Chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy

We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleotides at the 3′-overhang region were easily prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified miR-143 duplexes carrying the urea/thiourea compounds in the 3′-end of each strand were able to inhibit the growth of human bladder cancer T24 cells.     

Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.     

Nucleic Acids Synthesis of Nuclear Polyhedrosis Virus in Cultured Embryonic Cells of Silkworm

Embryos of the silkworm, Bombyx mori L., were dispersed by trypsin and the dissociated cells were cultured for infection with nuclear polyhedrosis virus (NPV) of the silkworm. The monolayer and suspension cultures were infected with NPV. RNA and DNA syntheses in the normal and NPV-infected cells were measured by incorporation of ³²P into RNA and DNA fractions. RNA and DNA syntheses in the cells after infection significantly increased over those in control cells (mock infection). The effects of actinomycin D, chloramphenicol and mitomycin C on RNA and DNA syntheses in infected cells were examined. The syntheses were inhibited by the antibiotics. It was suggested that the cellular DNA synthesis was inhibited by the viral infection, because the mitomycin C-resistant DNA synthesis was found in the normal cells but not in the infected cells treated with mitomycin C. The rate of DNA synthesis induced by NPV was immediately dropped to that of control cells by addition of chloramphenicol, while the RNA synthesis induced by NPV was not affected for 6 hr after the addition of chloramphenicol. If the antibiotic did not affect the size of precursor pools, this event suggested that the RNA polymerase concerned with viral RNA synthesis was more stable than the DNA polymerase participating in the viral DNA synthesis. The viral DNA as templates for RNA and DNA syntheses was decomposed by mitomycin C.     

Kinetic Studies on Xylanase Induction by β-Xyloside in Streptomyces sp.

Xylanase induction by β-xyloside was investigated in non-growing conditions using non-induced mycelia of Streptomyces sp. No. 3137 harvested from glucose medium. The mycelia started to produce xylanase without lag time when β-xyloside was added. The rate of xylanase synthesis was dependent on the concentration of β-xyloside added to the inducing culture medium. The induction constants of various β-xylosides were calculated from the Lineweaver-Burk plots; those of methyl-, isopropyl-, butyl- and ethylencyanohydrin-β-d-xylosides were 10.53 mm, 3.83 mm, 0.55mm and 0.25 mm, respectively. Some α-xylosides repressed xylanase synthesis. The rate of xylanase synthesis decreased suddenly after the addition of α-xyloside. The inhibition constants of methyl-, ethyl- and isopropyl-α-d-xylosides were 8.80 mm, 12.50 mm and 33.33 mm, respectively. The xylanase induction was also repressed by glucose. However, this repression was completely restored after consuming additional glucose.     

A Novel Synthetic Procedure for “Reverse” C-Nucleosides

The Colletotrichum lagenarium PKS1 gene was expressed in the heterologous fungal host, Aspergillus oryzae, under the starch-inducible α-amylase promoter to identify the direct product of polyketide synthase (PKS) encoded by the PKS1 gene. The main compound produced by an A. oryzae transformant was isolated and characterized to be 1,3,6,8-tetrahydroxynaphthalene (T4HN) as its tetraacetate. Since the PKS1 gene was cloned from C. lagenarium to complement the nonmelanizing albino mutant, T4HN was assumed to be an initial biosynthetic intermediate, and thus the product of the PKS reaction, but had not been isolated from the fungus. The production of T4HN by the PKS1 transformant unambiguously identified the gene to encode a PKS of pentaketide T4HN. In addition, tetraketide orsellinic acid and pentaketide isocoumarin were isolated, the latter being derived from a pentaketide monocyclic carboxylic acid, as by-products of the PKS1 PKS reaction. Production of the pentaketide carboxylic acid provided insights into the mechanism for the PKS1 polyketide synthase reaction to form T4HN.     

Application of Nitrate Reductase for the Determination of Nitrate in Meat and Fishery Products

A rapid and specific method is described for the determination of nitrate in meat and fishery products.

Nitrate separated from foods by extraction with 1/50Ν sodium hydroxide and ultrafiltration was readily reduced to nitrite by the use of respiratory nitrate reductase (NR) from Escherichia coli K-12. The nitrite so obtained can be determined by the specific diazotation-coupling reaction method.

The use of an enzymatic reaction resulted in quantitative reduction of nitrate, and the method was relatively free of interferences. Recoveries of 10 and 100 ppm of nitrate from 5 samples of meat and fishery products ranged from 92.8 to 97.8% for 10 ppm and 97.8 to 99.4% for 100 ppm with a detection limit of 0.5 ppm.     

Effects of Alloxan Diabetes and Hypophysectomy on Growth,and Plasma and Liver Free Amino Acids of Rats Fed Excess Glycine Diets

Three chitinases (EC 3.2.1.14) were purified from yam, Dioscorea opposita THUMB, by fractionation with ammonium sulfate, chromatographies on DEAE-Cellulose and DEAE-Sephadex A-50, chromatofocusing and gel filtration on Bio-Gel P-60. The purified enzymes (E-l, E-2 and E-3) showed single bands on sodium dodecylsulfate polyacrylamide gel electrophoresis, and the molecular weights were estimated to be 33,500. The pIs were 4.05 (E-l), 4.0 (E-2) and 3.8 (E-3). All enzymes were glycoproteins and the neutral sugar contents were 3.6% (E-l), 3.6 (E-2) and 0.9% (E-3). The N-terminal amino acids of E-l and E-3 were the same and determined to be histidine. All enzymes hydrolyzed glycolchitin, but not p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside or Micrococcus lysodeikticus cell walls. E-l and E-3 were stable in the pH range of 5 ~ 11, and below 60°C. These enzymes showed two optimum pHs around 3.5 and 8.0 or 8.5 with glycolchitin as substrate.     

Oxidation of Free Methionine and Methionine Residues in Protein Involved in the Browning Reaction of Phenolic Compounds

The oxidation of methionine to its sulfoxide, as a possible cause of decrease in the biological value of red clover during drying with aeration, was examined using various model systems, in the presence or absence of polyphenol oxidase. The effects of catalase were also examined. Results indicated hydrogen peroxide as a possible intermediate that directly oxidizes methionine. The methionine oxidation can be one of the causes of the decrease in biological value of red clover during drying, beside the known damage of lysine in the same process.