Acetobacter xylinum subsp. sucrofermentans BPR2001, a cellulose-producing bacterium, that was newly isolated from a natural source, produced large amounts of the water-soluble polysaccharide, acetan. UDP-glucose is known to be the direct precursor in the synthetic pathways of both cellulose and acetan. We attempted to breed mutant strains and succeeded in obtaining one, BPR3001A, which produced 65% more bacterial cellulose and accumulated 83% less acetan than the parent strain, BPR2001. The cellulose formed was found to be structurally ordered, with higher degrees of polymerization and crystallinity and larger crystallite size than those produced by BPR2001 and other conventional strains. Furthermore, a processed dry sheet of this cellulose exhibited a higher Young’s modulus than that of the wild strain. The ordered structure of the cellulose obtained was probably due to the decreased amount of acetan which may reflect the ribbon assembly of cellulose fibrils without prevention of hydrogen bonding between microfibrils.
Cellulose triacetate prepared from bacterial cellulose of Acetobacter xylinum subsp. sucrofermentans BPR3001A showed a higher degree of polymerization and higher mechanical strength than that from the cotton linter. The fine fibrils of bacterial cellulose required only a short time for acetylation which preserved the high degree of polymerization.
Two monoterpentriols were isolated from the stipe segment without either the pileus or apical growth zone of the harvested fruiting body of F. velutipes. The structures of these monoterpentriols were elucidated as (1R, 2R, 4R, 8S)-(−)-p-menthane-2,8,9-triol (1) and its 8-epimer (2) based on a spectroscopic analysis and stereoselective chemical transformation from an authentic monoterpene alcohol. These monoterpentriols showed growth-promoting activities on the excised stipe with the pileus segment, which had been excised from a fruiting body just under the growth zone before the middle stage of the rapid growth period, at concentrations below 10 ppm, while the growth of the segment was inhibited at concentrations of 100 ppm and above.
A novel phenolic metabolite, ornatipolide, was isolated from the Boletus ornatipes fungus. Its structure was established by a combination of spectroscopic and chemical methods, and by an X-ray crystallographic analysis.
Cell-free extracts from ripening seeds of Arctium lappa L. catalyzed the enantioselective formation of (-)-pinoresinol, (-)-lariciresinol and (-)-secoisolariciresinol from achiral coniferyl alcohol in the presence of NADPH and H2O2. The enantioselectivity of the lignan formation was opposite to that of the (+)-secoisolariciresinol formation catalyzed by cell-free extracts from petioles of the same plant species.
Embryogenesis was induced from regenerated carrot plantlets in a shorter time and at a higher frequency than that observed previously from hypocotyl explants of carrot seedlings (Masuda et al. J. Plant Physiol. 145, 531 (1995)). Serial observations of embryogenesis from single cells to globular embryos via cell clusters were possible in the conditioned medium. The ability to produce embryos seemed to be acquired during the transition from torpedo-shaped embryos to regenerated plantlets, when differentiation of the epidermal cells of regenerated plantlets occurred.
A recombinant β-galactosidase, which was obtained from the β-galactosidase C gene of Bacillus circulans and cleaves the non-reducing end galactosyl residue of β(1→3)-linkages selectively, was immobilized using CNBr-Sepharose. Although the effect of pH was not changed by the immobilization, the thermostability and stability in the presence of DMF were increased. Optimization of the transglycosylation using para-nitrophenyl β-D-galactopyranoside as a donor and benzyl-α-D-N-acetylgalactosaminide as an acceptor afforded a β(1→3)-linked disaccharide derivative with 62% molar yield in a gram scale synthesis. Using the methyl-analogue as an acceptor, 53% of the acceptor was converted to the respective β(1→3)-disaccharide.
The leukemia inhibitory factor (LIF) is a secretory glycoprotein and a pluripotent growth factor which acts in diverse cell systems. LIF has been reported to be heavily glycosylated. In this paper, we examine the transient expression of rat LIF (rLIF) in COS7 cells and its glycosylation by a PNGaseF treatment and lectin blot. rLIF expression in COS7 cells resulted in seven molecular species being produced with zero to six N-glycosyl moieties. Mutated rLIF proteins with substitutions at the seven possible N-glycosylation sites were also expressed. An analysis of the molecular weight of the mutated rLIF confirmed the six N-glycosylation sites. Bioassays of mouse leukemia cell lines were performed to analyze the contribution of the glycosyl moieties to their functions. We found that the glycosyl moieties at each of the N-glycosylation sites were not essential to their function of the protein, but the reduced functions to promote the proliferation of DA-1a cells that had been observed for some mutants suggests a biochemical role for the in vitro function.
Mannan-binding protein (MBP) is a calcium-dependent mammalian serum lectin important in first-line host defense. MBP belongs to the collectin family, which is characterized by an NH2-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain (CRD). We have expressed a recombinant human MBP, consisting of the short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain, and the CRD, in Escherichia coli. The truncated MBP was capable of forming trimers by association of the neck domain and could bind sugar with a specificity similar to that of the native form. Results of hemagglutination inhibition (HI) assay of influenza A virus showed that the truncated MBP inhibited hemagglutination less strongly, although the native MBP induced the HI phenomenon. These results suggest that an oligomeric structure is an advantage for MBP to have full biological activity against influenza A virus.
An endo-β-N-acetylglucosaminidase specific for plant glycoprotein oligosaccharides was purified from the culture fluid of a fungus. The Mr of the purified enzyme was 89,000. This enzyme was stable at pH 5.5-7.0, up to 30°C, and showed the highest activity at pH 6.0. Among sugar chains tested, xylose-containing sugar chains (M3X, M3FX, and M2FX) were the most favored substrates. Oligomannose type (M3, M5, and M9) and hybrid type (GNM3) sugar chains were hydrolyzed much more slowly than xylose-containing sugar chains, and a complex type sugar chain (GN2M3) was not hydrolyzed at all by the enzyme. Moreover, the enzyme released sugar chains from native horseradish peroxidase and stem bromelain, which were not hydrolyzed by other endo-β-N-acetylglucosaminidases (Endo H, D, and F). The enzyme could transfer the xylose-containing sugar chain from bromelain to DNS-Asn-GlcNAc-Fuc.
Three forms of α-amylases, designated Amyl I, Amyl II, and Amyl III were purified to a homogenous state by several column chromatographies from a koji culture in wheat bran of a strain of black mold, which was isolated in Indonesia and identified as Aspergillus awamori. They have molecular weights of 49,000, 63,000, and 97,000 by SDS-PAGE, respectively, and the optimum pHs were 4.0 for Amyl I and 5.5 for both Amyl II and Amyl III on soluble starch. Amyl I hydrolyzed malto-tetraose, -pentaose, -hexaose, -heptaose, and β- and γ-cyclodextrin to produce maltose and maltotriose as major products but not or hardly hydrolyzed maltose, isomaltose, maltotriose, isomaltotriose, α-cyclodextrin, or raw corn starch. On the other hand, both Amyl II and Amyl III hydrolyzed maltotriose as well as all the maltooligosaccharides described above and α-, β-, and γ-cyclodextrin, and even raw corn starch as well as heat-gelatinized corn starch to produce maltose as a major product and glucose and maltotriose as minor products, but they did not hydrolyze maltose, isomaltose, or isomaltotriose. The limit hydrolyses of soluble starch with three kinds of enzymes were 33% for Amyl I, 35% for Amyl II, and 38% for Amyl III, the reaction products had α-anomeric forms by NMR analysis, and the blue color reaction with I2 disappeared completely at about 18% of hydrolysis of the starch for all enzymes.
We isolated a genomic DNA harboring a cytosolic ascorbate peroxidase gene (ApxSC) from a genomic library of the strawberry (Fragaria x ananassa). Restriction mapping and sequence analyses showed that the DNA is composed of 2.5 kb of the full-length ApxSC gene, 3.7 kb of the 5′-upstream region, and 0.5 kb of the 3′-downstream region. The ApxSC genomic DNA contains 10 exons and 9 introns, which is similar to the structure of pea ApxI. A primer extension analysis suggested that the transcription of ApxSC gene was started at three start sites with different degrees. The promoter region of ApxSC gene contains a sequence or structure distinct from other reported plant ascorbate peroxidase genes, though with several known functional elements such as a TATA box.
Staurosporine is a potent inhibitor of protein kinase C. To identify the genes that functionally interact with the Pkc1 pathway of the yeast Saccharomyces cerevisiae, we screened for the genes that cause induced staurosporine sensitivity when overexpressed from a galactose-inducible promoter. The novel gene ISR1 encodes a predicted protein kinase with the highest sequence similarity to mammalian Raf in the kinase domain. Drug sensitivity induced by ISR1 overexpression is specific to staurosporine. Although ISR1 disruption causes no obvious phenotype, it does exacerbate the phenotypes of a temperature-sensitive allele (stt1-1) of PKC1, but not of the mpk1 and bck1 mutants of the Mpk1 MAP kinase pathway. These results suggest that Isr1 functions in an event important for growth in a manner redundant with a Mpk1-independent branch of the Pkc1 signalling pathways.
Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous “serine protease inhibitors” produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35-41% similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a “His-Glu-Xaa-Xaa-His” motif of SGMPII was identified as the residue modified by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2, an active-site-directed irreversible inhibitor of thermolysin-like metalloproteases. Based on the sequence comparison of SGMPII and other bacterial metalloproteases, we discuss the structural basis for the differences in substrate specificity and stability between SGMPII and other thermolysin-like proteases. A possible SSI-binding locus of SGMPII is also proposed.
Hepatocyte growth factor (HGF) is most likely a physiological hepatotrophic factor that triggers regeneration of the injured liver. Histamine may also be important in the pathophysiology of the injured liver. Previously we showed that histamine production was increased in liver macrophages of mice injected with CCl4, a well-known hepatotoxin. Therefore, it is likely that the biological actions of histamine in repairing processes of the injured liver are mediated by HGF. This study was aimed at examining the effects of histamine on production of HGF using, as a model, the human promyelocytic leukemia cells, HL-60. 12-o-Tetradecanoylphorbol-13-acetate (TPA) markedly stimulated HGF production and release from the cells; the maximal amount of HGF was released at a concentration of 3 ng/ml of TPA. Histamine significantly stimulated the TPA-induced HGF production and release in these cells, depending on incubation time and its dose. These actions of histamine were abrogated by a H2 receptor antagonist, ranitidine.
We obtained information on the full length tobacco NADPH-cytochrome P450 oxidoreductase (P450 reductase) by a combination of the cDNA clone pCTR1 and the genomic DNA clone pGTR1. The deduced primary structure consisting of 713 amino acid residues contained sequences corresponding to FMN, FAD, and NADPH-binding regions. Based on this information, we prepared the full-length cDNA pFTR of tobacco P450 reductase by RT-PCR and expressed it in the yeast Saccharomyces cerevisiae. The transformed yeast cells carrying pFTR produced the corresponding mRNA and protein, and had increased cytochrome c reductase activity in the microsomes. An in vitro reconstitution system of the yeast microsomal fractions expressed tobacco P450 reductase and rat P450 1A1 showed an increased 7-ethoxycoumarin O-deethylase activity. These results indicated that tobacco P450 reductase expressed in the yeast microsomes coupled with rat P450 1A1 resulting in an increased monooxygenase activity.
Cysteine protease activity in mycelial culture increased 7.7-fold after fruit body formation in Pleurotus ostreatus, using the Leu pNA (LPNA) cleavage assay. The enzyme was purified from fruit bodies and its Mr was 97,000 by gel filtration and 48,500 by SDS-PAGE, indicating that it is a dimer. The enzyme was sensitive to iodoacetic acid, p-chloromercuribenzoate, N-ethylmaleimide, and HgCl2. The sequence of the first 9 N-terminal amino acids of cysteine protease was ASGLXXAIL.
The methanol extract from Machilus thunbergii showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver metabolizing enzymes. The methanol extract from M. thunbergii was successively re-extracted with chloroform, butanol and water. A suppressive compound in the chloroform extract fraction was isolated by SiO2 column chromatography and identified as meso-dihydroguaiaretic acid by GC-MS, and 1H- and 13C-NMR spectroscopy. Meso-dihydroguaiaretic acid inhibited of the SOS-inducing activity of Trp-P-1 in the umu test. Gene expression was suppressed by 62% at less than 0.18 μmol/ml, the ID50 value being 0.08 μmol/ml. Compound 1 was also assayed with aflatoxin B1 (AfB1) and showed a suppressive effect.
Jasmonates and related compounds were found to elicit the seed germination of the important root parasites, clover broomrape (Orobanche minor Smith) and witchweed [Striga hermonthica (Del.) Benth]. The stimulation of seed germination by the esters was more effective than by the corresponding free acids, and methyl jasmonate (MJA) was the most active stimulant among the compounds tested.
Incubation of Escherichia coli γ-glutamylcysteine synthetase with L-glutamic acid γ-monohydroxamate and ATP caused slow but irreversible inhibition of the enzyme, and more than 90% activity was lost in three days. The enzyme was not inactivated when ATP was absent or L-aspartic acid β-monohydroxamate was substituted for L-glutamic acid γ-monohydroxamate, suggesting that the inactivation process reflected a mechanism-based reaction of L-glutamic acid γ-monohydroxamate and ATP.
Transfection of nearly senesced human fibroblasts with plasmids encoding HPV16 E6 protein or dominant-negative p53 mutants greatly increased their colony-forming ability. Isolated colonies with these plasmids showed extension of lifespan compared to those with a control plasmid. These data demonstrate that p53 plays a major role in senescence in normal human fibroblasts.
The two staphylococcal bi-component toxins, leukocidin and γ-hemolysin share LukF [Kamio et al, FEBS Lett., 321, 15-18 (1993)]. This report identifies the pivotal amino acid residues in the N-terminal region of LukF for the leukocytolytic and hemolytic activities in the presence of LukS and Hlg2, respectively, measuring the toxin activiy of a series of LukF mutants with truncated N-terminals. The data obtained showed that the LukF mutant TF21, lacking 20 amino acid residues at the N-terminus of LukF, failed to have any hemolytic activity and had less 10% leukocytolytic activity than that of the intact LukF, while 16-residue truncations retained both toxin activities without loss. The LukF mutants lacking 18- through 19-residue segments from the N-terminus showed low toxin activity on both target cells. All mutants having no toxin activity were also not capable of binding to the human erythrocytes. It can thus be concluded that the 3-residue segment, L18Y19K20 of LukF is crucial for the biological activity of the toxin.
The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC50 values for histamine release were 30 μg/ml and 25 μg/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.
α-Tocopherol was reacted with the phosphatidylcholines (PCs), 1-palmitoyl-2-linoleoyl-3-sn-PC (PLPC), 1-palmitoyl-2-linolenoyl-3-sn-PC, 1-palmitoyl-2-arachidonoyl-3-sn-PC (PAPC) and 1-stearoyl-2-arachidonoyl-3-sn-PC, in the presence of the free radical initiator, 2,2′-azobis(2,4-dimethylvaleronitrile), at 37°C. The addition products of α-tocopherol with the PC peroxyl radicals were isolated and identified as 8a-(PC-dioxy)-α-tocopherones, in which the peroxyl radicals derived from each PC molecule attacked the 8a-position of the α-tocopheroxyl radical. The antioxidative efficiency of α-tocopherol against the peroxidation of PLPC and PAPC in liposomes was assessed by the formation of the reaction products of α-tocopherol. When α-tocopherol was oxidized in the presence of the water-soluble free radical initiator, 2,2′-azobis(2-amidinopropane) dihydrochloride, epoxy-α-tocopherylquinones were mainly produced together with 8a-(PC-dioxy)-α-tocopherones and α-tocopherylquinone. The yield of α-tocopherylquinone was increased by treating each sample with dilute acid which indicates the presence of tocopherone precursors other than the 8a-(PC-dioxy)-α-tocopherones. The same products were also detected from iron-dependent peroxidation, although the yields were very low.
Methyl linoleate containing β-carotene was autoxidized or photooxidized at 37°C in the bulk phase, and the oxidation products of β-carotene were analyzed by high-performance liquid chromatography. Formyl β-carotenes, β-carotene 5,6-epoxide, and cyclic ethers of β-carotene were detected as the oxidation products during the peroxidation of methyl linoleate initiated by a free radical initiator. These products, which were also detected in the methyl linoleate autoxidized without an initiator, were detectable only in much smaller amounts than the consumed β-carotene. In the chlorophyll-sensitized photooxidation process, the products were β-carotene 5,8-endoperoxide and β-carotene 5,6-epoxide. α-Tocopherol partially inhibited the formation of the 5,6-epoxide, but had no effect on the main product, the 5,8-endoperoxide. These results indicate that β-carotene reacted with singlet oxygen to form the 5,8-endoperoxide as the primary product during the photooxidation of methyl linoleate, and that β-carotene trapped lipid-peroxyl radicals to form oxygenated products which decomposed immediately during the autoxidation process.
The effects of a total bone extract (TBE), a new mineral preparation made from bovine bone rendered soluble by lactic acid and citric acid under decompression, on the bone metabolism and apparent calcium absorption were examined for an ovariectomized (OVX) rat model of osteoporosis. The apparent calcium absorption from TBE was significantly higher than that from calcium carbonate. There were no significant differences in serum biochemical indices. Although there were no significant differences in the dry weight, ash and calcium content in the tibia, or in the bone mineral density of the femur, TBE feeding increased the cortical thickness index of the femur. A positive effect of TBE on bone formation is thus suggested.
The major allergen (Tur c 1) in the muscle of the gastropod, Turbo cornutus, was isolated by Sephacryl S-300, Mono Q HR 5/5 and TSKgel Phenyl-5PW RP column chromatography. ELISA showed Tur c 1 to react strongly with sera from three individuals sensitive to both mollusks and crustaceans. SDS-PAGE showed Tur c 1 to produce a major band corresponding to a molecular mass of 35 kDa under the reduced condition. Its amino acid composition was characterized by the abundance of Glx, followed by Leu, Ala and Lys in decreasing abundance, and the absence of Trp. In addition to these properties, the determined partial amino acid sequence identified Tur c 1 to be a tropomyosin, as in the case of the known mollusk and crustacean allergens. However, the results of competitive ELISA inhibition experiments suggest that Tur c 1 has an IgE-binding epitope in the C-terminal region which is dissimilar to those proposed for Cra g 1 (the oyster Crassostrea gigas allergen) and Pen i 1 (the shrimp Penaeus indicus allergen).
The effects of three seafoods, shrimp, squid and octopus, on lipid metabolism were investigated in mice fed on 0.1% and 1.0% cholesterol-supplemented diets in the first experiment. One of each of these seafoods and casein were added to the basal diet at levels of 15% and 5%, respectively, as proteins. Casein served as the sole protein source of the control diet. The serum cholesterol concentration was significantly lower in the mice fed on shrimp and squid in the 0.1% cholesterol diet and on any seafood in the 1.0% cholesterol diet when compared with that in the mice fed on the control diet. The liver cholesterol concentration was significantly lower in all seafood groups given the 0.1% cholesterol diet, and in the squid and octopus groups given the 1.0% cholesterol diet. In the second experiment, the effect of these seafoods on lipid metabolism was compared with that of their defatted products in mice fed on a 0.2% cholesterol diet. Defatting resulted in an increase in the serum cholesterol and triglyceride levels in the shrimp and squid groups. The hepatic cholesterol concentration in all the seafood groups was significantly lower than that in the control group, and defatting did not influence the liver cholesterol concentration. Fecal total steroid excretion was higher in all the seafood groups when compared with that in the control group, and was not modified by the removal of fats. Thus, shrimp, squid and octopus exerted hypolipidemic activity; the serum cholesterol-lowering activity of shrimp and squid was attributed to their lipid fraction, whereas the non-lipid fraction of shrimp, squid and octopus contributed to a reduction of hepatic cholesterol and an increase of fecal steroid excretion.
The behavior of the dielectric properties of gelatin in the frequency range from 103 Hz to 107 Hz was investigated and compared with that of the globule protein, bovine serum albumin (BSA), desalted gelatin and BSA being used. Dielectric relaxation was observed for both the gelatin and BSA solutions. The relaxation data were fitted well by the Cole-Cole equation; the Cole-Cole parameter (β) and the relaxation time (τ) were obtained. For the BSA solutions, τ was proportional to the solution viscosity (η) at 40°C and 25°C, and the values of β at 40°C were similar to those at 25°C. For gelatin solution, τ was proportional to η at 40°C, but was not proportional to η at 25°C. In addition, the values of β at 25°C were smaller than those at 40°C. These results indicate that the rotation of gelatin and/or polarization of submolecular groups in the coil state greatly contributed to the dielectric relaxation at 40°C; on the other hand, the formation of cross-linking junctions consisting of helix structures would have affected the dielectric relaxation at 25°C.
The effects of two polyunsaturated fatty acids, 18:4n-3 and 16:4n-3 purified from the marine algae, Undaria pinnatifida and Ulva pertusa, on icosanoid production in MC/9 mouse mast cells were assessed. Both fatty acids suppressed the production of leukotriene B4 (LTB4), leukotriene C4 (LTC4), and 5-hydroxyeicosatetraenoic acid (5-HETE). The order of the suppressive activity for the two marine algae-derived fatty acids and three other common polyunsaturated fatty acids was as follows; 22:6n-3=18:4n-3=18:3n-3>20:5n-3=16:4n-3 for LTB4; 22:6n-3=18:4n-3=18:3n-3>16:4n-3>20:5n-3 (no suppression) for LTC4; 22:6n-3=18:4n-3>18:3n-3>20:5n-3=16:4n-3 for 5-HETE.
The reaction of 2,3-diketo-L-gulonic acid (DKG), which is one of the important intermediate products in the degradation of L-ascorbic acid (ASA) in both food and biological systems, in an aqueous solution was studied. The formation of a small amount of the γ-lactone, dehydro-L-ascorbic acid (DASA), from DKG was observed. This strongly suggests the chemical possibility of a reverse reaction in DASA hydrolysis which has been long believed to be irreversible.
Part of the salt in soy sauce formed no eutectic H2O•2NaCl crystals at sub-zero temperatures and remained in a freeze-concentrated product. NMR line width of 23Na was broader in the concentrated soy sauce than in the material. A broad line width of 23Na was also observed in an aqueous solution of NaCl and a non-diffusible soy sauce fraction. The data indicate that part of the salt in soy sauce interacted with its non-diffusible fraction and that such bound salt formed no eutectic crystals.
In order to evaluate the positional specificity for a glucoside group in the hydrolysis of flavonoid glucosides in the rat small intestine, β-glucosidase activity was measured with the quercetin monoglucosides, quercetin-3-O-β-D-glucopyranoside (Q3G), quercetin-4′-O-β-D-glucopyranoside (Q4′G) and quercetin-7-O-β-D-glucopyranoside (Q7G), as well as with quercetin-3-O-rutinoside (rutin) and p-nitrophenyl-β-D-glucopyranoside (NPG) by using the HPLC technique. Enzymes were prepared from rat small intestinal mucosa of the duodenum, jejunum and ileum, among which the enzyme activity of the jejunum was highest for all the glycosides tested. Q4′G was the richest substrate for a β-glucosidase solution among these glycosides, while rutin and NPG were both poor substrates. This suggests that dietary flavonoid glucosides are primarily hydrolyzed and liberated aglycones in the jejunum.
Two hours after an intraperitoneal injection of ferric nitrilotriacetate to rats at a dose of 15 mg of Fe/kg of body weight, the level of lipid hydroperoxides, which was determined by a specific method involving chemical conversion into 1-naphthyldiphenylphosphine oxide and HPLC, had increased significantly in the liver (from 190.1±58.8 of the control to 467.1±175.8 pmol/mg of protein) and kidney (from 181.8±52.3 of the control to 405.9±22.7 pmol/mg of protein). These results demonstrate that oxidative stress was transiently increased by an iron overload in these tissues.
A two-dimensional mapping analysis was performed by HPLC for 4 kinds of standard galactosyllactoses (GLs, trisaccharide) which were assumed to be produced from lactose (galactopyranosylβ1→4 glucopyranose) in yogurt during the fermentation of lactic acid bacteria. After the pyridylamination of GLs, they were analyzed by HPLC in the reverse-phase (RP) and anion-exchange (AE) modes. The retention times of each peak obtained were converted to glucose units (GU) in RP mode for the pyridylaminated isomaltooligosaccharides (G1-3) and to relative retention time (RRT) in AE mode against pyridylaminated-isomaltotriose, and then the address data [GU, RRT] were plotted on a graph. This two-dimensional mapping method was found useful for a rapid qualitative evaluation of the chemical structure of trisaccharides formed in yogurt.
A detection technique for the genetically engineered food, glyphosate-tolerant soybean (GTS), was designed. Commercial soybeans imported from North America were cultured in pots and genomic DNA was isolated from their leaves. To detect the genes, promoter and terminator, involved in the expression of glyphosate tolerance, PCR was done using the genomic DNA and chemically synthesized primers specific to the genes. DNAs with predicted sizes were amplified and confirmed by DNA sequencing to be the genes responsible for the expression of glyphosate tolerance. Glyphosate-tolerant soybeans were found to form approximately 1.1% of the commercial soybeans, when commercially available soybeans were cultivated and number of soybeans resistant to glyphosate was found. This level is somewhat lower than an estimated value announced officially on the basis of the cultivation area of the glyphosate-tolerant soybeans.
In a mixture containing γ-glutamyl donor (donor) and γ-glutamyl acceptor (acceptor), the glutaminase of Pseudomonas nitroreducens IFO 12694 simultaneously catalyzed a γ-glutamyl transfer reaction and hydrolysis of the donor. The variation of the activities responding to the concentration of glutathione and glycylglycine indicated that the enzyme might be classified in a group of glutaminases that shows hydrolysis prior to transfer reaction. On the other hand, the results with glutamine and ethylamine or methylamine indicated that the enzyme was active in the transfer reaction with suppressed hydrolysis of glutamine, and suggested the possibility of using the reaction for producing γ-glutamylethylamide (theanine) or γ-glutamylmethylamide (γ-GMA). In fact, in a mixture containing high concentrations of substrates (0.7 M glutamine, 1.5 M ethylamine or methylamine) and 0.5 unit/ml glutaminase (borate buffer pH 11), 270 mM (47 g/L) theanine or 250 mM (38 g/L) γ-GMA was formed in 7 h of incubation at 30°C.
l-Menthol was glucosylated by the α-glucosidase (EC 22.214.171.124) of Saccharomyces cerevisiae using maltose as the glucosyl donor. When 50 mg of l-menthol and 1.6 M maltose in 10 mM citrate-phosphate buffer (pH 5.5) were incubated at 45°C, l-menthyl α-D-glucopyranoside (α-MenG) was α-anomer-selectively formed as a product. The specificity of the α-linkage was confirmed by 13C-NMR analysis. In the reaction mixture after 2 h, α-MenG was mainly accumulated in a crystalline form and the concentration of dissolved α-MenG was constant at 1.4 mM. The molar conversion yield of α-MenG produced based on the supplied l-menthol was maximally 30.7% at 48 h of reaction.
UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40% higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.
Volatile compounds from two South-East Asian fermented soybean foods, Chungkuk-jang (CKJ) and Itohiki-natto (natto), were analysed by gas chromatography-mass spectrometry (GC-MS), gas chromatography (GC), and GC-sniffing. A total of 112 compounds were identified. A large amount of ethanol was detected from CKJ, while acetone and methyl isobutyrate were major components of natto. The characteristic odor compounds of CKJ were some ethyl esters of short chain fatty acids, diallyl disulfide, and several natto-like odor compounds were identified as ammonia, 2,5-dimethylpyrazine, and 2-methylbutanoic acid.