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.     

Purification and Properties of Allothreonine Aldolase from Maise Seedlings

In order to elucidate the structure-activity relationship of griseofulvin (1), (±)-6′-demethyl analog (3), 2′-demethoxy-6′-demethyldihydro analog (4), (±)-dechloro-6′-ethyl analog (5), (±)-dechloro-6′-epi-ethyl analog (6), (±)-6′-ethyl analog (7) and (±)-6′-epi-ethyl analog (8) were synthesized by a Diels-Alder cycloaddition of alkylidene ketones (16, 17, 18, 19 and 20) with modified 1,3-butadienes (21 or 22). Their biological activities were examined against fungi.     

Extracellular Production of l-Lysine α-Oxidase in Wheat Bran Culture of a Strain of Trichoderma viride

A mold strain Y244-2 capable of producing l-lysine α-oxidase, a new enzyme catalyzing the α-oxidative deamination of l-lysine, was identified as Trichoderma viride. Among strains belonging to the genus Trichoderma tested, only Trichoderma viride Y244-2 produced the enzyme in wheat bran culture. The maximum enzyme production by the mold grown on wheat bran was observed after 10 and 14 days incubation with and without NaN0₃, respectively. Addition of NaN0₃, NH₄N0₃, adenine, purine nucleosides, l-histidine, glycine or l-glutamine to wheat bran stimulated the production of the enzyme. In the liquid culture, the enzyme was produced extracellulary under the aerobic conditions, although the production was much lower than that in the wheat bran culture.     

Purification of 7-Keto-8-aminopelargonic Acid-7,8-Diaminopelargonic Acid Aminotransferase,an Enzym Involved in Biotin Biosynthesis,from Brevibacterium divaricatum

Vitamin B₆ is synthesized by green Cytisus scoparius callus and green Phellodendron amurense callus cultured on Linsmaier and Skoog Agar-medium with 10^?5m of ±-naphthaleneacetic acid (NAA) and 10^?6 m of 6-benzyladenine (BA). Even when thiamine and inositol were omitted from this medium, the growth and vitamin B₆ content of Cytisus scoparius callus did not change. Vitamin B₆ contents of clones of the calluses varied and were unstable during long-term subculture. Clonal selection was repeated to obtain stable strains with high vitamin B₆ content, and the vitamin B₆ content of one strain of green Cytisus scoparius callus became 4-times higher than that of the green leaves.     

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.     

Substrate Specificity of Nuclease P1

Nuclease P₁ cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.     

Hydrolysis of Proteins by Successive Incubation with Proteinase and Aminopeptidases Mixture

1. A trial test was attempted of complete hydrolysis of peptides and proteins into amino acids by enzymes. “Neutral proteinase” of Bacillus subtilis or “Alkalophilic proteinase” of a Streptomyces sp. was used for preliminary digestion of substrate, and a mixture of three aminopeptidases of Bacillus subtilis was employed for subsequent hydrolysis of proteinase digest.

2. The oxidized insulin B chain was hydrolyzed completely by the method. Several proteins including enzymes which contained no or less cystine and cysteine were also hydrolyzed almost completely.

3. On the other hand, certain glycoproteins were hydrolyzed to leave a few glycopeptides in which all glycomoieties of the proteins were retained. The implications of the results are discussed.