Phytuberol,from Japanese Domestic Tobacco,Nicotiana tabacum cv. Suifu

Carcinogenesis is believed to be induced through the oxidative damage of DNA, and antioxidants are expected to suppress it. So, the polyphenolic antioxidants in daily foods were investigated to see whether they protect against genetic damage by active oxygen. In the evaluation, we used a bioassay and a chemical determination, a Salmonella mutagenicity test for mutation by a N-hydroxyl radical from one of the dietary carcinogens 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the formation of 8-hydroxyl (8-OHdG) from 2′-deoxyguanosine (2′-dG) in a Fenton OH-radical generating system. Thirty-one antioxidants including flavonoids were compared in terms of radical-trapping activity with bacterial DNA and 2′-dG. Antioxidants inhibited the mutation but the IC₅₀ values were in the mM order. Against 8-OHdG formation, only α-tocopherol had a suppressive effect with an IC₅₀ of 1.5 μM. Thus, except α-tocopherol, the dietary antioxidants did not scavenge the biological radicals faster than bacterial DNA and intact 2′-dG, indicating that they failed to prevent oxidative gene damage and probably carcinogenesis.     

Isolation of New Tobacco Constituents, 8,9-Dihydro-8,9-dihydroxymegastigmatrienone,from Japanese Domestic Suifu Tobacco

The tetradecapeptide of a renin substrate, DRVYIHPFHLLVYS, was used as a substrate for assaying several fungal aspartic and acidic proteinases in the acidic pH range. Aspartic and acidic proteinases froll) Phycomycetes, Mucor and Rhizopus, and Deuteromycotina, Aspergillus and Penicillium, cleaved the tetradecapeptide at its tyrosyl⁴-isoleucyl⁵ (Y⁴-I⁵),histidyI⁶-proly⁷ (H⁶_P⁷) and leucyl¹¹-valyl¹² (L¹¹-V¹²) bonds in the acidic pH range, while acidic proteinases type B and type A-I from Scytalidium lignicolumn, and those from Cladosporium and Basidiomycetes, Pycnoporus sanguineus, and the yeast, Rhodotorula glutinis; showed slightly different specificities towards the tetradecapeptide. Pepsin primarily cleaved the valy³-tyrosyl⁴ (V³-Y⁴) and leucyl¹⁰-leucyl¹¹ (L¹⁰-L¹¹) bonds. All of the aspartic and acidic proteinases of fungal origin tested in the present study have different specificities from that of pepsin.     

Mechanism of Action of Showdomycin

The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.

The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphosphates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the inhibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.

N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloromercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.

The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.     

4-Hydroxy-β-damascone and 4-Hydroxy-dihydro-β-damascone from Cigar Tobacco

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

Volatile Gas Components Contribute to Cigarette Smoke-induced DNA Single Strand Breaks in Cultured Human Cells

Thermostable purine nucleoside phosphorylases, PUN PI and PUNPII, have been purified from Bacillus stearothermophilus JTS 859. The characterization of PUNPI was reported previously. [Hori et al.₉ Agric. Biol. Chem. 53, 2205 (1989)] PUNPII had a molecular weight of 113,000, consisting of 4 identical subunits (M_w 28,000). The isoelectric point was 5.3. The Michaelis constants for inosine, guanosine, and adenosine were 0.22, 0.34, and 0.075 mm, respectively. The optimal temperature of the reaction was 70°C. The enzyme was stable at 70°C. Although other reported purine nucleoside phosphorylases were SH-enzymes, PUNPII was not a SH-enzyme because the enzyme reaction was not inhibited by PCMB and iodoacetic acid, the optimal pH of the enzyme reaction was from 7.0 to 11.0, and the enzyme did not contain cysteine.

PUNPII and PUNPI were different in several points. Not PUNPI but PUNPII could catalyze the phosphorolysis of adenosine. Specific activity of PUNPI and II for inosine were 405 and 50.6 μmol/min/mg protein at 60°C, respectively. PUNPI was stable at 80°C. PUNPII was stable at 70°C, but was denatured at 80°C.     

Degradation of Barley Glucan and Lichenan by a Bacillus pumilus Enzyme

A bacterial strain was isolated from soil, which rapidly degraded purified barley β-glucan as well as lichenan. The strain belonged to Bacillus pumilus, and some authentic strains of this species were also shown to hydrolyze the gluean. An enzyme active on the above substrates but not on laminaran and on CM-cellulose was partially purified from the culture fluid. This enzyme, about 27,000 in molecular weight, was found to cleave a β-(1 → 4) linkage adjacent to a β-(1 → 3) in the polymers. It was suggested that only an enzyme of this type should be called a ‘lichenanase’ and discriminated from cellulases and laminaranases.     

Expression of Arabidopsis thaliana xylose isomerase gene and its effect on ethanol production in Flammulina velutipes

To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes.