Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.     

Temperature dependence for anammox bacteria enriched from freshwater sediments

The anoxic ammonium oxidation (anammox) process has been regarded as an attractive alternative process to treat wastewater containing high ammonium concentrations. By the implementation of anammox process at moderately low temperatures (<25°C), the anammox process will be applied to more various industrial wastewater treatments. In this study, we established enrichment cultures of anammox bacteria from freshwater sediments by using an up-flow column reactor equipped with porous polyester nonwoven fabric at moderately low temperatures. Their nitrogen conversion rates reached 0.07-0.26kg-N/m(3)/d. Phylogenetic analysis based on 16S rRNA gene from enrichment cultures revealed the presence of various anammox bacteria affiliated with unknown anammox bacteria as well as known anammox candidates, i.e., Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia fulgida, Candidatus Scalindua wagneri. Anammox bacterial populations were influenced by enrichment conditions, i.e., seed sediments and temperature.     

Tiliroside, a glycosidic flavonoid, inhibits carbohydrate digestion and glucose absorption in the gastrointestinal tract

Scope Recent studies have reported that tiliroside, a glycosidic flavonoid, possesses anti‐diabetic activities. In the present study, we investigated the effects of tiliroside on carbohydrate digestion and absorption in the gastrointestinal tract. Methods and results This study showed that tiliroside inhibits pancreatic α‐amylase (IC₅₀ = 0.28 mM) in vitro. Tiliroside was found as a noncompetitive inhibitor of α‐amylase with K_i values of 84.2 μM. In male ICR mice, the increase in postprandial plasma glucose levels was significantly suppressed in the tiliroside‐administered group. Tiliroside treatment also suppressed hyperinsulinemia after starch administration. Tiliroside administration inhibited the increase of plasma glucose levels in an oral glucose tolerance test, but not in an intraperitoneal glucose tolerance test. In human intestinal Caco‐2 cells, the addition of tiliroside caused a significant dose‐dependent inhibition of glucose uptake. The inhibitory effects of both sodium‐dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) inhibitors (phlorizin and phloretin, respectively) on glucose uptake were significantly inhibited in the presence of tiliroside, suggesting that tiliroside inhibited glucose uptake mediated by both SGLT1 and GLUT2. Conclusion These findings indicate that the anti‐diabetic effects of tiliroside are at least partially mediated through inhibitory effects on carbohydrate digestion and glucose uptake in the gastrointestinal tract.     

A method for the determination of acrylamide in a broad variety of processed foods by GC-MS using xanthydrol derivatization

A novel GC-MS method was developed for the determination of acrylamide, which is applicable to a variety of processed foods, including potato snacks, corn snacks, biscuits, instant noodles, coffee, soy sauces and miso (fermented soy bean paste). The method involves the derivatization of acrylamide with xanthydrol instead of a bromine compound. Isotopically labelled acrylamide (d?-acrylamide) was used as the internal standard. The aqueous extract from samples was purified using Sep-Pak? C?? and Sep-Pak? AC-2 columns. For amino acid-rich samples, such as miso or soy sauce, an Extrelut? column was used for purification or extraction. After reaction with xanthydrol, the resultant N-xanthyl acrylamide was determined by GC-MS. The method was validated for various food matrices and showed good linearity, precision and trueness. The limit of detection and limit of quantification ranged 0.5-5 and 5-20?μg?kg?1), respectively. The developed method was applied as an exploratory survey of acrylamide in Japanese foods and the method was shown to be applicable for all samples tested.     

Immunomodulatory effect of mushrooms on cytotoxic activity and cytokine production of intestinal lamina propria leukocytes does not necessarily depend on β-glucan contents

We evaluated the effects of seven mushroom extracts (Grifola frondosa, Pholiota nameko, Panellus serotinus, Hypsizygus marmoreus, Pleurotus cornucopiae, Armillaria mellea, and Flammulina velutipes) on cytotoxic activity and cytokine production of lamina propria leukocytes (LPLs) isolated from rat small (S) and large (L) intestinal mucosa. Boiling water extracts from seven species of mushrooms showed no direct cytotoxicity against the YAC-1 target cells. However, prominent increases of cytotoxicity were observed in S- and L-LPLs co-cultured with P. serotinus extract. Cytokine production (TNFα, IFNγ, IL-12 p70, and IL-4) of S- and L-LPLs was stimulated in response to P. cornucopiae extract. Mushroom extracts contributed to target cell adhesion and/or cytokine production in the effector cells. The promotion of cytotoxic activity in S- and L-LPLs was not necessarily related to β-glucan content of the mushroom.     

Applicability of DNA barcode for identification of fish species

DNA barcoding is a species identification technique, which uses a very short DNA sequence from a region of approximately 650 base-pairs in the 5'-end of the mitochondrial cytochrome c oxidase subunit I gene as a marker to identify species of mammals and fishes. The applicability of DNA barcoding for identification of fish species consumed in Japan was studied. Among thirty-one fresh or processed fishes were obtained from the market, two samples could not be identified due to lack of data in the Barcode of Life Data (BOLD) database. However, BLAST-search of 16S rRNA genes in the National Center for Biotechnology Information (NCBI) database and the PCR-RFLP method published by the Food and Agricultural Materials Inspection Center (FAMIC) were found to be applicable to identify these 2 fishes. The results show that the DNA barcoding technique is potentially useful as a tool for confirming the proper labeling of fish species in the Japanese market.     

Inter-laboratory validation studies of biological method for determination of streptomycin in royal jelly

Inter-laboratory validation studies were conducted in 6 laboratories to validate the biological method for determination of streptomycin in royal jelly. Streptomycin spiked at the level of 0.2 and 1.0 ppm was analyzed. Mean recoveries were 89 and 96%, reproducibility relative standard deviations (RSD(R)) were 15.0 and 14.0%, HORRAT(R) values were 0.7 and 0.9. Samples containing residues at the levels of 0.25 and 0.80 ppm were analyzed. Mean recoveries were 113 and 99%, RSD(R) were 15.0 and 10.4%, and HORRAT(R) values were 0.8 and 0.6. The determination limit was 0.1 ppm. These results show that this method has good performance.