This work evaluates the protective effects of Chinese herbs against ONOO⁻-induced biomolecule damage. Thirty-two Chinese traditional herbs were preliminarily screened for their ONOO⁻-scavenging activity by the fluorometric method. The potency of scavenging activity was in the following order: Apis cerana Fabricius (Propolis) > Rosmarinus officinalis L (Rosemary) > Pseudolarix amabilis (Nelson) Rehd. (Pine Bark PE) > Echinacea Moenck. (Echinacea); the remaining twenty-eight herbs performed unsatisfactorily for their scavenging activity. The four extracts with high ONOO⁻ scavenging activity were selected for further characterization of their inhibiting effects on DNA single breaks, tyrosine nitration and LDL oxidation. A further study of the origin of this scavenging activity on the major active components showed cichoric acid and rosemary acid to both have strong ability to scavenge ONOO⁻. These scavengers might be developed as therapeutic drugs for preventing ONOO⁻-involved diseases.

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Seven coumarins, including a new O-terpenoidal coumarin, named anisumarin (1), were isolated from Clausena anisum-olens Merr. The structure of this new coumarin was identified as 7-{(E)-4-[(4-acetoxymethyl-1,5-dihydro-5-oxo)-2H-furylium-2-yl]-3-methyl-2-butenoxy}-8-methoxycoumarin on the basis of an extensive spectroscopic analysis.

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To develop an economical, easy technique for producing recombinant E2 glycoprotein (rE2) of classical swine fever virus (CSFV) as a candidate immunogen, a bi-cistronic baculovirus/larvae expression vector was constructed using p10 promoter, an internal ribosome entry site, and the gfp gene. Trichoplusia ni larvae were successfully infected with the occluded recombinant baculovirus via feed, and the characteristics of rE2 were confirmed by immunoblot and glycosylation stain. rE2 at a concentration of 0.6–0.8 mg/ml without degradation was obtained from hemolymphs of infected larvae that emitted high levels of green fluorescence. Immunization assays indicated that mice and piglets immunized with rE2-containing hemolymph elicited high titers of anti-CSFV E2 antibodies with virus-neutralizing activity. This is the first study to indicate that baculovirus/T. ni larvae-expressed rE2 can be served as a vaccine candidate. This system provides an economical alternative for the production of vaccine components in the veterinary industry.

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Diabetic nephropathy is characterized as the progressive development of renal insufficiency in a setting of hyperglycemia. Previous studies indicate that reactive oxygen species (ROS) play an important role in high glucose-induced renal injury. Cilostazol was reported to lower the production of superoxide significantly in situ. We hypothesized that cilostazol administration in streptozotocin-induced diabetic rats exerts effects via improving oxidative stress. Male Sprague–Dawley rats were fed with cilostazol (5 mg/kg or 25 mg/kg) for 12 weeks after streptozotocin-induced diabetes mellitus. The results showed that cilostazol decreased reactive oxygen species activity significantly in the kidneys of diabetic rats and improved the urine albumin/creatinine ratio. Cilostazol can also improve the levels of serum cholesterol, triglyceride, and LDL-cholesterol. Additionally, diabetes-caused increased glomerular size, TGF-β, and NF-κB decreased under treatment with cilostazol in diabetic rats. Our results indicate that cilostazol has beneficial effects in early diabetic nephropathy.

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A neutralizing epitope fragment of ApxIIA toxin (ApxIIA#5) of the Korean Actinobacillus pleuropneumoniae serotype 2 strain was expressed and immobilized on the cell surface of Saccharomyces cerevisiae for efficient vaccine development. Expression of ApxIIA#5 was confirmed by Western blot analysis using cell-wall proteins, and the surface display of ApxIIA#5 was further visualized under confocal microscopy. Quantitative ELISA revealed that the recombinant ApxIIA#5 directed to the cell surface consisted of approximately 16% cell-wall proteins, estimated to be 35 mg of ApxIIA#5 protein per liter of cultured cells. An immunoassay revealed that antigen-specific antibodies against ApxIIA#5 were present in the sera of mice fed recombinant ApxIIA#5-expressing yeast, but not in mice fed the wild-type nor the vector-only transformant. Moreover, the mice fed the recombinant epitope-expressing yeast were protected from injection of a lethal dose of A. pleuropneumoniae.

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To develop a gene therapeutic method for human cytomegalovirus (HCMV), the IE86 specific short hairpin (sh) RNA expressing vector was constructed and subsequently transfected into MRC-5 cells. After infection of these cells with HCMV AD169, expression of IE86 was reduced strikingly as compared to the control. In addition, the inhibitory effect corresponded to a decrease in viral DNA replication and the virus-induced cytopathic effect. Measurement of the virus yield demonstrated that infection of cells expressing IE86-specific shRNA resulted in suppression of the formation of infectious viral progeny. These observations indicate that IE86 can be used as an effective target against HCMV infection using RNA interference (RNAi) technology, which provides new possibilities for anti-HCMV studies.

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One of the biochemical pathways affected by aging in all organisms is protein synthesis. Previous reports from our laboratory have indicated that the elongation step is specially affected by aging as a consequence of alterations in elongation factor-2 (eEF-2). In the present work, we studied in vitro the effectiveness of several individual nutritional antioxidants in protecting the levels of hepatic eEF-2 subjected to oxidative stress induced by cumene hydroperoxide. The in vitro system employed consisted of rat liver homogenates treated with cumene hydroperoxide. The antioxidants used in this study were lipoic acid, coenzyme Q10, tethrahydrofolic acid, and N-tert-butyl-alpha-phenylnitrone. The results indicate that the antioxidants have different capacities to prevent eEF-2 loss, folic acid being the most effective. A comparison between the antioxidants used and their potential pro-oxidant activity is also discussed, on the basis of the oxidative stress parameters measured.

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With reference to the local conformation of a protein, it is interesting to differentiate the individual fluorescence properties of included tryptophan residues without modification. The fluorescence spectrum of bitter gourd trypsin inhibitor (BGTI) was separated into two emission bands by the quenching-resolved fluorescence method. One emission band was given as a fraction with the Stern-Volmer quenching constant, 44.9×10⁻³ M⁻¹, against the fluorescence quenching by KI, and it showed an emission maximum intensity at 341 nm. The fluorescence quenching constant of the other band was 1.58×10⁻³ M⁻¹, and the maximum wavelength was found at 337 nm. These separated emissions were due to the fluorescence of Trp54 and Trp9 of BGTI. The quenching resolved-fluorescence spectrum was effectively applied to the precise description of the polar circumstances surrounding the Trp residues in the unfolding intermediate state of BGTI. The results suggested that the molten globule-like state of BGTI adopted such a peculiar conformation that the helix domain including Trp9 was packed more densely while the other loop domain partially unfolded.

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The occurrence of denitrification and nitrate respiration among eukaryotes has been established during the last few decades. However, denitrification-related eukaryotic genes have been isolated from only a few fungi, and eukaryotic denitrification (or nitrate respiration) is still inadequately understood. In this study, we identified genes that were up-regulated under denitrifying conditions in the fungus Cylindrocarpon tonkinense using the suppression subtraction hybridization technique, and the expression patterns of these genes were characterized by Northern analysis. We identified copper-containing nitrite reductase, cytochrome P450 nitric oxide reductase, flavohemoglobin (Fhb), and formate/nitrite transporter homolog genes as possibly involved in fungal denitrification. Our results concerning the involvement of Fhb and formate/nitrite transporter perhaps provide new insight into the fungal denitrification system.

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At the terminal step of heme biosynthesis, ferrochelatase (FECH) catalyzes the insertion of Fe²⁺ into protoporphyrin to form heme. It is located on the inner membrane of the mitochondria of animals. The enzyme inserts divalent metal ions, including Fe²⁺, Co²⁺, and Zn²⁺, into porphyrins in vitro. We have reported that it can remove Fe²⁺ from heme. To characterize the iron-removal reverse activity of FECH, we examined its properties in porcine liver and muscle mitochondria, and isolated porcine FECH cDNA. The amino acid sequence of porcine FECH showed high homology with bovine (91%), human (85%), mouse (87%), and rat (76%) equivalents. It was expressed in Escherichia coli, and purified, and the kinetic properties of the zinc-chelating and iron-removal activities were examined. Both activities peaked at 45 °C, but different optimal pH values, of 7.5–8.0 for zinc-ion insertion and 5.5–6.0 for the reverse reaction were found. The K_m values for mesoporphyrin IX and Zn²⁺ were 6.6 and 1.1 μM, respectively, and the K_m for heme was 5.7 μM. The k_cat value of the forward reaction was about 11-fold higher than that of the reverse reaction, indicating that the enzyme preferably catalyzes the forward reaction rather than the iron-removal reaction. Reverse activity was stimulated by fatty acids and phospholipids, similarly to the case of the forward reaction, indicating that lipids play a role in regulating both enzyme activities.

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Juvenile hormone epoxide hydrolases (JHEHs) are a family of enzymes that hydrolyze juvenile hormones (JHs). They are important in terms of organ-specific regulation and irreversible degradation. In contrast to three JHEH genes (jheh) in Drosophila melanogaster and five jheh in Tribolium castaneum, only one jheh gene has been reported to date in lepidopteran insects. By searching a genome database of the silkworm, KAIKOBLAST, five JHEH-related genes (jheh-r), in addition to Bmjheh, were found. Developmental changes in mRNA expression were brought about revealing several unique patterns for each of jheh-r as to developmental stages and organ-specificity. Recombinant proteins of JHEH-r were expressed using a baculovirus system to evaluate their enzymatic activities. Three of the five JHEH-r recombinant proteins had JH hydrolytic activities. This is the first report on lepidopteran jheh-related genes and also provides the comprehensive analysis of multiple jheh-related genes in an insect species with respect to their functions in enzyme activities.

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Osthole, a natural coumarin derivative, is extracted from the fruit of Cnidium monnieri Cusson. Breast cancer is one of the most commonly diagnosed cancers and the leading cause of death in women. Recent studies have shown that Osthole has anti-tumor activity. However, the effects of Osthole on the migration and invasion of cancer cells have not yet been reported. Here, we found that Osthole is effective in inhibiting the migration and invasion of breast cancer cells by wound healing and transwell assays. Luciferase and zymography assays revealed that Osthole effectively inhibits matrix metalloproteinase-2 promoter and enzyme activity, which might be one of the causes that lead to the inhibition of migration and invasion by Osthole. This is the first report on the inhibitory function of Osthole in migration and invasion in breast cancer cells. Our findings indicate a need for further evaluation of Osthole in breast cancer chemotherapy and chemoprevention.

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To endure considerable fluctuations in temperature, plants need precise regulation of temperature-controlled gene expression. In this study, the involvement of TAS1 trans-acting siRNA (tasiRNA) in temperature-controlled gene expression was examined in Arabidopsis. The accumulation of TAS1 tasiRNA was downregulated at 4 °C. Concomitant with the reduction of TAS1 tasiRNA-mediated cleavage, expression of At1g51670, a target of TAS1 tasiRNA, was upregulated at 4 °C in the wild type but not in a dicer-like enzyme (DCL) 4 mutant (dcl4-2), which is impaired in tasiRNA biogenesis. The expression of At4g29760 and of At5g18040, further TAS1 tasiRNA targets, was upregulated both in the wild type and in dcl4-2 at 4 °C. However, after shifting the temperature to 22 °C, low-temperature-induced expression of At4g29760 rapidly dropped in the wild type, but not in dcl4-2. Thus TAS1 tasiRNA acted as a sweeper for the clearance of excess amounts of At4g29760 transcripts. Our data suggest that differential regulation of TAS1 tasiRNA targets is involved in temperature-controlled gene expression.

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The aim of this study was to investigate a new allergen of Salsola kali, Sal k 4, and to investigate the predictive value of the conserved conformational regions in cross-reactivity with other plant-derived profilins. The Sal k 4-coding sequence was cloned, expressed, and purified by one-step Ni²⁺ affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted conserved conformational regions among rSal k 4 and other plant-derived profilins. Immunodetection and inhibition assays using 30 individual sera from S. kali allergic patients indicated that purified rSal k 4 might be the same as that in the crude extract. The results of inhibition assays among rSal k 4 and other plant-derived profilins were in accordance with the homology of the predicted conserved conformational regions. Amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins might depend on the predicted conserved conformational regions.

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Saturation mutagenesis was performed on three non-catalytic residues, Asn71, Leu108, and Gly177, in and near the active site of Arabidopsis thaliana GSTZ-1 (AtGSTZ-1). Forty unique mutants with more than 10% activity increases, were obtained. Of these, 12 resulted in large activity improvement and were purified for further characterization. Remarkably, 11 of them contained mutations at Leu108, suggesting that Leu108 plays an important role in dichloroacetic acid-dechlorinating (DCA-DC) activity. Kinetic analysis revealed that multiple mutations at these residues increased k_cat/K_m toward DCA and GSH by as much as 2.2- and 5.8-fold, respectively. Since the catalytic residues were not involved in mutagenesis, the activity enhancements were presumably due to structural change in the active site rather than to a change in catalysis. Our results also suggest that the specific shape of the active site in AtGSTZ-1 is essential to its unique DCA-DC activity.

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We used directed evolution to enhance the thermostability of formaldehyde dehydrogenase from Pseudomonas putida. At 50 °C, the wild-type enzyme was inactivated within 30 min, but the variants obtained retained 80% activity for at least 300 min. At room temperature (30 °C), the variants obtained retained <80% activity for at least 500 h (21 d).

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We analyzed the gene and protein expression of serologically defined colon cancer antigen 8. Gene expression was upregulated in the maturing rat testis, and was localized to the spermatocytes. Protein was detected in the spermatids and at the sites of mRNA expression. Specific expression of colon cancer antigen 8 was observed in the maturing rat testis.

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The use of peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to identify and phenotypically characterize toluene-degrading bacteria via biomarkers of degradation and taxonomical classification. Pseudomonas putida F1, P. mendocina KR1, and Burkholderia sp. JS150 were grown on toluene, extracted, electrophoretically separated, and analyzed by MALDI-TOF MS. Catabolic enzymes were identified and results substantiated using tandem MS.

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We found that the small ubiquitin-related modifier (SUMO) conjugation reaction can be visualized simply by incubating green fluorescent protein (GFP)-SUMO-1 with permeabilized cells in the presence of ATP for 15 min. Neither special equipment for protein purification nor highly developed skills for recombinant technologies is required, making the assay potentially applicable to large-scale drug-screening strategies for the identification of drugs that can inhibit or enhance SUMOylation.

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The gene expression and enzyme activity of superoxide dismutase, catalase, and ascorbate peroxidase in the space-grown barley were not significantly different from those of the ground-grown barley. Cu²⁺ reducing and radical scavenging activities in an extract of the space-grown barley were lower than those of the ground-grown barley by 0.7 fold, suggesting that the space environment does not induce oxidative stress, and reduces antioxidant capacity in plants.

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The development of the protein body in the late stage of seed maturation is poorly understood, because electron-microscopy of mature cereal endosperm is technically difficult. In this study, we attempted to modify the existing method of embedding rice grain in resin. The modified method revealed the ultrastructures of the mature protein body in dry cereal grains.

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To clarify the involvement of seven Arabidopsis homologs of rat L-gulono-1,4-lactone (L-GulL) oxidase, AtGulLOs, in the biosynthesis of L-ascorbic acid (AsA), transgenic tobacco cells overexpressing the various AtGulLOs were generated. Under treatment with L-GulL, the levels of total AsA in three transgenic tobacco cell lines, overexpressing AtGulLO2, 3, or 5, were significantly increased as compared with those in control cells.

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Ascorbate peroxidase (APX) of chloroplasts plays a pivotal role in detoxifying the hydrogen peroxide (H₂O₂) produced through photoexcitation of the photosystem, but it easily loses its catalytic activity under attack by excess H₂O₂. Here we report that the H₂O₂-tolerance of APX localized in the stroma of chloroplasts was improved by removing a unique 16-amino-acid-residue loop not found in H₂O₂-tolerant isoforms of cytosol and glyoxysome.

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Although coloration in plants is ascribable to both the accumulation of anthocyanin pigments in vacuoles and to the acidification of vacuolar pH, the environmental factors causing the decrease in vacuolar pH are unknown. We found that blue-light irradiation of buckwheat seedlings using light-emitting diodes caused reddening on the surface of the hypocotyls. It has also been reported that light stimulation induces an accumulation of anthocyanin pigments. However, here we confirmed for the first time on the basis of real-time PCR analysis that light stimulation simultaneously triggers expression of the genes coding for subunit A of vacuolar H⁺-ATPase (V-ATPase) and vacuolar H⁺-pyrophosphatase (V-PPase).

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Screening of mRNA export factors in Saccharomyces cerevisiae and Drosophila melanogaster has identified a number of mRNA processing factors involved in multiple mRNA processing steps. However, only limited information is available on human cells. Here we established a screening system searching for mRNA processing factors in human cells by combining the luciferase reporter system and fluorescence in situ hybridization, which evaluates the nuclear/cytoplasmic distribution of bulk poly(A)⁺ RNA. This system makes it possible to search for the compounds affecting mRNA processing from the various resources.

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Proteose peptone (PP) is a heat-stable and acid-soluble protein in milk whey. We reveal in this study the IgE production-suppressing activity of the PP fraction in bovine milk. The PP fraction suppressed IgE production by human myeloma cell line U266 cells by depressing the IgE mRNA expression. The suppressive activity of the PP fraction was facilitated by trypsin digestion. An oral administration of the PP fraction significantly decreased the levels of total and ovalbumin (OVA)-specific IgE in the serum collected from OVA-sensitized mice. However, the serum levels of other Ig classes in OVA-sensitized mice were not affected by the intake of the PP fraction. The PP fraction suppressed the mRNA expression level of IgE in mice splenocytes collected from OVA-sensitized mice. Moreover, the B cell population in the spleen was decreased, while the T cell population was increased by administering the PP fraction. These results suggest that the PP fraction modified the B/T cell balance.

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The amounts and compositions of polyphenol in persimmon leaves and persimmon leaf tea were investigated. The predominant polyphenols in fresh leaves were water-soluble, and the contents reached a maximum (2.40% w/w) in June, and then gradually decreased. Separation of them followed by thiolytic degradation revealed that the major components were unique proanthocyanidin oligomers consisting of four heterogeneous extension units, including epigallocatechin-3-O-gallate. Persimmon leaf tea also contained similar proanthocyanidins with similar compositional units. Oral administration of starch with polyphenol concentrate of persimmon leaf tea resulted in a significant and dose-dependent decrease in the blood glucose level in Wistar rats. This effect is considered to be due to inhibition of pancreas α-amylase. These results indicate that persimmon leaf tea containing peculiar proanthocyanidins has a significant role in suppressing blood glucose elevation after starch intake, and that the best harvest time is June.

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Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-M_r glycoprotein fraction (F1) from cow’s milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 °C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.

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The effect of sulfated modification on polysaccharides from Hypsizigus marmoreus was examined by determining their molecular structures and bioactivities. The sulfation, which was implemented by using an orthogonal array design, produced polysaccharides with varying degrees of substitution (DS) ranging from 0.11 to 1.06. The sulfated polysaccharides exhibited a lower average molecular weight (M_w) and considerably higher radius of gyration (R_g) than those of native polysaccharide, suggesting that the conformation of the sulfated polysaccharides had been changed towards a more extended type. The inhibitory activity toward cancer cell growth was enhanced by treating with the sulfated polysaccharides by up to 34%, as compared to the native polysaccharide. In addition, treating with the sulfated polysaccharides increased the nitric oxide (NO) and cytokine (IL-1β and TNF-α) release to levels comparable to those detected in the positive control, lipopolysaccharide (LPS), suggesting that the sulfated polysaccharides might have strong immunomodulatory activity.

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The effects of dietary Yamabushitake mushroom (Hericium erinaceus) on lipid metabolism were examined. C57BL/6J mice were fed a high-fat diet containing hot-water extract (HW-E) and an ethanol extract (EtOH-E) of Yamabushitake mushroom. Administration of HW-E or EtOH-E with a high-fat diet for 28 d resulted in a significant decrease in body weight gain, fat weight, and serum and hepatic triacylglycerol levels. Our in vitro experiments indicated that EtOH-E acts as an agonist of peroxisome proliferator-activated receptor α (PPARα). Quantitative analyses of hepatic mRNA levels revealed that EtOH-E administration resulted in up-regulation of mRNA for a number of PPARα-regulating genes in spite of the fact that the gene expression of PPARα did not change. These results suggest that EtOH-E improves lipid metabolism in mice fed a high-fat diet, and that these effects were mediated by modulation of lipid metabolic gene expression, at least in part via activation of PPARα.

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To determine the bioavailability of tocotrienol complex with γ-cyclodextrin, the effects of tocotrienol/γ-cyclodextrin complex on tocotrienol concentration in rat plasma and tissues were studied. Rats were administered by oral gavage an emulsion containing tocotrienol, tocotrienol with γ-cyclodextrin, or tocotrienol/γ-cyclodextrin complex. At 3 h after administration, the plasma γ-tocotrienol concentration of the rats administered tocotrienol/γ-cyclodextrin complex was higher than that of the rats administered tocotrienol and γ-cyclodextrin. In order to determine the effect of complexation on tocotrienol absorption, rats were injected with Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoprotein by lipoprotein lipase, and then administered by oral gavage an emulsion containing tocotrienol, tocotrienol with γ-cyclodextrin, or tocotrienol/γ-cyclodextrin complex. The plasma γ-tocotrienol concentration of the Triton-treated rats administered tocotrienol/γ-cyclodextrin complex was higher than that of the other Triton-treated rats. These results suggest that complexation of tocotrienol with γ-cyclodextrin elevates plasma and tissue tocotrienol concentrations by enhancing intestinal absorption.

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We examined the acute and sub-chronic toxicity of D-allose in rats. In the acute toxicity test, the calculated LD₅₀ value was 20.5 g/kg. In the sub-chronic toxicity test, no difference was found among the four groups in most of the serum chemical and hematological test results. These results suggest that D-allose is not toxic to rats.

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Fourteen-week-old db/db mice showed significant higher blood glucose levels than 7 week-old mice. The mRNA levels of IL-1β and S100a4/a6/a9 in peripheral leukocytes were higher in 14 week-old mice than in 7 week-old mice. Together, inflammatory cytokine/cytokine-like factor mRNA levels in peripheral leukocytes were associated with progression of diabetes in db/db mice.

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We administered Acetobacter malorum NCI1683 (S24), containing a high concentration of dihydroceramide (7.2 mg/g of dry cell weight), consecutively to aged rats (male Crlj:Wistar rats, 22 months old). The ingestion of Acetobacter malorum for 89 d significantly extended the memory retention in passive avoidance tests, increased the release of acetylcholine with depolarization of brain synaptosomes and decreased the causative agents of neurodegenerative diseases in the cerebral cortices.

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An EtOH extract of fruits of Piper longum was found to exhibit a potent inhibitory effect against α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16 mouse melanoma cells. Bioassay-directed fractionation led to the isolation of prenylated phenolic compounds bakuchiol, bavachin, and isobavachalcone. These compounds and the crude extract of the fruits of P. longum may have suppressive effects against pigmentation by melanin in the skin.

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In order to establish the molecular taxonomy of the genus Rhizopus, all described species of the genus were collected and the nucleotide sequences of the internal transcribed spacer of the rRNA gene (rDNA ITS), actin, and translation elongation factor 1α (EF-1α) were determined. Quantitative real-time PCR revealed that R. americanus had a R. stolonifer-type ITS sequence as the dominant sequence type, although it had three different types of ITS sequences in a single genome. Phylogenetic analysis and gene genealogy concordance phylogenetic species recognition (GCPSR) identified eight species in the genus, whereas recent morphological taxonomy includes 10 species. R. niveus is proposed to be re-classified as R. delemar, and R. sexualis and R. americanus are re-classified as R. stolonifer.

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The purpose of this study was to investigate the effects of heat-killed Lactobacillus plantarum strain b240 (b240) on systemic infection by Salmonella enterica serovar Typhimurium (S. Typhimurium) and to determine the mechanism by which b240 protects against infection. Mice were administered either b240 or saline orally for 3 weeks, and then inoculated with S. Typhimurium. The mice treated with b240 were significantly protected against S. Typhimurium as compared to those fed saline. Moreover, translocation of S. Typhimurium into each organ tested in the mice that received b240 tended to be less than in the control mice. An important mechanism of protection against infection was demonstrated by the ability of b240 to inhibit both binding by and invasion of S. Typhimurium into cells. These results indicate that nonviable lactic acid bacteria also play important roles in preventing infection by enteric pathogens.

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Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant ΔadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. ΔadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting ΔadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on ΔadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

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The methylotrophic yeast Pichia methanolica possesses two genes, PmDAS1 and PmDLP1, whose amino acid sequences show high similarity to dihydroxyacetone synthase (DAS), the formaldehyde-fixing enzyme for methanol metabolism within the peroxisome. The PmDAS1 and PmDLP1 genes encode 709 and 707 amino acid residues respectively, and PmDas1p contains a type-1 peroxisomal targeting signal (PTS1), while PmDlp1p does not. Upon phylogenetic analysis, PmDas1p fit into the DAS group with other DASs, while PmDlp1p was grouped with the DAS-like proteins (DLP) of non-methylotrophic yeasts and fungi, a branch of the phylogenetic tree independent of the DAS and transketolase (TK) groups. While expression of PmDAS1 restored the methylotrophic growth of the Candida boidinii das1Δ strain, the PmDLP1 and PmDAS1−ΔPTS1 genes did not. Taken together, these results indicate that PmDAS1 encodes a functional DAS and has an indispensable role in methanol metabolism, and that PmDlp1p share a common, as yet uncharacterized function in P. methanolica as well as in non-methylotrophic yeasts and fungi.

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