The wild type of Selenomonas ruminantium subsp. lactilytica, which is a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, requires one of the normal saturated volatile fatty acids with 3 to 10 carbon atoms for its growth in a glucose medium; however, no such obligate requirement of fatty acid is observed when the cells are grown in a lactate medium. This bacterium is characterized by a unique structure of the cell envelope and a novel lysine decarboxylase and its regulatory protein. In the first part of this article, we will refer to the chemical structure of phospholipid and lipopolysaccharide in the cell membranes of this bacterium compared with that from the general Gram-negative bacteria for understanding their biological functions. S. ruminantium has neither free nor bound forms of Braun lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria. However, S. ruminantium has cadaverine, which links covalently to the peptidoglycan as a pivotal constituent for the cell division. In the second part of this article, we will refer to the chemical structure of the cadaverine-containing peptidoglycan, its biosynthesis, and the biological function. In the third part of this article, we will depict the molecular cloning of the genes encoding S. ruminanitum lysine decarboxylase (LDC) and its regulatory protein of 22-kDa (22-kDa protein; P22) which has similar characteristics to that of antizyme of ornithine decarboxylase in eukaryotic cells, and the molecular dissection of these proteins for understanding the regulation of cadaverine biosynthesis. Finally, we will illustrate a proposed structure of the cell envelope, a processes of biosynthesis of the cadaverine-containing peptidoglycan layer, and the LDC degradation mechanism in S. ruminantium, on the basis of the analyses of the cell envelope components, the results from the in vitro experiments on the biosynthesis of the peptidoglycan layer, and the current status of the knowledge on LDC and P22 in this organism.
A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU β-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.
Anteiso fatty acids having 16 to 29 carbon atoms were labeled with the chiral fluorescent conversion reagents, (1R,2R)- and (1S,2S)-2-(2,3-anthracenedicarboximido)cyclohexanol. The diastereomeric esters of anteiso acids having up to 20 carbon atoms were separated into two peaks in an ODS column under low column-temperature conditions, while those having more than 21 carbon atoms were not separated. A C30 column made it possible to separate diastereomeric esters up to C29 anteiso acid. It was possible to predict the absolute configuration of each acid by the elution order of the derivatives.
A simple new assay was designed for lipoxygenase inhibitors. This assay was used to find the novel lipoxygenase inhibitor, tetrapetalone A (1). Tetrapetalone A (1), C26H33NO7, was isolated from Streptomyces sp. USF-4727 strain. Its planar structure was determined by spectroscopic evidence and by methylating with diazomethane to show the presence of a novel tetracyclic skeleton and a β-D-rhodinosyl moiety. The stereochemistry of 1 was investigated by the coupling constant in the 1H-NMR spectrum, NOE correlations, modified Mosher’s method and derivation. We have reported the structural elucidation of 1 in our previous paper. However, further investigation gave another structure for 1, which is described in this paper. Tetrapetalone A showed similar inhibitory activity against soybean lipoxygenase to the two well-known lipoxygenase inhibitors, kojic acid and NDGA, while methylated tetrapetalone A (2) showed little inhibitory activity, even at a concentration of 1 mM.
The fungal diterpene, aphidicolin, is a well-known specific inhibitor of DNA polymerase α. Terpenoids are an important class of natural products. However, identification of the biosynthetic gene cluster in terpenoids is relatively rare compared with another important class of natural products, polyketides. To explore a reliable identification method for the biosynthetic gene cluster in fungal diterpenoids, cloning of the biosynthetic gene cluster of aphidicolin was employed. The application of a simple PCR method for genome walking based on the sequence of cDNA encoding aphidicolan-16β-ol synthase (ACS) allowed us to analyze a 15.6-kb region of the Phoma betae genomic DNA. Six ORFs, PbGGS, ACS, PbP450-1, PbP450-2, PbTP, and PbTF were found in this region, and respectively expected to encode geranylgeranyl diphosphate synthase, diterpene synthase, two cytochrome P-450s, the transporter and transcription factor. Their amino acid sequences and introns were deduced by a corresponding cDNA analysis. This study shows that simple PCR-based genome walking without constructing a genomic DNA library is useful for identification of a small gene cluster. We propose a general strategy for the cloning the biosynthetic genes of fungal diterpenoids by using fungal GGS.
(1R)-trans-Norchrysanthemic acid fluorobenzyl esters are synthesized and their structure-activity relationships are discussed. These esters show outstanding insecticidal activity against mosquitoes. In particular, the 2,3,5,6-tetrafluoro-4-methoxymethylbenzyl analog (metofluthrin) exhibits the highest potency, being approximately forty times as potent as d-allethrin in a mosquito coil formulation when tested against southern house mosquitoes (Culex quinquefasciatus). Metofluthrin also exhibits a significant vapor action at room temperature.
Nine furofuran compounds having a different type of oxidation were synthesized from one common intermediate in a short series of steps, and the antioxidant activity was evaluated. It was found that the tertiary hydroxy group on the furofuran ring affected the degree of antioxidant activity and that the structure, except for the phenolic part, was important for the antioxidant activity.
New acetylenic nematicidal compound, penipratynolene (1), methy (2′R)-4-(2′-hydroxy-3′-butynoxy)benzoate, together with two known compounds, 6-methoxycarbonylpicolinic acid (2) and 2,6-pyridinedicarboxylic acid (3), were isolated from the culture filtrate of Penicillium bilaiae Chalabuda. The structures of 1–3 were established by spectroscopic methods. The absolute configuration of 1 was confirmed by using a modified version of Mosher’s method. Compounds 1–3 showed nematicidal activity of 77%, 52%, and 98%, respectively, by a bioassay at 300 mg/l with the root-lesion nematode Pratylenchus penetrans.
The new prenylflavonoid, isonymphaeol-B (1), together with three known compounds, nymphaeol-A (2), nymphaeol-B (3), and nymphaeol-C (4), were isolated from propolis collected in Okinawa, the southern-most prefecture of Japan. The structure of each compound was determined by spectral methods, including mass spectrometry and 2D NMR. Each compound had 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging activity.
Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670–27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.
The basidiomycete Lentinula edodes (Le.) cytochrome P450, Le.CYP1 was functionally expressed in Saccharomyces cerevisiae. The microsomal fraction containing Le.CYP1 was prepared from the recombinant yeast and the Le.CYP1 was analyzed. The 7-ethoxycoumarin and benzo(a)pyrene were found to be the substrates of Le.CYP1 enzyme. Le.CYP1 converted 7-ethoxycoumarin to 7-hydroxycoumarin.
A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg−1 which corresponds to 2927.15 Ug−1 of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65°C and pH 9, respectively. It was stable up to 65°C at pH 7 and active over a wide pH range (pH 6–11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application.
We isolated and identified 10 α-amylase isoforms by using β-cyclodextrin Sepharose affinity column chromatography and two-dimensional polyacrylamide gel electrophoresis from germinating rice (Oryza sativa L.) seeds. Immunoblots with anti-α-amylase I-1 and II-4 antibodies indicated that 8 isoforms in 10 are distinguishable from α-amylase I-1 and II-4. Peptide mass fingerprinting analysis showed that there exist novel isoforms encoded by RAmy3B and RAmy3C genes. The optimum temperature for enzyme reaction of the RAmy3B and RAmy3C coding isoforms resembled that of α-amylase isoform II-4 (RAmy3D). Furthermore, complex protein polymorphism resulted from a single α-amylase gene was found to occur not only in RAmy3D, but also in RAmy3B.
The effects of anisodamine on the Ca2+-ATPsae of sarcoplasmic reticulum (SR) were investigated by using differential scanning calorimetry to measure the ability of anisodamine to denature the transmembrane domain and the cytoplasmic domain. Anisodamine significantly altered the thermotropic phase behaviors of the transmembrane domain of purified Ca2+-ATPase. Specifically, the melting temperature of the transmembrane domain moved toward lower temperatures with the concentrations of anisodamine increasing and the thermotropic phase peak was abolished at 10 mM, indicating that the stabilized structure of the transmembrane domain in the presence of Ca2+ could be destabilized by anisodamine. Decreases of the intrinsic fluorescence and increases of the extrinsic fluorescence of ANS, a fluorescent probe, showed the exposure of tryptophan and hydrophobic region, respectively, suggesting again that anisodamine caused a less compact conformation in the transmembrane domain. A marked inhibition of the Ca2+ uptake activity of SR Ca2+-ATPase was observed when the addition of anisodamine. The drug did not affect the cytoplasmic domain of the enzyme and only slightly decreased the ATPase activity of the enzyme at concentrations up to 10 mM. This was likely due to the destabilized protein transmembrane domain. To sum up, our results revealed that anisodamine interacted specifically with the transmembrane domain of SR Ca2+-ATPase and inhibited the Ca2+ uptake activity of the enzyme.
We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.
G-type lysozyme is a hydrolytic enzyme sharing a similar tertiary structure with plant chitinase. To discover the relation of function and structure, we analyzed the primary structure of new G-type lysozyme. The complete 185 amino acid residues of lysozyme from rhea egg white were sequenced using the peptides hydrolyzed by trypsin, V8 protease, and cyanogen bromide. Rhea lysozyme had sequence similarity to ostrich, cassowary, goose, and black swan, with 93%, 90%, 83%, and 82%, respectively. The six substituted positions were newly found at positions 3 (Asn), 9 (Ser), 43 (Arg), 114 (Ile), 127 (Met), and 129 (Arg) when compared with ostrich, cassowary, goose, and black swan lysozymes. The amino acid substitutions of rhea lysozyme at subsite B were the same as ostrich and cassowary lysozymes (Ser122 and Met123). This study was also constructed in a phylogenetic tree of G-type lysozyme that can be classified into at least three groups of this enzyme, namely, group 1; rhea, ostrich, and cassowary, group 2; goose, black swan, and chicken, and group 3; Japanese flounder. The amino acid sequences in assembled three α-helices found in this enzyme group (Thammasirirak, S., Torikata, T., Takami, K., Murata, K., and Araki, T., Biosci. Biotechnol. Biochem., 66, 147–156 (2002)) were also highly conserved, so that they were considered to be important for the formation of the hydrophobic core structure of the catalytic site and for maintaining a similar three-dimensional structure in this enzyme group.
When plasma membranes were prepared from tubers of Helianthus tuberosus L. (Jerusalem artichoke) frozen at a sublethal temperature (−10°C), the levels of some plasma membrane proteins, named frost-susceptible proteins (FSPs), decreased [Uemura, M., et al., Plant Physiol., 80, 187–195 (1986)]. The aim of this study was to characterize the response of FSP120, which is named FSP-3 in a previous report, to freezing treatment by immunoblotting. Levels of FSP120 in the plasma membranes of tubers decreased after sublethal freezing, whereas no degraded products were detected in the microsomes or the soluble fraction. The amount of FSP120 in the crude extract of frozen tubers remained at a comparable level to that of the unfrozen tubers. These results suggest that FSP120 might be released from plasma membranes during freezing treatment of the tubers of Jerusalem artichoke.
In the isotope ratio (Ir) analysis using GC-MS, several variable factors in sampling incidental to any food analysis were investigated for yuzu fruit. The Irs of ten monoterpene hydrocarbons in yuzu essential oils from each of six fruiting positions of three trees were measured. The sign test following t-test of all the Ir values demonstrated that there was no significant difference between both sampling years of 2001 and 2002. There was also no significant variation in the Ir values among the three trees and six fruiting positions in the individual two years.
To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.
We previously reported that expression of Sla1ΔC, a truncated form of Sla1, induces ectopic meiosis in heterothallic fission yeast and this was possibly due to the inhibition of Pat1 kinase by Sla1ΔC. Here we found mei2 mRNA and the Mei2 protein accumulated and stability of the Mei2 protein increased when Sla1ΔC was expressed. The former two results are considered to be the consequence of de-repression of Ste11, which is the transcription factor of mei2 and negatively regulated by Pat1 kinase. The latter result reflects the consequence of deregulation of Mei2 by Pat1 kinase. In addition, Ste11 accumulated in the nucleus when Sla1ΔC was expressed. All these data consistently support the idea that the action of Sla1ΔC is to inactivate Pat1 kinase.
Glycosylated ascorbic acids were synthesized by using the transglycosylation activity of Bacillus stearothermophilus maltogenic amylase with maltotriose to show effective antioxidative activity with enhanced oxidative stability. The modified ascorbic acids comprised mono- and di-glycosyl transfer products with an α-(1,6)-glycosidic linkage. The antioxidative effects of the glycosyl derivatives of ascorbic acid on the lipid oxidation of cooked chicken breast meat patties were compared, and the synergistic effect when combined with α-tocopherol was determined in terms of thiobarbituric acid-reactive substances (TBARS) and volatiles production during storage. The results indicate that the glycosylated ascorbic acids had very effective antioxidative activity in preventing lipid oxidation, and were better in their synergistic effect in comparison to authentic ascorbic acid, with maltosyl-ascorbic acid being the most effective. Volatiles production was highly correlated with the TBARS values in the lipid oxidation of cooked meat. The antioxidative effect preventing the production of volatiles was particularly strong on pentanal, fairly strong on propanal and butanal, and not at all on ethanal. Propanal, pentanal, and the total volatiles thus provided a good representation of the lipid oxidation status of cooked chicken meat.
We investigated a useful chemical index for an excessive nicotinamide intake and how this excessive nicotinamide intake affects the tryptophan-nicotinamide metabolism in rats. Weaning rats were fed on a tryptophan-limited and nicotinic acid-free diet containing no, 0.003%, 0.1%, 0.2%, or 0.3% nicotinamide for 21days. Urine samples were collected on the last day and analyzed the intermediates and metabolites on the tryptophan-nicotinamide pathway. Nicotinamide N-oxide, nicotinic acid and nicotinuric acid, metabolites of nicotinamide, were detected when nicotinamide at more than 0.1% had been taken. An intake of nicotinamide of more than 0.1% increased the urinary excretion of quinolinic acid, an intermediate on the pathway. Nicotinamide N-oxide and nicotinuric acid increased with increasing dietary concentration of nicotinamide. These results show that the measurements of nicotinamide N-oxide and nicotinuric acid in urine would be useful indices for an excessive nicotinamide intake.
Tryptophan was isolated from rat feces as an active compound against ovalbumin permeation in an in vitro Caco-2 cell model. Tryptophan dose-dependently inhibited ovalbumin permeation with accompanying increase in transepithelial electric resistance, and its inhibitory activity reached a plateau at 10 mM. Brown Norway rats were sensitized by intragastric administration of ovalbumin together with or without tryptophan. Antibody levels specific to ovalbumin in the sera and proliferative responses of spleen mononuclear cells to ovalbumin were significantly lower in rats administered ovalbumin plus tryptophan than those administered ovalbumin alone. These results suggest that tryptophan suppresses oral sensitization to ovalbumin, probably via suppression of ovalbumin absorption from the intestinal tract.
Powdery encapsulation of shiitake flavors, extracted from dried shiitake, was investigated by spray drying. Flavor retention increased with an increase in drying air temperature and solid content, and decreased with an increase in dextrose equivalents of maltodextrin. A heat-treatment of the extract liquid made the lenthionine concentration increase, but did not influence the concentrations of the other flavors. The formation of lenthionine with heat-treatment could be described by the consecutive unimolecular-type first order reaction. Lenthionine content in a spray-dried powder prepared with the heated extracted liquid significantly increased. α-Cyclodextrin was the most suitable encapsulant of α-, β-, and γ-cyclodextrins to prepare the spray-dried powder, including lenthionine. The flavor retentions were markedly increased by using of α-cyclodextrin and maltodextrin in combination as an encapsulant.
To elucidate the mechanism for hyperlipidemia in the hepatoma-bearing state, changes in some parameters related to the lipid metabolism and serum tumor necrosis factor-α (TNF-α) level were examined in Donryu rats that had been subcutaneously implanted with an ascites hepatoma cell line of AH109A. These parameters were also examined in rats that had been given a single injection of lipopolysaccharide (LPS), a model for acute infection with TNF-α secretion into the blood circulation. The serum triglyceride and total cholesterol (Ch) levels were significantly higher in both the hepatoma-implanted and LPS-injected rats than in normal rats. The level of adipose tissue lipoprotein lipase was decreased by hepatoma implantation and LPS injection, while the hormone-sensitive lipase activity was increased by the same treatments. Fatty acid (FA) oxidation and Ch synthesis were also stimulated by both treatments. The serum TNF-α level was noticably elevated by hepatoma implantation and greatly by the LPS injection. This LPS injection increased hepatic FA synthesis. The serum high-density lipoprotein Ch level and hepatic Ch 7α-hydroxylase activity were not changed by the LPS injection. Hepatoma implantation led to hyperlipidemia and elevated the serum TNF-α level, as did the LPS injection.
Nine triterpene acids, viz., six of the ursane type, ursolic acid (1), corosolic acid (2), 3-epicorosolic acid (3), pomolic acid (4), tormentic acid (5) and hyptadienic acid (6), and three of the oleanane type, oleanolic acid (7), augustic acid (8) and 3-epimaslinic acid (9), among which 1 constituted the most predominant triterpene acid, were isolated and identified from ethanol extracts of the leaves of red perilla [Perilla frutescens (L.) Britton var. acuta Kudo] and green perilla [P. frutescens (L.) Britton var. acuta Kudo forma viridis Makino]. These eight compounds, 1, 2, 4–9, were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 μg/ear) in mice. All the compounds tested showed a marked anti-inflammatory effect, with a 50% inhibitory dose (ID50) of 0.09–0.3 mg per ear. In addition, an evaluation against the Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA showed five compounds, 1–3, 5 and 9, with a potent inhibitory effect on EBV-EA induction (91–93% inhibition at 1×103 mol ratio/TPA). Furthermore, compound 5 exhibited strong antitumor-promoting activity in an in vivo two-stage carcinogenesis test of mouse tumor by using 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator and TPA as a promoter.
The decomposition process of maltose in subcritical water was studied using a tubular reactor in the temperature range of 180 to 260°C and at 10 MPa. The formation of glucose and 5-hydroxymethyl-2-furaldehyde during the maltose decomposition was also observed. The decomposition rate of maltose was faster at higher temperatures. The rate was approximated by first-order kinetics during the early stage of the decomposition, but was accelerated and deviated from these kinetics at the later stage. The effluent pH decreased as the residence time in the reactor increased and the decrease of pH affected the maltose decomposition rate and glucose formation. Low pH of a feed solution accelerated maltose decomposition. A good correlation was obtained between the pH of the effluent and the rate constant of the first-order kinetics.
We previously reported that oral administration of luteolin can inhibit serum tumor necrosis factor (TNF)-α production and several inflammatory and allergic models. We investigated here the effect of various flavonoids which resemble luteolin in structure. Lipopolysaccharide (LPS)-induced TNF-α production from macrophages was inhibited by treatment with flavone (luteolin, apigenin, and chrysin), flavonol (quercetin and myricetin), flavanonol (taxifolin), and anthocyanidin (cyanidin chloride) in vitro. Most of these, however, did not affect mice when administered orally. Serum TNF-α production was inhibited only by luteolin or apigenin, and only luteolin or quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema. These results suggest that the structure of luteolin: 3′,4′,5,7-tetrahydroxyflavone, is most suitable for the oral anti-inflammatory activity and that existence or disappearance of a hydroxy group may cause a loss of efficiency.
In this study, we have examined the anti-inflammatory actions of royal jelly (RJ) at a cytokine level. When supernatants of RJ suspensions were added to a culture of mouse peritoneal macrophages stimulated with lipopolysaccharide and IFN-γ, the production of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1, was efficiently inhibited in a dose-dependent manner without having cytotoxic effects on macrophages. This suggests that RJ contains factor(s) responsible for the suppression of proinflammatory cytokine secretion. We named the factor for honeybees RJ-derived anti-inflammatory factor (HBRJ-AIF), and further investigated the molecular aspects of it. Size fractionation study showed that HBRJ-AIF is composed of substances of low (<5 kDa) and high (>30 kDa) molecular weights, with the former being a major component. Chromatographic analysis showed that MRJP3 is one candidate for the HBRJ-AIF with high molecular weights. Thus, our results suggest that RJ has anti-inflammatory actions through inhibiting proinflammatory cytokine production by activated macrophages.
Sprouts of several plants (10 families and 28 species) were cultivated in a high selenium environment, and the chemical species of selenium in these selenium-enriched sprouts were identified by using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). Cultivation of sprouts of kaiware daikon (type of radish) with 5.0 μg/ml or 10.0 μg/ml of selenium as selenite inhibited the growth. However, no abnormalities in the shape or color were apparent even in the sprouts exposed to 10.0 μg/ml of selenium. The selenium concentration in the sprouts of most plants examined was higher than that from environmental exposure. Among the types of selenium that were accumulated, a large part (69–98%) was extractable in 0.2 M HCl. Chemical analysis of selenium in the HCl extract showed that the main selenium species in all the sprouts examined was Se-methylselenocysteine. In addition to Se-methylselenocysteine, selenomethionine, non-metabolized selenite, γ-glutamyl-Se-methylselenocysteine and an unknown selenium compound were also detected in several high-selenium sprouts. Since higher anticarcinogenic activities of these monomethylated selenoamino acids have been observed, it is anticipated that such selenium-enriched sprouts will be used as a foodstuff for cancer prevention.
A water-soluble and low-molecular-weight fraction (SB) was obtained from tomato paste. The effects of SB on the formation of advanced glycation end-products (AGE) in protein glycation were studied by the methods of specific fluorescence, ELISA and a Western blot analysis, using the anti-AGE antibody after incubating protein with sugar. The results suggest that SB had strong inhibitory activity, in comparison with aminoguanidine as a positive control, and that the inhibitory mechanism of SB differed from that of aminoguanidine to involve trapping of reactive dicarbonyl intermediates in the early stage of glycation. SB contained an antioxidant, rutin, which showed potent inhibitory activity. The results also suggest that rutin chiefly contributed to inhibiting the formation of AGE, and that other compounds in SB may also have been related to the activity.
The SMXA-5 strain, a new mouse model for type 2 diabetes, is a recombinant inbred strain derived from non-diabetic SM/J and A/J strains. As dietary fat is a key component in the development of diabetes, we compared the glucose tolerance and diabetes-related traits among the SMXA-5, SM/J, and A/J strains while feeding a high-fat diet for 10 weeks. SMXA-5 fed on a high-fat diet showed an increased serum insulin concentration. Judging from the hyperinsulinemia in SMXA-5, this strain showed insulin resistance, an inability of peripheral tissues to respond to insulin, which was strengthened by feeding with a high-fat diet. When fed on a high-fat diet for 5 weeks, the SMXA-5 mice showed severely impaired glucose tolerance. On the other hand, SM/J showed mildly impaired glucose tolerance, even when fed on a high-fat diet for 10 weeks. These results indicate that SMXA-5 would be available for use as a diabetic model susceptible to a high-fat diet.
The distribution of minerals in quinoa (Chenopodium quinoa Willd.) seed was examined using energy dispersive X-ray microanalysis (EDX) in combination with scanning electron microscopy (SEM). Phosphorus, K, and Mg coincided in localization in embryonic tissue. Since phytin globoids have been known to localize in protein bodies in embryonic cells of quinoa seed, it is thought that P is attributed to phytic acid and that K and Mg form to phytate. Calcium and K were present in the pericarp, where the cell wall is thickly developed, suggesting that these minerals are associated with pectin. Sulfur occurred in embryonic tissues, which would be derived from sulfur amino acid residues of storage proteins concentrated in the tissues. Abrasion of quinoa seeds resulted particularly in decrease in Ca content.
We recently established a novel co-culture assay system using activated inflammatory cells and AS52 Chinese hamster ovary cells, and demonstrated that reactive oxygen and nitrogen species (RONS) generated from activated inflammatory leukocytes induce mutations in the gpt recorder gene in AS52 cells. In this study, we examined the inhibitory effects of 19 agents with antioxidative properties on RONS generation in cultured inflammatory cells and on mutagenesis in AS52 cells co-cultured with activated inflammatory cells. The results demonstrate that there is a linear correlation between the ability of these agents to suppress RONS production in activated inflammatory cells and to inhibit mutation in AS52 cells.
In this study, we ascertained whether the parathyroid hormone (PTH) dominantly regulated the effects of high phosphorus (P) intakes on urinary excretion of P and bone metabolism in rats. To maintain serum PTH level equally, parathyroidectomy (PTX) and sham-operated rats were constantly exposed to rPTH(1–34) and fed both control (0.3% P) and high P (1.2% P) diet for 7 days, respectively. Urinary excretions of P and C-terminal telopeptides of type I collagen were significantly increased in both PTX and sham rats by the high P diet. These results suggest that high P diet increased urinary P excretion while promoting bone resorption regardless of PTH-dependent regulation.
Blue-M1 is a blue pigment formed from xylose and glycine in the Maillard reaction. Previous work revealed that Blue-M1 scavenged hydroxyl radicals, and prevented the autoxidation of linoleic acid in vitro. We investigated the protective effect of Blue-M1 for 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH)-induced toxicity in COS-1 cells. COS-1 cells were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37°C for 24 h. Blue-M1 decreased the AAPH-induced toxicity in COS-1 cells, and this effect was dose-dependent. Furthermore, COS-1 cells were treated with diphenyl-1-pyrenylphosphine (DPPP), as a reagent for the detection of lipid peroxide, and then were cultured in AAPH containing DMEM medium with or without Blue-M1 at 37°C for 6 h. Blue-M1 prevented the AAPH-induced peroxidation of cell membrane on COS-1 cells, and this effect was also dose-dependent. These results suggest that Blue-M1 prevents the oxidative cell injury. Therefore, Blue-M1 will be an antioxidant, which protect against the oxidative stress in living systems.
DPR, a fragment peptide of enterostatin (VPDPR) having hypocholesterolemic activity, was introduced into the three homologous sites, EPR, DYR, and DPI, in the soybean β-conglycinin α′ subunit by site-directed mutagenesis. The modified β-conglycinin was expressed in Escherichia coli and recovered in the soluble fraction. After purification on ion-exchange HPLC, the modified β-conglycinin was digested by trypsin to release integrated DPR. The yield of DPR from 1 mole of the modified β-conglycinin was 1.2 mole.
We examined the effects of bisphenol A (BPA) on immune cells and it was shown that BPA upregulated the proliferation of murine splenocytes stimulated with Concanavalin A (ConA). The upregulating effects of BPA were removed with depleting Mac1+ cells from the splenocytes. This study provides evidence for the first time that Mac1+ cells were required for enhancement of splenocytes proliferation caused by bisphenol A.
Dynamic change in microbial flora was monitored with an oxygen electrode. The 1st phase microorganisms, which first grew well in LB medium, were followed by the 2nd phase microorganisms, which supposedly assimilated microbial cells of the 1st phase and their metabolites. In a similar way, a change in microbial flora was observed from the 1st phase to the 4th phase in 84 hr. Based on this observation, prolonged enrichment culture was done for as long as two months to increase the ratio of existence of rare microorganisms. From these culture liquids, four slow-growing bacteria (provisionally named Shinshu-ah1, -ah2, -ah3, and -ah4), which formed scarcely visible small colonies, were isolated. Sequence analysis of their 16S rDNA showed that Shinshu-ah1 had 97% homology with Bradyrhizobium japonicum and uncultured alpha proteobacterium clone blaii 16, Shinshu-ah2 91% with Rasbo bacterium, Alpha proteobacterium 34619, Bradyrhizobium genosp. P, Afipia felis and an unidentified bacterium, Shinshu-ah3 99% with Methylobacterium mesophilicum, and Shinshu-ah4 95% with Agromyces ramosus DSM 43045. Phylogenetic study indicated that Shinshu-ah2 had a possibility to form a new family, Shinshu-ah1 a new genus, and Shinshu-ah4 a new species.
Point mutation of Gly1250Ser (1250S) of the yeast fatty acid synthase gene FAS2 confers cerulenin resistance. This mutation also results in a higher production of the apple-like flavor component ethyl caproate in Japanese sake. We mutated the 1250th codon by in vitro site-directed mutagenesis to encode Ala (1250A) or Cys (1250C) and examined cerulenin resistance and ethyl caproate production. The mutated FAS2 genes were inserted into a binary plasmid vector containing a drug-resistance marker and a counter-selectable marker, GALp-GIN11M86. The plasmids were integrated into the wild-type FAS2 locus of a sake yeast strain, and the loss of the plasmid sequences from the integrants was done by growth on galactose plates, which is permissive for loss of GALp-GIN11M86. These counter-selected strains contained either the wild type or the mutated FAS2 allele but not the plasmid sequences, from which FAS2 mutant strains were selected by allele-specific PCR. The FAS2-1250C mutant produced a higher amount of ethyl caproate in sake than FAS2-1250S, while FAS2-1250A produced an ethyl caproate level intermediate between FAS2-1250S and the parental Kyokai no. 7 strain. Interestingly, these mutants only showed detectable cerulenin resistance. These ‘self-cloning’ yeast strains should be acceptable to the public because they can improve sake quality without the presence of extraneous DNA sequences.
σmarY1 is the LTR of the retroelement marY1 from the homobasidiomycete Tricholoma matsutake. Upon integration through transformation, pLC1-hph carrying a σmarY1 derivative, σ*marY1, conferred the hygromycin-resistant phenotype stronger than the vector without σ*marY1 on Lentinula edodes. Based on the densitometric analysis after Southern hybridization, a copy number of the former construct integrated in the genome is much higher than that of the latter. We conclude that σmarY1 allows multicopy DNA integration and will be useful in the genetic research on this fungal group.
A hyperthermophilic cellulase derived from Pyrococcus horikoshii was successfully produced with the Bacillus brevis host-vector system. The production of the recombinant enzyme was increased about 20-fold (to a level of 100 mg per liter) by the insertion of certain amino acid such as alanine and peptides like AEEAADP between the carboxyl end of signal peptide and the N-terminus of the mature cellulase. These recombinant cellulases had the same characteristics as that of the cellulase expressed in Escherichia coli.
A mixture of steamed soybean and boiled rice with seeded Aspergillus oryzae was naturally fermented without addition of yeasts or Lactobacilli, and kept matured for 12 months at room temperature. Chemical analysis of this rice-koji miso sample for lipid changes during maturation showed that triacylglycerol was gradually decomposed into free fatty acid, with distinct formation of fatty acid ethyl ester which, six months after the start of fermentation, came to account for 35.0% of total lipid. The ester was constituted primarily with linoleic acid (ca. 50%) and oleic acid (ca. 20%), no appreciable change in this proportion being observed during maturation. Also, the proportion was unique in that this did not reflect the fatty acid composition in a mixture of the two materials. It is possible to monitor the maturation of the rice-koji miso by following up the increase with time in fatty acid ethyl ester.