The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 μl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 °C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.
The germination stimulants for root parasitic plants Striga and Orobanche produced by cotton (Gossypium hirsutum L.) were examined in detail. Seeds of cotton were germinated and grown on glass wool wetted with sterile distilled water in sterile filter units. The root exudate was collected daily and extracted with ethyl acetate. Each of these ethyl acetate extracts was analyzed directly by high-performance liquid chromatography linked with tandem mass spectrometry (LC/MS/MS). The results demonstrate that cotton roots exuded strigol and strigyl acetate, but no other known strigolactones such as orobanchol and alectrol. The production of strigol was detected even in the root exudate collected during the first 24 h of incubation and reached a maximum 5–7 days later. The average exudation of strigol and strigyl acetate during the incubation period was ca. 15 and 2 pg/plant/day, respectively, indicating that strigol mainly contributed to germination stimulation by the cotton root exudate.
Olivil-type lignans, an enantiomeric type of natural olivil, were synthesized for the first time to evaluate the relationship between the structure of olivil and its antioxidant activity. A comparison of the antioxidant activity with that of other synthesized tetrahydrofuran lignans indicated reduced activity with the tertiary hydroxy group. A different effect of the two phenolic groups of olivil on the antioxidant activity was also observed.
A five-step and scalable synthesis of racemic cytoxazone, a novel cytokine modulator, was accomplished in a total yield of 51% from p-methoxycinnamyl alcohol without any protective groups. The keystep was the new one-pot azidohydroxylation procedure by the combined use of NaN3–H2O2–CH3CN. The epoxidation of an olefin by means of an in situ-formed iminohydroperoxide worked well, accompanied by the concomitant regioselective ring opening reaction of the resulting highly reactive epoxide with an azide ion.
The tyrosinase inhibitory activity of methanol extracts of the leaves of 39 plant species growing on the seashore of Iriomote island (Okinawa, Japan) was investigated. The extracts of Hibiscus tiliaceus, Carex pumila, and Garcinia subelliptica showed potent activity among them. The inhibitors in the extract of Garcinia subelliptica were purified by assay-guided fractionation to give two biflavonoids. These were known compounds (2R,3S-5,7,4′,5″,7″,3′′′,4′′′-heptahydroxy flavanone[3-8″] flavone and 5,7,4′,5″,7″,3′′′,4′′′-heptahydroxy[3-8″] biflavanone), although their strong inhibitory activity toward tyrosinase is revealed for the first time in this work. One of these biflavonoids (2R,3S-5,7,4′,5″,7″,3′′′,4′′′-heptahydroxy flavanone[3-8″] flavone) showed much stronger activity (IC50 2.5 μM) than that of kojic acid (IC50 9.1 μM) when L-tyrosine was used as the substrate.
Four possible stereoisomers of 3-hydroxy-4-methyltetradecanoic acid were enantioselectively synthesized by using Sharpless epoxidation and a subsequent epoxide-ring opening reaction with trimethylaluminum as the key steps. The absolute configuration of the β-oxyacid component of antifungal cyclodepsipeptides W493 A and B was consequently determined as 3S,4R.
In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 °C and pH 10–11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and α-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15–25% NaCl respectively using Suc–Ala–Ala–Phe–pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.
Cotton woven fabrics which were previously dyed with a reactive dye were treated with a commercial cellulase preparation. Dyeing with a reactive dye for cotton apparently inhibited the weight loss activity and saccharification activity of cellulase. In addition, dyed cotton was treated with highly purified cellulases which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type cellulases were inhibited more than endo-type cellulase by dyeing in the case of saccharification activity. CBH I was severely inhibited by dyeing as compared with CBH II or EG II from the viewpoint of morphological changes in the fiber surface. Dyes on the cellulose substrates severely influenced CBH I in spite of the rare modification, because CBH I hydrolyzed cellulose with true-processive action. The change in the activity of each cellulase component on dyed cotton can affect the synergistic action of cellulases.
Apoptin is derived from chicken anemia virus (CAV) and known to induce tumor specific apoptosis but not normal cells. The aim of this study was to use increased expression of apoptin by the Myc–Max response element (MMRE) and SV40 enhancer in small-cell lung cancer (SCLC) gene therapy. To investigate the possibility of the utilization of the MMRE, apoptin, and SV40 promoter/enhancer in targeted cancer gene therapy, adenovirus vector expressing apoptin controlled by the MMRE, and SV40 promoter/enhancer was constructed. Ad-MMRE-apoptin-enh infected SCLC cells were significantly suppressed and induced apoptosis more than those of Ad-apoptin or Ad-apoptin-enh. Infection with Ad-MMRE-apoptin-enh of normal cells did not increase apoptosis. About 85% of SCLC tumors show overexpression of the myc family, so the increased expression of apoptin by MMRE and SV40 enhancer can be used in targeted SCLC gene therapy. These results indicate that apoptin expression was increased by the MMRE and SV40 promoter/enhancer, and that this strategy can be used in SCLC targeted cancer gene therapy.
Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor or stem cells. The aim of this study was to use increased telomerase promoter activity in small-cell lung cancer (SCLC) gene therapy. The hTERT promoter and Myc–Max response elements (MMRE) in pGL3-Control vector containing SV40 enhancer resulted in strong expression of the luciferase gene only in telomerase positive and myc overexpressing SCLC cell line but not in normal human cell line. To investigate the possibility of the utilization of the MMRE, hTERT promoter, and SV40 enhancer in targeted SCLC gene therapy, adenovirus vector expressing HSV-TK controlled by the MMRE, hTERT promoter, and SV40 enhancer for the induction of telomerase positive and myc-overexpressing cancer specific cell death was constructed. SCLC cells infected with Ad-MMRE-hT-TK-enh were significantly suppressed and induced apoptosis more than those of Ad-hT-TK or Ad-hT-TK-enh infected cells. Telomerase and c-myc are activated in 60∼80% of SCLC, so the increased activity of telomerase promoter can be used for targeted SCLC gene therapy. These results show that the MMRE, hTERT promoter, and SV40 enhancer can be used in SCLC targeted cancer gene therapy.
RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C. japonica protoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptis alkaloid research are discussed.
In this study, the isolation and characterization of a phytochrome A (PHYA) homologous cDNA (OmPHYA) in the non-photosynthetic holoparasitic plant Orobanche minor are described. The present findings provide the first report of the presence of a PHYA homolog in the holoparasite. This study found that OmPHYA is of similar size to the other PHYAs of green plants and shows 72, 77, and 77% amino acid sequence identity with PHYA in Arabidopsis, potato, and tobacco respectively. The OmPHYA contains a conserved chromophore attachment cysteine at position 323. Although OmPHYA shows high sequence identity with other PHYAs in green plants, 13 amino acid substitutions located in both the N and C-terminal domains are observed (a total of 26 amino acids). OmPHYA is encoded by a single gene within the O. minor genome. The abundance of the OmPHYA transcript as well as nuclear translocation of OmphyA occurs in a light-dependent manner.
Optically active styrene oxide derivatives are versatile chiral building blocks. Stereoselective reduction of phenacyl halide to chiral 2-halo-1-phenylethanol is the key reaction of the most economical synthetic route. Rhodotorula glutinis var. dairenensis IFO415 was discovered on screening as a potent microorganism reducing a phenacyl halide to the (R)-form of the corresponding alcohol. An NADPH-dependent carbonyl reductase was purified to homogeneity through four steps from this strain. The relative molecular mass of the enzyme was estimated to be 40,000 on gel filtration and 30,000 on SDS-polyacrylamide gel electrophoresis. This enzyme reduced a broad range of carbonyl compounds in addition to phenacyl halides. Some properties of the enzyme and preparation of a chiral styrene oxide using the crude enzyme are reported herein.
Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5–6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using β-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3 + (GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s−1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53–65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B.
Hyperthermostable β-glucosidase from Pyrococcus furiosus was enclosed in gelatin gel by cross-linking with transglutaminase. Gelatin-immobilized β-glucosidase was considerably more thermostable than the native enzyme. Lyophilized immobilisate was stored at 90 °C for 1 month without loss of activity. The immobilized β-glucosidase catalyzed transglucosylation of 5-phenylpentanol with 10.0 equivalent of cellobiose at pH 5.0 and 70 °C for 12 h to afford 5-phenylpentyl β-D-glucopyranoside in 41% yield. The immobilized enzyme was more effective than the native one in transglucosylation. The gelatin-immobilized Pfu-β-glucosidase recovered from the first run of the reaction was reusable on successive runs.
We have determined the structures of N-glycans linked to major allergens in the mountain cedar (Juniperus ashei) pollen, Jun a 1. First, two kinds of the pollen glycoallergen (Jun a 1-A and Jun a 1-B) were purified from partially purified Jun a 1 by cation exchange chromatography. The N-glycans were liberated by hydrazinolysis from the two glycoallergens and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC from Jun a 1-A and Jun a 1-B respectively. The structures were determined by a combination of exo- and endo-glycosidase digestions, two dimensional sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS) analysis. Structural analysis indicated that Lewis a epitope (Galβ1–3(Fucα1–4)GlcNAcβ1–) occurs in the N-glycans of the pollen allergens.
Clostridium thermocellum xylanase Xyn10C (formerly XynC) is a modular enzyme, comprising a family-22 carbohydrate-binding module (CBM), a family-10 catalytic module of the glycoside hydrolases, and a dockerin module responsible for cellulosome assembly consecutively from the N-terminus. To study the functions of the CBM, truncated derivatives of Xyn10C were constructed: a recombinant catalytic module polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBM and CM (rCBM–CM). The recombinant proteins were characterized by enzyme and binding assays. Although the catalytic activity of rCBM–CM toward insoluble xylan was four times higher than that of rCM toward the same substrate, removal of the CBM did not severely affect catalytic activity toward soluble xylan or β-1,3-1,4-glucan. rCBM showed an affinity for amorphous celluloses and insoluble and soluble xylan in qualitative binding assays. The optimum temperature of rCBM–CM was 80 °C and that of rCM was 60 °C. These results indicate that the family-22 CBM of C. thermocellum Xyn10C not only was responsible for the binding of the enzyme to the substrates, but also contributes to the stability of the CM in the presence of the substrate at high temperatures.
Neoglycolipids composed of disaccharide glycoside and phospholipid were designed and prepared as mimetics of lactosylceramide. The lactosyl- and N-acetyllactosaminyl-phospholipids (Lac-DPPA and LacNAc-DPPA) were enzymatically synthesized from lactose and LacNAc respectively by cellulase-mediated condensation with 1,6-hexanediol, followed by conjugation of the resulting glycosides and dipalmitoylphosphatidyl choline (DPPC) mediated by Streptomyces phospholipase D. Alternatively, allyl β-lactoside was ozonolyzed to give an aldehyde, which was condensed with dipalmytoyl phosphatidyl ethanolamine to afford a second type of glycolipid (Lac-DPPE). NMR spectroscopy indicated that the neoglycolipids behave differently in different solvent systems. X-ray diffraction clearly showed that multilamellar vesicles (MLVs) of Lac-DPPE and Lac-DPPA-MLV are in the bilayer gel phase at 20 °C, whereas those of Lac-DPPE-MLV were in the lamellar liquid-crystalline phase at 50 °C. Differential scanning calorimetry showed that Lac-DPPE-MLV had complex thermotropic behavior depending on the incubation conditions. After a long incubation at 10 °C, endothermic transitions are observed at 39.6, 42.3 °C, and 42.9 °C. These neoglycolipids have the ability to trap calcein, a chelating derivative of fluorescein, in MLVs and showed specific binding to lectin in plate assays using fluorescently labeled compounds.
Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.
Some lectins are known to stimulate interleukin-8 (IL-8) productions in human colon carcinoma Caco-2 cells. Since IL-8 may cause deleterious effects, we examined this stimulatory activity of Aralia cordate lectin (ACL) and Wasabia japonica lectin (WJL), both of which we isolated recently. The results indicate that ACL exhibited strong stimulatory activity for IL-8 protein production, while WJL showed marginal activity. The activity of ACL was associated with high enhancement of IL-8 gene expression. The effect of ACL was abolished almost completely in the presence of brefeldin A, indicating that internalization into cells is necessary for ACL to exert activity. The findings suggest that ingestion of a large amount of raw vegetable Aralia cordate might cause unfavorable effects on the colon.
We constructed a protein expression vector with an improved enoA promoter that harbored 12 tandem repeats of the cis-acting element (region III) of Aspergillus oryzae. The improved promoter yielded reporter β-glucuronidase (GUS) activity approximately 30-fold of the original promoter. Northern blot analysis confirmed that GUS expression was increased at the transcriptional level. The transformant harboring seven copies of the novel vector showed more than 100,000 U/mg GUS protein, which was approximately 30% of all the cell-free soluble proteins.
Y-700, 1-[3-cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid, is a newly synthesized inhibitor of xanthine oxidase. This study found that feeding of Y-700 suppressed the development of colonic aberrant crypt foci, precursor lesions of colon cancer, and cell proliferation in 1,2-dimethylhydrazine-treated mice, accompanied by reduced serum urate. These results suggest that Y-700 is a useful agent for the prevention of colon tumorigenesis and that xanthine oxidase plays an important role in the development of colon cancer.
Tn5-derived mutants of the γ-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimobilis UT26 were genetically characterized, and an endogenous insertion sequence (IS) which belongs to the IS1380 family was identified. The IS, named ISsp1, existed as multi copies in UT26, and its transposition appeared to be activated during the process of Tn5-mutagenesis. It was found that transposon mutagenesis can cause endogenous mutations.
Plant Kunitz-type protease inhibitors contain a conserved Asn residue in the N-terminal region. To investigate the role of Asn residue in protease inhibitory activities, Erythrina variegata trypsin inhibitor a (ETIa), E. variegata chymotrypsin inhibitor (ECI), and their mutants, ETIa-N12A and ECI-N13A, were used. Both mutants exhibit weaker inhibitory activities toward their cognate proteases than the wild-type proteins and were readily cleaved at reactive sites. Furthermore, kinetic analysis of the interactions of the mutated proteins with their cognate proteases by surface plasmon resonance (SPR) measurement indicated that replacements of the Asn residue mainly affected dissociation rate constants. The conserved Asn residues of Kunitz-type inhibitors play an important role in exhibiting effective inhibitory activity by stabilizing the structures of the primary binding loop and protease-inhibitor complex.
Expression of AtCIPK14 (an ArabidopsisCBL-interacting protein kinase 14) was induced by metabolic sugars. Two A/T-rich sequences similar to elements involved in sugar-inducible expression of other genes were found within the −183 bp 5′ region of the AtCIPK14 promoter that was responsible for the sugar induction. Histochemical analysis using a reporter gene indicated vascular-specific expression of AtCIPK14.
The effects of modified cyclodextrins (CDs) hydroxypropyl-β-CD and methyl-β-CD were studied in vitro on cDNA-expressed human cytochrome P-450 (CYP) activities (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). The modified CDs inhibited the activities of CYP2C19 and CYP3A4 while enhancing CYP2C9 activity by 140 to 176% relative to the control values at lower concentrations. In addition, methyl-β-CD inhibited CYP1A2 and CYP2D6 at higher concentrations.
The temperature dependence of regeneration of bacteriorhodopsin (bR) from its apoprotein, bacterio-opsin (bO), and all-trans retinal was investigated using two different procedures to probe the structural properties of bO at high temperatures. Regeneration experiments performed at 25 °C after incubation of bO within the temperature range of 35–75 °C indicate that irreversible thermal unfolding begins at 50 °C. When bO is incubated for one hour and mixed with retinal at the same elevated temperatures, however, a greater extent of regeneration to bR occurs, even at temperatures ranging from 50 to 65 °C. These experimental results indicate that regeneration of bR occurs from thermally unfolded bO and suggest dynamic structural fluctuation of bO in the unfolded state.
Estragon and thyme extracts showed potent inhibitory activities against chemical mediator release from rat basophilic leukemia RBL-2H3 cells. 7-Methoxycoumarin was isolated from estragon, and 5,4′-dihydroxy-6,7,3′-trimethoxyflavone, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone and luteolin were isolated from thyme as active components. Structure-activity relationship studies among the active isolates and their related compounds indicated that the oxygen-containing functional group at the 7-position of the coumarin structure was advantageous for the inhibitory activity and that methylation of the hydroxyl group at the 4′-position of the flavone structure was disadvantageous. It was also found that coumarin derivatives inhibited an earlier step than intracellular calcium release and proteinkinase C activation, while flavones inhibited a later step or both earlier and later steps.
We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H–1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4′ of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.
We compared the effects of non-gelatinized rice and corn starches on the life-span of ICR mice. Six groups of male ICR mice consisting of 30 animals each were maintained on purified experimental diets containing either corn or rice starch and different amounts of soybean oil (6, 12 or 24%) throughout their life-time. Plots of the survival rates of the mice indicate that rice compared to corn starch conferred a longer life-span to ICR mice, although a significant difference due to the starch type was only observed in the mice fed on the 24% fat diet (p=0.012). A divergent effect of rice and corn starches on the survival rate was apparent when observations were combined with respect to the starch type regardless of the dietary fat level (p=0.005). In addition, two-way ANOVA data indicate that the mean survival time was longer for the mice given rice starch (593–645 days) than for those fed corn starch (538–580 days) (p=0.011). However, no significant difference in these parameters due to dietary fat levels was observed. The results of our study indicate that starch type is one of the determinants of longevity in mice.
The effect of oral administration of partially hydrolyzed water-soluble corn husk arabinoxylan (CHAX), the average molecular weight of which is about 53 kDa, on immunopotentiating activity was investigated in mice. Oral administration of CHAX to healthy mice significantly augmented the production of interleukin (IL)-2 and interferon (IFN)-γ with a slight increase in IL-4 in mitogen-induced proliferation of spleen cells. Natural killer (NK) cell activity in spleen cells from mice, which were transplanted tumor and administrated CHAX, was augmented about 2-fold. In model mice of atopic dermatitis, the average ear thickness of mice administrated CHAX was induced by dinitrophenyl-fluorobenzene (DNFB) after injection of an anti-dinitrophenyl (DNP)-IgE monoclonal antibody was much smaller than that in control animals. These results suggest that CHAX has the ability to increase the level of immunopotentiating activity without causing over response of immunological reaction even if it is administrated orally to mice.
A metal-chelating substance in brewed coffee was separated and characterized by its chemical structure. This substance was a brown polymer. The contents of sugars, amino acids and phenolics in the substance were evaluated. This polymer contained small amounts of sugars and amino acids in its partial structure. After being decomposed by alkaline fusion, the decomposition products were identified by HPLC and GC–MS. Several phenolics were detected in the decomposed products. To characterize this substance, various types of model compounds were prepared by roasting chlorogenic acid, sucrose, and (or) protein with cellulose powder. Among these model compounds, the polymer-forming ability was highest in the model prepared from all four of materials, but the metal-chelating ability was the highest in the model prepared from chlorogenic acid and cellulose. These results suggest that this metal-chelating substance was a melanoidin-like polymer formed by the decomposition and polymerization of sugars, amino acids and phenolics.
We examined the effects of intake of Korean foxtail millet protein (FMP) on plasma levels of lipid, glucose, insulin, and adiponectin in genetically type 2 diabetic KK-Ay mice. When mice were fed a normal FMP diet or a high-fat-high-sucrose diet containing FMP for 3 weeks, in both experiments plasma concentrations of high-density lipoprotein cholesterol (HDL-cholesterol) and adiponectin increased remarkably in comparison with a casein diet group, whereas concentrations of insulin decreased greatly and that of plasma glucose was comparable to that in the casein diet group. Considering the role of adiponectin, insulin, and HDL-cholesterol in diabetes, atherosclerosis, and obesity, it appears likely that FMP may improve insulin sensitivity and cholesterol metabolism through an increase in adiponectin concentration. Therefore, FMP would serve as another beneficial food component in obesity-related diseases such as type 2 diabetes and cardiovascular diseases.
Sesamin, a major lignan in sesame seeds, has multiple functions such as cholesterol-lowering and anti-hypertensive activities. To investigate the effect of sesamin on gene expression in the liver, a DNA microarray analysis was carried out. The ingestion of sesamin dissolved in olive oil up-regulated the expression of 38 genes, 16 of which encode proteins possessing a lipid-metabolizing function, and 16 of which encode proteins possessing a xenobiotic/endogenous substance metabolizing function. In particular, sesamin significantly increased the expression of β-oxidation-associated enzymes in peroxisomes and auxiliary enzymes required for degradation, via the β-oxidation pathway, of unsaturated fatty acids in mitochondria. The ingestion of sesamin also resulted in an increase in the gene expression of acyl-CoA thioesterase involved in acyl-CoA hydrolase and very-long-chain acyl-CoA thioesterase. Interestingly, it induced the expression of the gene for aldehyde dehydrogenase, an alcohol-metabolizing enzyme. These results suggest that sesamin regulates the metabolism of lipids, xenobiotics, and alcohol at the mRNA level.
Among the lipophilic extracts of seven traditional edible mushrooms, the acetone extract of Sarcodon aspratus markedly inhibited the growth of HL60 human leukemia cells and induced apoptosis after 24 h incubation. The major active component was identified as ergosterol peroxide by NMR and ESI-MS analysis. Ergosterol peroxide completely inhibited growth and induced apoptosis of HL60 cells at a concentration of 25 μM.
Apoptosis induced by fucoxanthin in HL-60 cells was associated with a loss of mitochondrial membrane potential at an early stage, but not with an increase in reactive oxygen species. Fucoxanthin treatment caused cleavages of procaspase-3 and poly (ADP-ribose) polymerase without any effect on the protein level of Bcl-2, Bcl-XL, or Bax. Apoptosis induction by fucoxanthin may be mediated via mitochondrial membrane permeabilization and caspase-3 activation.
An increase in plasma ovalbumin concentrations after intragastric administration of ovalbumin was suppressed by concomitant freeze-dried kefir in BALB/c mice. Serum levels of ovalbumin-specific immunoglobulin G and proliferation of splenic mononuclear cells in mice immunized orally with ovalbumin were suppressed by feeding freeze-dried kefir. We propose that kefir reduces intestinal permeation of food antigen, which contributes to suppression of oral sensitization.
chsA and chsC are genes encoding class II and I chitin synthases of Aspergillus nidulans respectively. In a previous study, chsA chsC double mutants showed various growth defects, suggesting that their cell wall architecture was disorganized and their cell wall integrity diminished. Here, we constructed chsA chsC chsD triple mutants and chsA chsC csmA triple mutants to investigate the role of the class IV and class V chitin synthases, ChsD and CsmA respectively, in maintaining the cell wall structure of the chsA chsC double mutant. The former triple mutant grew a little slower than the chsA chsC double mutant, but the two showed similar phenotypes. In contrast, the latter triple mutant exhibited severe growth defects, particularly under low osmotic conditions. The levels of the csmA transcript of the wild-type strain and chsA or chsC single mutants were markedly elevated under low osmotic conditions, while that of the chsA chsC double mutants was high even under such conditions. These and other results suggest that the function of csmA is important for the maintenance of cell wall integrity and the polarized growth of the chsA chsC double mutant.
When supplemented to the culture medium of mushroom Coprinus cinereus, rice husks soaked beforehand in methanol stimulated mycelia growth up to a concentration of 80 mg/ml dose-dependently, whereas the non-treated stimulated mycelia growth up to 20 mg/ml. This result suggests the existence of both stimulatory and inhibitory substances in rice husks. Since momilactone A (MLA) is recognized as one of the phytoalexins in rice husks, its biological activity against mycelia growth was tested. Momilactone A inhibited mycelia growth at 5 μg/disc, whereas the methanol extract of husks did so at 1 mg/disc, wherein 0.2 μg of MLA was estimated by LC/MS/MS. Thus the phytoalexins including MLA should inhibit mycelia growth. Rice husks stimulated mycelia growth in some edible mushroom species such as Grifola frondosa (maitake), Lentinus edodes (shiitake), Pleurotus eryngii (eringi), and P. ostreatus (hiratake). Our findings might lead to the development of new profitable cultivation methods for mushrooms using rice husks.
Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV–visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.
We investigated the optimum culture conditions for the production of a novel enzyme, N-substituted formamide deformylase, which acts mainly on N-benzylformamide, in Arthrobacter pascens F164. The highest enzyme activity was obtained when this strain F164 was cultivated in a synthetic medium with N-benzylformamide as sole nitrogen source. This deformylase was found to be an inducible enzyme depending on N-benzylformamide.
Transformation system for Escherichia coli based upon introduction of plasmid DNA by natural phospholipids has been developed. Transformants are easily obtained by treatment with natural phospholipids such as phosphatidylethanolamine, phosphatidylcoline, and phosphatidylserine, where the presence of MgCl2 or CaCl2 is essential. This method of transformation is applicable not only for small plasmid pHSG399 (2.3 Kb) but also for giant plasmid R6K (100 Kb).
Pradimicin, a mannose-binding antifungal antibiotic, induces apoptosis-like cell death in Saccharomyces cerevisiae. Previously we found that the substitution of the 74th amino acid from glycine to cysteine in Ypd1 yields a mutant resistant to pradimicin. In this study, the involvement of a membrane-spanning osomosensor, Sln1, which is located upstream of Ypd1, was investigated. A mutant, sln1 ΔNG, that lacks the putative N-glycosylation sites in the extracellular domain became resistant to pradimicin. On the other hand, the null mutants of Ssk1, Pbs2, and Hog1, which are located downstream of the Sln1 cascade, were sensitive to pradimicin as well as the wild-type strain. In conclusion, pradimicin exerts its fungicidal action with the involvement of Sln1, but the downstream branch, Ssk1 and the HOG pathway, is not involved.
Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.