A new tetraglycosyl flavonol, 3-O-[2-O-xylosyl-6-O-(3-O-glucosyl-rhamnosyl) glucosyl] kaempferol was isolated from pale purplish-pink petals of Wabisuke camellia cv. Tarokaja with three known flavonols. It was named urakunoside after the species name of Tarokaja, Camellia uraku. Urakunoside was a major flavonol component in the Tarokaja petals, but was not detected in petals of Tarokaja’s presumed ancestor species.
We identified a new radical scavenger, 10T024A (C15H12N2O4), from a culture of the Streptomyces sp. Spectroscopic elucidation indicated that this compound is a new phenazine derivative. 10T024A showed radical-scavenging activity with an ED50 of 125 μM. Moreover, it showed prostaglandin D2 (PGD2) and leukotriene B4 (LTB4) release suppressive activity in rat basophilic leukemia (RBL-2H3) cells, at IC50: 8 μM and 10 μM respectively.
Synthesis of tetrasaccharide portion of ganglioside HPG-1 is described. The tetrasaccharide sequence, Fuc-α(1,8)-Neu5Gc-α(2,4)-Neu5Ac-α(2,6)-Glc, was successfully assembled by a linear strategy, in which the 1,5-lactamized sialyl galactose acceptor and the 8-O-Lev-N-Troc-sialic acid donor were exploited as key units.
Catalase is the major H2O2-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)6-tagged proteins in Escherichia coli. The recombinant (His)6-polypeptides were enriched to apparent homogeneity and characterized. With H2O2 as substrate, the highest catalase kcat value (20±1.71×10−3 min−1) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, β-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats.
Experimental evidence in vivo as to the functional roles and binding properties to cadmium (Cd) of type-2 plants metallothionein (MT) has been limited thus far. We investigated the biological role of metallothionein from Colocasia esculenta (CeMT2b) in Escherichia coli and tobacco, and developed a new model for the relationship between Cd tolerance and Cd-binding ability. Heterologous expression of CeMT2b in Escherichia coli greatly enhanced Cd tolerance and accumulated Cd content as compared to control cells. The molecular weight of CeMT2b increased with Cd, and CeMT2b bound up to 5.96±1 molar ratio (Cd/protein). Under Cd stress, transgenic tobacco plants displayed much better seedling growth and high Cd accumulation than the wild type. The presence of an extra CXC motif in CeMT2b contributed to the enhanced Cd-tolerance. The present study provides the first insight into the ability of type-2 plant MT to bind physiological Cd.
Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.
An extracellular thermostable xylanase produced by Saccharopolyspora pathumthaniensis S582 was purified 167-fold to homogeneity with a recovery yield of 12%. The purified xylanase appeared as a single protein band on SDS–PAGE, with a molecular mass of 36 kDa. The optimal temperature and pH of the xylanase were 70 °C and 6.5. The enzyme was stable within a pH range of 5.5–10.0. It retained its activity after incubation at 50 °C for 2 h. Its half lives at temperatures of 60 and 70 °C were 180 and 120 min respectively. Hydrolysis of beechwood xylan by the xylanase yielded xylobiose and xylose as major products. The enzyme acted specifically on xylan as an endo-type xylanase, and exhibited a Km value of 3.92 mg/mL and a Vmax value of 256 μmol/min/mg. Enzyme activity was completely inhibited by Hg2+, and was stimulated by Rb+ and Cs+. The xylanase gene was cloned from genomic DNA of Saccharopolyspora pathumthaniensis S582 and sequenced. The ORF consisted of 1,107 bp and encoded 368 amino acid residues containing a putative signal peptide of 23 residues. This xylanase is a new member of family (GH) 10 that shows highest identity, of 63.4%, with a putative xylanase from Nocardiopsis dassonvillei subsp. dassonvillei.
A secretory glycoprotein named Ψ-factor that we have purified and cloned from Dictyostelium discoideum is prespore cell-inducing factor. To address its functional significance, it is necessary to examine the attached sites and structures of its glycans as well as its protein structure. Here we identified and isolated a tryptic glycosylated peptide with the 71st to 89th amino acids of Ψ-factor that contained the consensus amino acid sequence for an N-linked glycan (N-T-T). MALDI-TOF mass spectrometry indicated that the major protonated molecular ions, [M+H]+, of the glycopeptide were present at m⁄z 3,806, the minor m⁄z 3,603 and 3,400 ions corresponding to the loss of one and two N-acetylhexosamines respectively. Digestion of it with N-glycosidase F gave a molecular mass of 1,766.9 for the whole glycan moiety, which accounts for its composition of five hexoses, four N-acetylhexosamines, and a deoxyhexose. Further digestion experiments on the basis of the substrate specificity of α-mannosidase and β-N-acetylhexosaminidase allowed us to elucidate the unique structure of the glycan, which contains a bisecting and an intersecting GlcNAc and a core α1,6-fucosyl moiety.
Eucalyptus bridgesiana, Cymbopogon martinii, Thymus vulgaris, Lindernia anagallis, and Pelargonium fragrans are five species of herbs used in Asia. Their essential oils were analyzed by GC-MS, and a total of 36 components were detected. The results of our study indicated that, except for the essential oil of P. fragrans, all of the essential oils demonstrated obvious antimicrobial activity against a broad range of microorganisms. The C. martinii essential oil, which is rich in geraniol, was the most effective antimicrobial additive. All of the essential oils demonstrated antioxidant activities on 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, β-carotene/linoleic acid assay, and nitric oxide radical scavenging assay. Furthermore, the T. vulgaris essential oil, which possesses plentiful thymol, exhibited the highest antioxidant activity. For P. acnes-induced secretion of pro-inflammatory cytokines, the essential oils of P. aeruginosa, C. martinii, and T. vulgaris reduced the TNF-α, IL-1β, and IL-8 secretion levels of THP-1 cells.
This investigation was designed to determine the effect of a novel soluble beta-glucan salecan on acute alcohol-induced hepatic injury in mice. Mice were given salecan (15 or 30 mg/kg) or PBS for 4 d. Ethanol (6 g/kg) was administered orally 1 h after the last injection. The animals were sacrificed at 10 h after alcohol administration. Pretreatment with salecan significantly ameliorated the hepatic damage induced by ethanol, as evidenced by markedly reduced serum aminotransferase activities and hepatocyte steatosis. Salecan administration remarkably alleviated the formation of thiobarbituric acid-reactive substances and counteracted glutathione depletion. The mRNA level of peroxisome proliferator activated receptor alpha, a major gene responsible for fatty acid oxidation, was significantly increased after salecan pretreatment. The expression of diacylglycerol acyltransferase 1, an important gene responsible for triacylglycerol synthesis, was markedly decreased after salecan was administrated. These findings suggest that salecan might represent a novel protective strategy against alcoholic liver injury.
Despite recent progress in fluorescence techniques employed to observe protein localization in living cells, the in vitro chloroplastic protein transport assay remains a useful tool for determining the destinations of proteins. Although an in vitro synthesized, radiolabeled precursor protein is frequently used as the transport substrate, we have developed a transport assay system with a non-radiolabeled precursor protein that carries an epitope tag and is overexpressed in Escherichia coli. Thus, a transported protein can be detected by immunoblotting (Inoue et al., Plant Physiol. Biochem., 46, 541–549 (2008)). Here, we propose another in vitro protein transport system that combines fluorescence techniques. We attempted to use two types of precursors: a green fluorescent protein (GFP)-fused precursor and a fluorescent dye-labeled one. Both were successfully imported into chloroplasts. However, the fluorescent dye-labeled precursor was more advantageous than the GFP-fused precursor in the in vitro system.
Two novel non-synonymous SNPs in the 2nd and 3rd exons of the porcine ApoR gene are reported. One was identified as a novel SNP significantly associated with multiple traits of pork meat quality. The data can provide a useful resource for developing a marker in the genetic improvement of pigs.
A designed self-folding RNA possessing two peptide-recognition motifs served as a template for the chemical ligation of two RNA-binding peptides under stoichiometric conditions. In this study, we investigated the turnover ability of this template RNA in facilitation of peptide ligation and found that the RNA exhibited modest turnover ability under conditions in which its 3D structure was marginally stable.
We investigated the roles of catalase (CAT) in abscisic acid (ABA)-induced stomatal closure using a cat2 mutant and an inhibitor of CAT, 3-aminotriazole (AT). Constitutive reactive oxygen species (ROS) accumulation due to the CAT2 mutation and AT treatment did not affect stomatal aperture in the absence of ABA, whereas ABA-induced stomatal closure, ROS production, and [Ca2+]cyt oscillation were enhanced.
In the model seed plant Arabidopsis thaliana, a sub-family of B-box containing transcriptional factors (BBXs), which is classified in the BBX-IV group based on the domain structure, contains two tandem B-box domains and plays crucial roles in early photomorphogenesis under the control of blue light receptors, cry1 and cry2. The results of an examination of light responsiveness of representative Physcomitrella BBX-IV genes and their heterologous expression in Arabidopsis suggested that the light signaling-related characteristics of the BBX-IV subfamily are evolutionarily conserved in a moss, which is a basal lineage of land plants.
A gene encoding a UDP-glucose dehydrogenase homologue was identified in the hyperthermophilic archaeon, Pyrobaculum islandicum. This gene was expressed in Escherichia coli, and the product was purified and characterized. The expressed enzyme is the most thermostable UDP-glucose dehydrogenase so far described, with a half-life of 10 min at 90 °C. The enzyme retained its full activity after incubating in a pH range of 5.0–10.0 for 10 min at 80 °C. The temperature dependence of the kinetic parameters for this enzyme was examined at 37–70 °C. A decrease in Kms for UDP-glucose and NAD was observed with decreasing temperature. This resulted in the enzyme still retaining high catalytic efficiency (Vmax⁄Km) for the substrate and cofactor, even at 37 °C. These characteristics make the enzyme potentially useful for its application at a much lower temperature such as 37 °C than the optimum growth temperature of 100 °C for P. islandicum.
Previous rotavirus infection studies used the focus reduction assay extensively to evaluate cellular responses to viral infection, but this technique has a number of limitations. In this study, we developed a simplified, accurate rotavirus infection assay to evaluate the effects of inhibitory substances on rotavirus infection in vitro by measurement of the fluorescence intensities of stained cells.
Fucoxanthin is a non-provitamin A carotenoid contained in brown seaweeds. We found that it suppressed interleukin-17 secretion from CD4+ T cells under IL-17-producing T (Th17) cell development conditions. By evaluating T cell differentiation in vitro, fucoxanthin and its metabolite fucoxanthinol inhibited T cell differentiation into Th17 cells. This suggests that fucoxanthin can improve inflammatory diseases due to Th17 cells.
Plasma hyaluronan-binding protein (PHBP) is a serine protease the activation of which is implicated in inflammation. Previous investigations have suggested the presence of catechol-binding sites in its proenzyme form, pro-PHBP. Here we found that compounds with plural catechol groups conjugated with strong electron-withdrawing groups, such as tyrphostin AG 537 (IC50=18 nM), were potent inhibitors of pro-PHBP activation.
SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.
Thioredoxin (TRX) catalyzes the reduction of disulfide bonds in proteins via the NADPH-dependent thioredoxin reductase system. Reducing the disulfide bonds of allergenic proteins in food by TRX lowers the allergenicity. We established in this study a method to prepare TRX-enriched extracts from the edible yeast, Saccharomyces cerevisiae, on a large and practical scale, with the objective of developing TRX-containing functional foods to mitigate food allergy. Treating with the yeast TRX-enriched extracts together with NADPH and yeast thioredoxin reductase enhanced the pepsin cleavage of β-lactoglobulin and ovomucoid (OM). We also examined whether yeast TRX can mitigate the allergenicity of OM by conducting immediate allergy tests on guinea pigs. The treatment with TRX reduced the anaphylactic symptoms induced by OM in these tests. These results indicate that yeast TRX was beneficial against food allergy, raising the possibility that yeast TRX-enriched extracts can be applied to food materials for mitigating food allergy.
Quillaja saponin (QS) was examined for its immunostimulating effect on mice and humans after oral administration. Mice fed QS for 24 h significantly increased in chemotactic and phagocytosis activities of peritoneal macrophages. This enhancing effect in both activities continued for 4-d after QS administration. Mice fed QS for 24 h prior to an interperitoneal challenge with Escherichia coli showed a higher survival rate than the control group. Peripheral blood analysis of volunteers showed significant increases in chemotactic and phagocytosis activities after oral administration of QS for 7 d. Furthermore, the volunteers did not show significant changes in immunoglobulin, transaminase, IL-1α, or TNF-α levels, or in serum albumin concentrations. Thus orally administered QS can effectively enhance the immune response through stimulation of macrophages without adverse effects.
Vitamin E deficiency from birth or infancy has recently been found to increase anxiety-like behavior in rodents. The present study was undertaken to elucidate the effect of dietary vitamin E deficiency on anxiety in adult rats in comparison with juvenile rats. Male Wistar rats, 3 or 10 weeks old, were divided into two groups and fed a control or vitamin E-deficient diet for 4 weeks. The results of behavioral analysis revealed that vitamin E-deficiency increased anxiety in both juvenile and adult rats. Plasma, liver, and brain α-tocopherol concentrations decreased significantly due to vitamin E deficiency in both age groups. Plasma corticosterone concentrations were higher in the vitamin E-deficient rats in response to the stress of a behavioral test. Based on these results, we conclude that dietary vitamin-E deficiency induces anxiety in adult rats as well as juvenile rats. This might be due to an elevated plasma corticosterone concentration.
Osteoporosis is a serious disease caused by decreased bone mass. There is constant matrix remodeling in bones, by which bone formation is performed by osteoblastic cells, whereas bone resorption is accomplished by osteoclast cells. We investigated the effect of a Japanese apricot (Prunus mume SIBE. et ZUCC.) extract on the proliferation and osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. An alkaline phosphatase (ALP) activity assay, cell proliferation assay, alizarin red staining and expression analysis of osteoblastic genes were carried out to assess the proliferation and osteoblastic differentiation. The water-soluble fraction of Prunus mume (PWF) increased the ALP activity, cell proliferation and mineralization. The gene expression of osteopontin and bone morphogenetic protein-2, which are markers in the early period of osteoblastic differentiation, were significantly enhanced by the PWF treatment. PWF therefore stimulated the proliferation and osteoblastic differentiation of cells and may have potential to prevent osteoporosis.
The intra- and extracellular levansucrase (LS) activities produced by Bacillus amyloliquefaciens were promoted by supplementing the sucrose medium with yeast and peptone as nitrogen sources. These activities were purified by polyethylene glycol (PEG) fractionation for the first time. PEGs of low molecular weight selectively fractionated the intracellular LS activity rather than the extracellular LS activity. Contrary to other LSs, B. amyloliquefaciens LSs exhibited high levan-forming activity over a wide range of sucrose concentrations. The optimum temperatures for the intra- (25–30 °C) and extracellular (40 °C) LS transfructosylation activities were lower than those for the hydrolytic activities (45–50 °C; 50 °C). In addition, the catalytic efficiency for the transfructosylation activity of intracellular LS was higher than that of extracellular LS. These differences between intra- and extracellular LSs reveal the occurrence of certain conformational changes to LS upon protein secretion and/or purification. This study is the first to highlight that B. amyloliquefaciens LSs synthesized a variety of FOSs from various saccharides, with lactose and maltose being the best fructosyl acceptors.
We have previously shown that medium-chain triglyceride (MCT) resulted in significantly less body fat mass than long-chain triglyceride (LCT) did in hypertriglyceridimic subjects. The possible mechanism for this was investigated by measuring and analyzing changes in the body fat, blood lipid profile, enzymatic level and activity of hormone-sensitive lipase (HSL) and its mRNA expression, and levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in white adipose tissue (WAT) of C57BL/6J mice fed for 16 weeks on an MCT or LCT diet. MCT induced lower body weight and body fat, and an improved blood lipid profile than LCT did. The enzymatic level and activity of HSL and its mRNA expression, and the levels of cAMP and PKA were significantly higher in WAT of mice fed with the MCT diet. No significant differences in the levels of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in WAT were apparent between the effects of MCT and LCT. It is concluded that lipolysis by the increased level and activity of HSL, which was induced by the activation of cAMP-dependent PKA in WAT, was partially responsible for the lower fat accumulation in C57BL/6J mice fed with MCT.
The heat inactivating effect of low-pressure carbonation (LPC) at 1 MPa against Escherichia coli was enhanced to 3.5log orders. This study aimed to investigate the mechanisms of this increase in heat inactivation efficiency. The increased inactivation ratio was found to be the result of LPC-induced heat sensitization. This sensitization was not due to any physical damage to the cells as a result of the treatment. Following the depletion of intracellular ATP, the failure of the cells to discard protons caused an abnormal decrease in the intracellular pH. However, in the presence of glucose, the inactivation ratio decreased. In addition, a further increase in inactivation of more than 2log orders occurred in the presence of the protein synthesis inhibitor chloramphenicol. Hence, the decreased heat resistance of E. coli under LPC was most likely due to a depletion of intracellular ATP and a decreased capacity for protein synthesis.
Utilizing phytochemicals in treating inflammation is becoming a viable alternative to pharmacological treatment. We have reported that fermented barley extract (FBE) effectively suppresses oxidative stress in chronically ethanol-fed rats. Here we report that FBE suppressed acute increases in oxidative stress as a response to lipopolysaccharide (LPS)-induced inflammation. Rats supplemented with FBE for 10 d showed decreases in plasma interleukin (IL)-1β, IL-6, and tumor necrosis factor-α by 25%, 34%, and 35% respectively after LPS challenge. Liver damage was significantly suppressed, as marked by a 44% decrease in plasma alanine aminotransferase. FBE supplementation sustained liver anti-oxidative enzymes, catalase, glutathione peroxidase, and superoxide dismutase, at transcriptional and enzymatic levels, thus suppressing oxidative stress markers such as plasma nitric oxide and 8-hydroxy-2′-deoxyguanosine, by 42% and 23% respectively. We concluded that active compounds in FBE effectively inhibited the propagation of inflammation by suppressing oxidative stress.
Previously, we identified methoxsalen (8-methoxy-2′,3′,6,7-furocoumarin) as the bioactive compound probably responsible for acetylcholinesterase (AchE) inhibition achieved by feeding crude extract of Poncirus trifoliate. To confirm the activity of methoxsalen, Institute of Cancer Research (ICR) mice were fed a control or a methoxsalen-supplemented diet for 4 weeks, and then learning and memory enhancing effects with respect to trimethyltin (TMT)-induced neurotoxicity were evaluated. The brain tissues of ICR mice were dissected after completion of the behavioral tests for biochemical analysis. Methoxsalen effectively reversed TMT-induced memory impairment on both Y-maze and passive avoidance tests. Brain AchE activity was inhibited by the oral consumption of all concentrations of methoxsalen. Moreover, the level of oxidative stress was significantly ameliorated in the groups on methodsalen containing diets. This is the first in vivo study conducted with methoxsalen in the field of AD research, and it indicates that further investigation of methoxsalen is warranted.
The effect of a plum ethanol extract (PEE) on immunity was analyzed. An oral administration of PEE increased the interleukin (IL)-12p40 concentration in the serum and T-cell ratio in the spleen. In vitro studies revealed that PEE stimulated IL-12p70 production in peritoneal macrophages and natural killer activity. These findings suggest that PEE enhanced the immune function by stimulating innate immune cells.
Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.
It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer’s disease.
The optimal conditions for the production of γ-aminobutyric acid (GABA) by the marine yeast isolate Pichia anomala MR-1 strain appeared to be completely different from those required for growth and biomass production of the yeast strain. By proper reaction temperatures and pH levels, the efficiency of GABA production was improved dramatically. Supplying enough energy for the reaction was another important technical element to improve the production efficiency of GABA. The MR-1 isolate was found to use various saccharides, including glucose and fructose, as well as intermediate metabolites in the tricarboxylic acid (TCA) cycle to promote GABA production. On the other hand, ethanol, various free amino acids, and organic acids were detected along with GABA in the reaction solution. These results suggest that production of GABA by the MR-1 strain is not due to a single enzymatic reaction. Rather it is a fermentation reaction, possibly involving a combination of intracellular metabolic pathways.
Arthrofactin is a biosurfactant produced by Pseudomonas sp. MIS38. We have reported that transposon insertion into spoT (spoT::Tn5) causes moderate accumulation of guanosine 3′,5′-bispyrophosphate (ppGpp) and abrogates arthrofactin production. To analyze the linkage of SpoT function and ablation of arthrofactin production, we examined the spoT::Tn5 mutation. The results showed that spoT::Tn5 is not a null mutation, but encodes separate segments of SpoT. Deletion of the 3′ region of spoT increased the level of arthrofactin production, suggesting that the C-terminal region of SpoT plays a suppressive role. We evaluated the expression of a distinct segment of SpoT. Forced expression of the C-terminal region that contains the ACT domain resulted in the accumulation of ppGpp and abrogated arthrofactin production. Expression of the C-terminal segment also reduced MIS38 swarming and resulted in extensive biofilm formation, which constitutes the phenocopy of the spoT::Tn5 mutant.
Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.
The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD+-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD+-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.
A novel bacterium, Massilia sp. BS-1, producing violacein and deoxyviolacein was isolated from a soil sample collected from Akita Prefecture, Japan. The 16S ribosomal DNA of strain BS-1 displayed 93% homology with its nearest violacein-producing neighbor, Janthinobacterium lividum. Strain BS-1 grew well in a synthetic medium, but required both L-tryptophan and a small amount of L-histidine to produce violacein.
We characterized high malic acid-producing strains of Saccharomyces cerevisiae isolated from sake mash. We compared the gene expression of these strains with those of the parental strain by DNA microarray, and found that stress response genes, such as HSP12, were commonly upregulated in the high malate-producing strains, whereas thiamine synthesis genes, such as THI4 and SNZ2, were downregulated in these strains.
Novel geldanamycin derivative, 4,5-dihydro-thiazinogeldanamycin (3), was characterized from the gdmP mutant in Streptomyces hygroscopicus 17997, besides expected 4,5-dihydro-geldanamycin (2). The presence of this compound would suggest an unknown post-PKS modification in geldanamycin biosynthesis. Compound 3 exhibited moderate anti-HSV-1-virus activity and higher water solubility than geldanamycin (1). Cysteine served as a precursor to synthesize 3, whose formation required obligatory enzymatic assistance.
Fe(III)-EDTA reductase was purified from Bacillus sp. B-3 isolated as a Fe(III)-EDTA-degrading bacterium. The purified enzyme showed a single protein band corresponding to a molecular mass of 19 kDa on SDS–PAGE, and had FMN as cofactor. It was alkali-thermostable. Its N-terminal amino acid sequence was identical with that of NADPH azoreductase from several species of Bacillus.
Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.