threonine aldolase activity as did s-SHMT of the same origin in addition to the usual SHMT activity.

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A thin porous silica membrane (average pore size of 3.3 mm) was prepared by the sol-gel method and used to separate the solute from supercritical carbon dioxide. The characteristics of solute permeation were investigated in respect of the adsorption properties of the solute, the desorption rate of the solute from the membrane being measured and the potential energy of solute near the silica surface being calculated by the molecular modeling technique. It was found that caffeine was strongly adsorbed to the surface and then slowly desorbed to form an adsorption layer, making the pores narrower and causing a molecular-sieving effect. Therefore, the rejection value was positive. On the other hand, the rejection value of n-octanoic acid, which was well adsorbed and rapidly desorbed, was negative. It is presumed that the molecules filled the pores due to their potential energy and were then forced to flow through the pores by the transmembrane pressure.

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Xyloglucan endotransglycosylase activity was identified and defined by transfer of a part of xyloglucan to reduced xyloglucan heptasaccharide ([³H]XXXGol) in an enzyme preparations from suspension-cultured poplar cells. Although the activity was distributed in buffer-soluble and buffer-insoluble fractions associated with cells and in the extracellular fraction, it was mostly recovered in the buffer-insoluble fraction, suggesting that the enzyme was bound to the cell wall. The affinity for acceptor XXXGol was increased at a higher concentration of donor xyloglucan with a constant V_max. The V_max for donor xyloglucan was increased at a higher concentration of the oligosaccharide without any change in affinity. These kinetic data suggest that the acceptor acts by combining with the enzyme independently of the donor. The velocity of the reaction decreased gradually as the heptasaccharide units was increased from two to four, suggesting that the xyloglucan endotransglycosylase reaction caused donor xyloglucan substantially to decrease in molecular size. The activity in buffer-soluble fraction was increased by ABA in auxin-starved cells, when cultured in MS medium containing various plant hormones. Nevertheless, the activity increased markedly at the exponential growth and decreased immediately at the stationary phase of cells in the presence of 2, 4-D. The activity of xyloglucan endotransglycosylase is developmentally regulated during growth but is not directly induced by plant hormones.

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In rice suspension culture, a large part (about 90% of total activity in the culture) of the chitinase activity was found in the medium. Two extracellular chitinases (which we named RCH-A and -B) were separated from the cell suspension by DEAE-cellulofine column chromatography. When cells were treated with N-acetylchitooligosaccharides (chitin oligosaccharides) for 3 days, extracellular chitinase activity increased about 3-fold over the control culture. After the treatment, another extracellular chitinase (named RCH-C) appeared in addition to increases in the levels of RCH-A and -B. Partial amino acid sequences of these enzymes indicated that RCH-A (33.5 kDa) and -B (34 kDa) were class Ib chitinases but RCH-C (27 kDa) was a class III chitinase. RCH-A and -B were capable of actively degrading water-insoluble chitin with high affinities, while RCH-C had less affinity for the substrate. However, when a water-soluble chitin derivative, 6-O-hydroxyethylchitin (glycolchitin) was used, RCH-C as well as RCH-A and -B degraded actively with a high affinity. A synergistic effect was observed when these three chitinases acted simultaneously in the hydrolysis of chitin.

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Treatment of 3T3-L1 adipocytes with either an oleanolic acid glycoside or a 20(S)-protopanaxatriol glycoside increased the secretion of lipoprotein lipase activity into the medium dose-dependently. At a concentration of 100 μg/ml, ginsenosides Ro, Re, Rg₁, and Rh₁ increased the secretion of lipase activity into the medium by 119, 107, 56, and 32%, respectively. The ratio of lipase activity in the medium to cellular lipase activity was 4.7% in control cells and 8.6% in ginsenoside Ro-treated cells, 8.3% in ginsenoside Re-treated cells, 7.0% in ginsenoside Rg₁-treated cells, and 6.3% in ginsenoside Rh₁-treated cells. Ginsenoside Rb₂, which is a 20(S)-protopanaxadiol glycoside, increased the secretion of lipase activity by 16% at 25 μg/ml, and the ratio of lipase activity in the medium to cellular lipase activity was higher in ginsenoside Rb₂-treated cells than in control cells. However, at 100 and 200 μg/ml, ginsenoside Rb₂ decreased the secretion of lipase activity in parallel with cellular lipase activity. Ginsenoside Rd also decreased the secretion of lipase activity in the same dose-dependent manner. Thus, the effective dose for the secretion of lipoprotein lipase activity with ginsenosides varies with their aglycone structure.

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The influence of ascorbic acid (AsA) on the proteins in raw meat paste (surimi) of walleye pollack during the formation of a gel by incubating at 40°C, or 40°C and 90°C was examined by SDS-polyacrylamide gel electrophoresis. AsA significantly enhanced the decrease in the amount of myosin heavy chain (MHC), by promoting the polymerization of MHC via disulfide bridging. Diethylenetriaminepentaacetic acid, a chelator, and tiron, a scavenger of the superoxide radical, both inhibited the promoting effect of AsA on MHC polymerization in surimi. Therefore, the superoxide radical generated via the metal-catalyzed autoxidation of AsA participated in the polymerization of MHC. The production of the cysteine (Cys) thiyl radical due to superoxide in a 10 mM Cys solution was confirmed by electron spin resonance spectroscopy, in which superoxide was generated by photo-activation of riboflavin. These results suggest that the polymerization of MHC by AsA proceeds through a radical-radical reaction of the thiyl radicals of Cys residues that are generated by superoxide radicals.

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Spin trapping of the hydroxyl (OH) radical formed by γ-irradiation in ice was performed by using 5, 5-dimethyl pyrroline N-oxide (DMPO) as a trapping agent. A frozen aqueous solution of DMPO was γ-irradiated at -70°C for three days, and ESR spectra were then measured at room temperature as a function of time after melting. A strong four-line absorption characteristic of the spin adduct (DMPO-OH) was observed and the intensity decreased with time, while no signal was observed when γ-irradiation was carried out at room temperature. The intensity of DMPO-OH increased linearly with radiation dose, but it was not changed by the standing time at -70°C after γ-irradiation, suggesting that the OH radical formed by γ-irradiation in ice was trapped by DMPO in the frozen state, DMPO-OH being stable for more than three days in contrast to the case at room temperature. The quantity of DMPO-OH was decreased in the presence of sodium L-ascorbate. The analysis of the intensity change shows that this solid-state spin trapping technique is useful to measure separately the OH radical scavenging and DMPO-OH scavenging abilities of antioxidants.

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The thermotropic effect of ganglioside GD1a on the phosphatidylcholine membrane immobilized on the porous cellulose acetate filter was investigated by ESR spin-labeling and DSC. The spin-labeled GD1a having 12-DOXYL-stearic acid instead of the long acyl chain of the ceramide portion (GD1a^*) was incorporated into the model membrane. An ESR examination of the membrane showed that GD1a^* undergoes an anisotropic rotation in the wide range of temperature -30-70°C. By monitoring the overall splitting (2T//) of spin-labeled phosphatidylcholine (PC^*), the model membranes were found to show decreased fluidity in accordance with the GD1a content. The phase transition temperature (Tm) of distealoyl-phosphatidylcholine (DSPC) model membranes could be estimated by the measurement of ESR and DSC. The effects of GD1a were found to be more significant in broadening the phase transition rather than in elevating the Tm of DSPC model membranes.

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The primary structure and base specificity of chicken liver RNase CL₁ which has been reported by Miura et al. [Chem. Pharm. Bull., 32, 4053-4060 (1984)] as poly U-preferential RNase, were extensively studied. The sequence study of this enzyme and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster and Drosophila melanogaster suggested that RNase CL₁ consists of three peptides with 17, 19, and 163 amino acid residues. The amino acid sequence of these three peptides were identified. The two small peptides are joined to the large peptide by disulfide bridges. The amino acid sequence of RNase CL₁ had 62 (31.2%) and 63 residues (31.6%) identical with oyster RNase and D. melanogaster RNase, respectively, and belongs to the RNase T₂ family RNase. Reassessment of the base specificity of RNase CL₁ found that it is guanylic acid, then uridylic acid-preferential, and not poly U preferential.

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The effects of 7-ketocholesterol on rat hepatocytes prepared by collagenase perfusion were examined. The viability of cells incubated with 100 μM 7-ketocholesterol was significantly lower than those with cholesterol, although the LDH activity in the cultured medium remained unchanged during the incubation. Hepatocytes treated with 7-ketocholesterol produced large amounts of ·NO and O₂^- in the early stage of incubation. Treatment of the hepatocytes with Carboxy-PTIO, which selectively scavenged ·NO, or with L-NMMA, an inhibitor of ·NO synthase, increased the cell viability. The addition of 7-ketocholesterol to the culture medium tended to increase the ratio of total sterol to phospholipid of the hepatocytes in a time-dependent manner without changing the content of phospholipid. No lipid peroxidation or oxidation of the cellular SH groups, protein SH and glutathione, was apparent. Vitamin E added 1 h before the addition of 7-ketocholesterol prevented the hepatocytes from cell death by suppressing the incorporation of 7-ketocholesterol into the hepatocytes and by scavenging O₂^-.

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Kiwifruit (Actinidia chinensis) contains a cysteine protease, actinidin, and it was suggested to contain two components, A1 and A2. However, the separation of two components was not shown, and the comparison of the two components has not been thoroughly done. We have now shown that actinidin can be separated into six proteases, named KP1, KP2, KP3, KP4, KP5, and KP6, by improved polyacrylamide gel electrophoresis at pH 4.0. Each kiwifruit protease was purified with two ion-exchange resins, Toyopearl-SuperQ and Bakerbond WP-PEI. Before the purification of kiwifruit proteases, excess p-chloromercuribenzoate was added to crude kiwifruit protease to prevent the autodigestion. Each kiwifruit protease had a molecular mass of 23, 500 and the same amino terminal sequences from the first to the thirteenth. They had different pI's. These six kiwifruit proteases were divided into two groups by the effects of DTT and Zn²⁺ on the activity. These results indicated that the six components must be A1, A2, and four previously unknown proteases. Thus we have separated the kiwifruit proteases which were thought to be two, into six components.

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Oxidative depolymerization of chitosan induced by oxygen radical-generating systems was studied. Chitosan, but not chitin, was susceptible to oxidative depolymerization by hydroxyl radical generated through Cu(II)-ascorbate and ultraviolet-H₂O₂ systems in time- and concentration-dependent manners. Superoxide, H₂O₂, and singlet oxygen did not cause depolymerization. Metal ion chelators inhibited depolymerization by Cu(II)-ascorbate system, suggesting that the formation of chitosan-copper ion complex is important in the oxidative depolymerization. The molecular weight of the initial product during depolymerization was similar to that of glucosamine. The results suggest that copper ion could tend to coordinate to the NH₂-groups at the terminal of chitosan and hydroxyl radical generated at its binding site cut off chitosan at the near position.

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Rapid alkalinization of the culture medium in response to an elicitor was observed before the accumulation of transcripts of the chitinase genes. A correlation between the increase in extracellular pH and the induction of the chitinase genes was investigated. N, N'-Dicyclohexylcarbodiimide, an inhibitor of proton channels such as H⁺-ATPase, and carbonyl cyanide m-chlorophenylhydrazone, a protonophore, also induced the expression of basic class I and acidic class II chitinase genes and the rapid alkalinization of the culture medium. Both the alkalinization of the culture medium and the induction of the basic class I chitinase genes in response to the elicitor were inhibited by fusicoccin. These inhibitory effects were also observed by the treatment with staurosporine, and by the treatments with La³⁺ ions or Gd³⁺ ions. By contrast, the elicitor-inducible accumulation of transcripts of the acidic class II chitinase genes was not prevented in the presence of these inhibitors.

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Genomic DNA encoding a β-D-glucosidase (EC 3.2.1.21), which has β-D-fucosidase activity, was cloned from Bifidobacterium breve clb. We sequenced a 1.9-kbp cloned DNA fragment that contained a single open reading frame encoding 460 amino acids with a calculated molecular mass of 51, 513 Da. A putative ribosome binding site was found 5 bp upstream of the initiation codon. The amino acid sequence of this β-D-glucosidase from Bifidobacterium breve clb had 46% identity with that of β-glucosidase from Microbispore bispore. The enzyme of Bifidobacterium breve clb was expressed in Escherichia coli. A cell-free extract prepared from the recombinant strain showed 80 to 90-fold more β-D-glucosidase activity than that from Bifidobacterium breve clb. The recombinant enzyme was purified to homogeneity from cell-free extracts of the recombinant strain using 4 column chromatographies. The recovery of enzyme from the recombinant strain was about 138-fold-higher than that of Bifidobacterium breve clb. The enzymatic properties were similar to those of Bifidobacterium breve clb. For application of this recombinant enzyme, we attempted to synthesize a disaccharide that seemed to be specifically assimilated by Bifidobacteria using the condensation activity of the enzyme.

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A survey of food components with α-glucosidase (AGH) inhibitory activity was conducted to identify a prophylactic effect for diabetes in food. Sardine muscle hydrolyzed by alkaline protease showed potent activity (IC₅₀=48.7 mg/ml) as well as green and oolong teas (IC₅₀=11.1 and 11.3 mg/ml, respectively). Furthermore, hydrolyzates prepared by various proteases gave differing AGH inhibitory activity. DEAE-Sephadex chromatography of the alkaline protease hydrolyzate eluted potent AGH inhibitors (IC₅₀=15.6 mg/ml) with a 50 mM phosphate buffer (pH 7.0) containing 0.3 M NaCl, and their subsequent separation by HPLC in an ODS column showed that there were some inhibitors possessing primary amino groups. This indicates that they would have been high anionic and peptidic compounds.

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Butanol-soluble neutral and acidic thearubigins were prepared by a combination of solvent extraction, fractional precipitation, and Toyopearl column chromatography. These pigments showed similar properties to those of Roberts' thearubigins on the paper chromatogram, and a convex broad band with several peaks on reversed-phase HPLC. The thearubigins were methylated, degallated, and chemically degraded with KMnO₄ under alkaline conditions. From the degradation products, methyl esters of 4-methoxy benzoic acid, 3, 4-dimethoxy benzoic acid, 3, 4, 5-trimethoxy benzoic acid, 3, 4-dimethoxy-1, 2-benzenedicarboxylic acid, 3, 4-dimethoxy-1, 5-benzenedicarboxylic acid, 3, 4-dimethoxy-1, 6-benzenedicarboxylic acid, 3, 4, 5-trimethoxy-1, 2-benzenedicarboxylic acid, 4, 5, 6-trimethoxy-1, 2, 3-benzenetricarboxylic acid, 4, 5, 4', 5'-tetramethoxy-2, 2'-diphenic acid, and 3, 4, 5, 3', 4', 5'-hexamethoxy-2, 2'-diphenic acid were detected by using GC-MS and GC-SIM. The butanol-soluble neutral thearubigins, which had been purified by Toyopearl column chromatography, were hydrolyzed by treating with HCl-BuOH. Cyanidin and delphinidin were identified in the reaction mixture. These results suggest that thearubigins are heterogeneous polymers of flavan-3-ols and flavan-3-ol gallates which have a bond at C4, C6, C8, C2', C5', and C6' in the flavan-3-ol units. In addition to the C4-C8 or C6 interflavanoid linkage, a C6'-C6' linkage was also found, and a possible partial structure for thearubigin is proposed.

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The effects of sodium hexametaphosphate concentration, incubation temperature, and pH on the extraction of polysaccharide from soybean okara (soybean curd waste) were examined. The results suggest that the soybean polysaccharides were almost completely extracted with 50 times their volume of a 2% hexametaphosphate solution at 100°C for 2 h, avoiding protein contamination. The viscosity, molecular weight, and sugar composition of the polysaccharides were compared with those of the commercially available samples. No differences were observed in the molecular weight or neutral sugar composition. However, it was found that the galacturonate distribution in the molecules of the two polysaccharides was different. The viscosity of the polysaccharide solution was changed by adding small amount of salt.

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Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24 was purified as two molecular forms of 84 and 82-kDa by SDS-PAGE. Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus. The DAP IV gene of Pseudomonas sp. WO24 was cloned and expressed in E. coli. The enzyme expressed in E. coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24%. The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1:1) was different from that of the native ones (about 1:4). The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity. The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others.

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Deoxy derivatives of p-nitrophenyl (PNP) α-D-mannopyranoside, PNP 2-deoxy-α-D-arabino-hexopyranoside, 3-deoxy-α-D-arabino-hexopyranoside, 4-deoxy-α-D-lyxo-hexopyranoside, and α-D-rhamno-pyranoside, were synthesized and hydrolytic activities of jack bean and almond α-mannosidases against them were investigated. These α-mannosidases scarcely acted on the 2-, 3-, and 4-deoxy derivatives, while the 6-deoxy one was hydrolyzed by the enzymes as fast as PNP α-D-mannopyranoside, which is a common substrate for α-mannosidase. These results indicate that the hydroxyl groups at C-2, 3, and 4 of the mannopyranoside are necessary to be recognized as a substrate by these enzymes, while that at C-6 does not have so a crucial role in substrate discrimination. Values of K_m and V_max of the enzymes on the hydrolysis of PNP α-D-rhamnopyranoside were obtained from kinetic studies.

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The NAD⁺-dependent 3-hydroxyisobutyrate dehydrogenase [EC 1.1.1.31] was purified to homogeneity from Pseudomonas putida E23. The enzyme was a tetramer (molecular mass, 120 kDa) consisted of identical subunits (molecular mass, 30 kDa). The enzyme was specific for NAD⁺ (K_m, 0.44 mM). The maximal activity was obtained at about pH 10. The enzyme was specific for the L-isomer of 3-hydroxyisobutyrate. In addition to L-3-hydroxyisobutyrate, L-serine, 2-methyl-DL-serine, and 3-hydroxypropionate were substrates. The K_m for L-3-hydroxyisobutyrate, L-serine, 2-methyl-DL-serine, and 3-hydroxypropionate were 0.12, 18, 44, and 83 mM, respectively. The enzyme was inhibited by p-chloromercuribenzoate, HgCl₂, and AgNO₃, but not by EDTA, α, α'-dipyridyl, and o-phenanthroline. The N-terminal 26 amino acid sequence was compared with the sequences deduced from the enzyme genes of rat liver and Pseudomonas aeruginosa.

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A lectin from Amaranthus hypochondriacus var. Mexico (AHML) was purified by affinity chromatography using asialofetuin-Sepharose 4B. AHML is specific for N-acetyl-D-galactosamine as are the other Amaranthus lectins. AHML has no carbohydrate moiety and requires no metal ion for the hemagglutination activity. The pI of AHML is 6.8. AHML has a native molecular mass of 45.0 kDa and is composed of homo-subunits having molecular masses of 36.8 kDa.

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The conditions of cultivation and the composition of medium for Desulfovibrio vulgaris Miyazaki F (DvMF) were examined to obtain cytochrome c₃ labeled with a stable isotope. The growth of DvMF was steady and reproducible under purging with N₂ and under pH control. DvMF was able to grow on a defined medium without natural products. The composition of medium containing a small amount of NH₄Cl as sole nitrogen source was established. Then, [U-¹⁵N]cytochrome c₃ was obtained during the culture of DvMF in a defined medium with ¹⁵NH₄Cl; it was confirmed by ¹H-¹⁵N HMQC. This is the first report of [U-¹⁵N]cytochrome c₃.

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Binding equilibria of Aspergillus niger glucoamylase with its several ligands were observed to analyze the binding modes of the ligands. Steady-state kinetic studies using p-nitrophenyl α-glucoside as a substrate showed that 1-deoxynojirimycin, which is a mixed type inhibitor for Rhizopus glucoamylase, was a competitive type inhibitor bound at the active site of the enzyme, but gluconolactone, which is also a mixed type inhibitor for Rhizopus glucoamylase, was a non-competitive type inhibitor forming a nonproductive ternary complex with the enzyme and the substrate. β-Cyclodextrin, which binds to the starch-binding domain of the enzyme, did not inhibit the enzyme activity, showing that there was no interaction between the catalytic domain and the starch-binding domain for the binding of the substrate and β-cyclodextrin. Isothermal titration calorimetry showed that one 1-deoxynojirimycin molecule and two β-cyclodextrin molecules bind to the catalytic domain and the starch-binding domain of the enzyme, respectively, and there is no significant interaction between the binding of these ligands.

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A highly regioselective (3-position) and efficient (quantitative yield) acylation of bile acids catalyzed by immobilized Candida antarctica lipase was established. Methyl cholate derivatives acylated with long-chain fatty acids (C₁₂-C₁₆) showed an inhibitory effect on the growth of some strains of Gram-positive and -negative bacteria (27-400 μg/ml). The anti-bacterial activity was slightly weaker than has been observed for methyl cholate, while the increased lipophilicity and lower melting points of the present derivatives are well suited for a potential germicide which would be safe and be topically applied. This enzyme-catalyzed transesterification is also demonstrated as an expeditious route to ursodeoxycholic acid, in respect of the regioselective introduction of acyl protecting groups on the hydroxyl groups of the intermediates. 7-Ketolithocholic acid, a known direct precursor of ursodeoxycholic acid, was obtained from cholic acid via chenodeoxycholic acid in a 460% yield and 9 steps.

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Corn coleoptile lectin was isolated using lactose/agarose affinity chromatograpy. The purified lectin, displaying a single band in polyacrylamide gel electrophoresis, was studied by a technique using dextran-raffinose biotinylated derivatives. Raffinose, lactose, lactulose, N-acetyl-D-galactosamine, glucose, and L-fucose were used to measure the inhibition and binding activity of the lectin. The inhibition pattern obtained agreed with previous hemagglutination inhibition data.

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A method for analyzing the EGCg concentration in human serum was developed by using high-performance liquid chromatography with electrochemical detection. EGCg was detected in human serum after the ingestion of 5 g of green tea powder (matsu-cha) dissolved in 200 ml of hot water. The concentration of EGCg in the serum reached the highest level about 2 h after ingesting the green tea, and then decreased.

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A plasmid, pCP53 (11.5 kilo base pairs long), was isolated from Lactobacillus helveticus CP53 and its restriction map was constructed. A chimeric plasmid, pCP53D (4.7 kb), which had a 2.0-kb HindIII fragment from pCP53 and a tetracycline resistance (Tc) gene from Streptococcus faecalis replicated and expressed tetracycline resistance in Lactobacillus casei CP680. Fifteen strains of L. helveticus were transformed with pCP53D by electroporation and selected for Tc resistance. Transformants were obtained from two out of 15 host strains, L. helveticus CP611 and CP791, with 5×10³ and 50 transformants per μg of DNA, respectively.

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Changes in the hepatic concentration of n-3 fatty acids, e.g., eicosapentaenoic acid and linolenic acid, were significantly reduced by their simultaneous administration with sesamin, whereas there was no such effect of sesamin for n-6 and n-9 fatty acids. However, there was no significant difference in lymphatic absorption between eicosapentaenoic acid (n-3) and arachidonic acid (n-6), irrespective of the presence or absence of sesamin.

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The elongation response of 17 near-isogenic dwarf lines of the Shiokari rice cultivar to gibberellins (GAs) was examined. Two strains, ID-18h and ID-19, were selected for further analysis of their endogenous GA levels. It is suggested that ID-18h dwarfism is caused by the deficiency of GA 3β-hydroxylation activity, and ID-19 dwarfism by the insensitivity of the elongation response to physiologically active GAs.

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A cosmid, pR4C1, composed of the actinophage R4 cos sequence and Streptomyces plasmid pIJ365, was encapsidated in R4 phage particles in vivo. [T. Morino et al., Mol. Gen. Genet., 198, 228-233 (1985)] In this report a cosmid derivative, pR4C4, is shown to be also encapsidated by heterogeneous actinophages, SPA10 and SPA38, and transferred to Streptomyces lividans. Use of this transduction and conservation of the DNA packaging mechanism are discussed.

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(1→3, 1→4)-β-Glucanase (EC 3.2.1.73), with a molecular weight of 34, 000 and an isoelectric point of 4.9, was purified to homogeneity from extracts of fresh rice bran. The enzyme specifically hydrolyzed (1→3, 1→4)-β-glucans such as barley β-glucan and lichenans, but laminarins and CM-cellulose were not substrates. Endproduct analysis using barley β-glucan as the substrate suggested that the enzyme is an endo-type (1→3, 1→4)-β-glucanase.

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A new antifungal alkaloid named fungerin was isolated as a fungal metabolite of Fusarium sp. The structure of fungerin was elucidated principally by NMR spectroscopy involving ¹⁵N-NMR studies using the pulsed field gradient (PFG)-HMBC technique at natural abundance.

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The frequency of appearance of aberrant crypt foci (ACF) in the distal colon was significantly lower in rats fed a high fiber (20% wheat bran) diet than in those fed a fiber-free one at 4 weeks after a single injection of 1, 2-dimethylhydrazine (DMH, 20 mg/kg), although crypt/ACF was high in the former relative to the latter. This result suggests that dietary wheat bran effectively serves as a regulator of ACF frequency at early stages after DMH injection.

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The antimutagenic effects of ajoene, which is an organosulfur compound derived from garlic, were investigated by the Ames test. Ajoene inhibited mutagenesis induced by both benzo[a]pyrene (B[a]P) and 4-nitro-1, 2-phenylenediamine (NPD) in a dose-dependent manner. In particular, NPD-induced mutagenesis was more effectively suppressed by ajoene than the B[a]P-induced type. Furthermore, the inhibition of mutagenesis by ajoene was more effective for transition-type mutations than for the frame shift type. HPLC analysis of B[a]P metabolism in the presence of the rat liver microsomal fraction (S-9) showed that ajoene dose-dependently inhibited the metabolic activation of B[a]P. This suggests that ajoene affected the metabolic enzymes in the S-9 fraction.

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Tobacco phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] cDNA was efficiently expressed in E. coli under the control of the lacZ promoter. The enzyme, purified to homogeneity, had the same catalytic activity, and was phosphorylated in vitro by maize PEPC kinase.

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Soluble collagen from fish (sardine) scales was yielded at about 5% with 0.5 M acetic acid after demineralization with EDTA, while a great portion of the collagen remained insoluble. The solubility of this insoluble collagen was about 20% at 45°C (denaturation temperature of soluble collagen) for 24 h. The remaining 80% of the insoluble collagen was denatured in the form of insoluble gelatin, and that may be an interesting food material.

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Reactions of tropolone (1a) and 3-isopropenyl-, 3-acetyl-, 3-acetamido-, and 5-bromotropolones 1b-e with 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide were carried out in the presence of silver carbonate at 80°C. Unsubstituted, 3'-/7'-isopropenyl-, 7'-acetyl-, 7'-acetamido-, and 5'-bromo-substituted 2'-troponyl 2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosides were respectively obtained.

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Indene was found to be microbially transformed to (-)-(1R, 2S)-cis-1, 2-indandiol (7.3% yield, 15% e.e.), (+)-(1R, 2R)-trans-1, 2-indandiol (3%) yield, 83% e.e.), (-)-(R)-indenol (0.1% yield, 89% e.e.), (+)-(S)-2-hydroxy-1-indanone (0.1% yield, 31% e.e.), 1-indanone (0.2% yield), and (+)-(S)-1-indanol (trace, 99% e.e.) by the Pyricularia zingiberi fungus.

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An N-linked sugar chain from 55-kDa royal jelly glycoprotein (RJGP), which maintains the high viability of rat liver primary cultured cell and is a different molecular species from 350-kDa RJGP [Kimura et al., Biosci. Biotech. Biochem., 59, 507-509 (1995)], has been identified. The sugar chains were released by hydrazinolysis followed by N-acetylation and pyridylamination. The structural analysis of the pyridylaminated sugar chain was done by a combination of sequential exo-mannosidase digestions, MALDI-TOF MS, and 500 MHz ¹H-NMR. For the carbohydrate moiety of 55-kDa RJGP, only one N-linked sugar chain has been detected. The structure has been found to be Manα1→2Manα1→6(Manα1→2Manα1→3)Manα1→6(Manα1→2Manα1→2Manα1→3)Manβ1→4GlcNAcβ1→4GlcNAc, which is a non-processed high mannose type structure.

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A new ciguatoxin congener, ciguatoxin-4A (CTX4A), was isolated from cultures of marine dinoflagellate Gambierdiscus toxicus, and its structure was elucidated to be 52-epiciguatoxin-4B on the basis of spectroscopic data. Chromatographic and spectral comparisons indicated that CTX4A was identical with a structurally unelucidated congener known as scaritoxin or SG1.

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