Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase

The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.     

Extracellular production of a heat-stable somatic antigen by Bacillus thuringiensis

Extracellular production of a heat-stable somatic antigen (HSSA) by Bacillus thuringiensis subsp. dendrolimus strain T84A1-A [flagellar (H) serotype 4a: 4b] was serologically detected. In Ouchterlony tests, the HSSA antiserum gave single precipitin lines against both untreated and heat-treated culture supernatants. These two precipitin lines fused completely. When colonies of strain T84A1-A were grown on nutrient agar plates containing the homologous HSSA antiserum, precipitin halos were formed around the colonies. Of 27 type strains of B. thuringiensis subspecies tested, only the type strains of B. thuringiensis subsp. sotto (H serotype 4a: 4b) and B. thuringiensis subsp. israelensis (H serotype 14) formed [precipitin halos on nutrient agar plates containing antiserum against the HSSA of strain T84A1-A.     

Phosphorylation of five histone H1 subtypes of L5178Y cells at the exponential growth and mitotic phases

Histone H1 of cells of L5178Y, a mouse lympholeukemic cell line, consists of five molecular species designated as H1-I, II, III, IV, and V. The phosphorylation of these H1 subtypes was examined at the exponential growth phase and during mitosis, by BioRex 70 column chromatography and two-dimensional polyacrylamide gel electrophoresis. In exponentially growing cells, the degree of phosphorylation was different for each subtype. H1-II was the most highly phosphorylated, 1.8 phosphate residues per molecule, followed by H1-IV/V, 1.4, I, 0.8, and III, 0.5. In the mitotic phase, H1-II was also the most highly phosphorylated 6.0 phosphate residues per molecule, H1-IV/V, 3.5, I, 2.7, and III, 1.2. The phosphorylation started simultaneously among the subtypes after colcemid addition, and phosphorylated H1 subtypes accumulated linearly. The rate of incorporation of 32P into each H1 subtype was almost constant during colcemid treatment. During 4 h after colcemid addition, the phosphate residues incorporated into H1 did not dephosphorylated. The H1 kinase activities increased to six times higher during the colcemid treatment.     

The branching of the inflorescence and vegetative shoot and taxonomy of the genusKummerowia (Leguminosae)

A study of the branching of the inflorescence and the vegetative shoot of the genusKummerowia, consisting ofK. stipulacea (Maxim.) Makino andK. striata (Thunb.) Schindler, has led to the following conclusions: (1) the inflorescences of both species are reduced compound cymes, (2) the branching system of the inflorescence ofKummerowia is not clearly different from that of the vegetative shoot and there are some transitional forms between both systems, and (3) the inflorescence ofKummerowia is different from the racemose inflorescences ofLespedeza andCampylotropis. Based on the differences found in the branching system of the inflorescence,Kummerowia is distinctly separated fromLespedeza andCampylotropis and is more correctly treated as a distinct genus from the latter two.     

Photochemical inactivation of human placental estradiol 17 beta-dehydrogenase in the presence of 2,3-butanedione

Estradiol 17 beta-dehydrogenase of human placenta was rapidly inactivated by 2,3-butanedione under u.v. light, and no protection against the inactivation was observed in the presence of sodium azide. Under ordinary laboratory illumination, the inactivation was biphasically progressed in time-dependent and concentration-dependent manners, while a partial protection from the inactivation was indicated by sodium azide. These results suggest that the inactivation mechanism of the dehydrogenase by 2,3-butanedione under laboratory illumination is different from that under u.v. light. Therefore, the inactivation under laboratory illumination proceeded by a reaction with excited singlet molecular oxygen (1 delta g or 1 sigma +g states), and that under u.v. light was caused by a reaction of substrate with triplet sensitizer. In the presence of NADP+, the inactivation of the enzyme by 2,3-butanedione was markedly reduced. The maximum protection by NADP+ was about 80% of the initial enzyme activity. Amino acid analysis of the enzyme treated with 2,3-butanedione under laboratory illumination showed that the modified enzyme contained considerably less of the following amino acids than the native enzyme: histidine, arginine, threonine, methionine, tyrosine and leucine. In addition, other dicarbonyl reagents, 1,4-dibromo-2,3-butanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, 16-oxoestrone, 1,2-cyclohexanedione, 2,4-pentanedione and glyoxal were found to decrease the dehydrogenase activity in various degree.     

Enhancement of ribonucleic acid synthesis by chromium(III) in mouse liver

The effect of Cr(III) administration on hepatic RNA synthesis in mice was studied. It was found that Cr accumulated in mouse liver. Forty-eight hours after intraperitoneal injection of CrCl3 (0.005-5 mg Cr/kg body weight) approximately 10% of the administered dose per g of tissue remained. The accumulated Cr was still retained 64 days after administration (5 mg Cr/kg) with only a slight decrease. Approximately 20% of the hepatic Cr was detected in the nuclei. By administering CrCl3. RNA synthesis in mouse liver was markedly enhanced without altering the pool size of nucleotides. This enhancement was dose-dependent and statistically significant at doses of 0.05 (p less than 0.05), 0.5 (p less than 0.01), and 5 mg Cr/kg (p less than 0.01), and remained so for at least 16 days after administration of 5 mg Cr/kg. The synthesis of DNA and protein in mouse liver were not significantly changed by CrCl3 administration. On the other hand, Cr(VI) administration did not enhance but rather inhibited RNA synthesis in mouse liver. These results suggest that Cr(III) specifically enhances RNA synthesis in mouse liver.     

The 25-kilodalton hemolytic protein affinity-purified from parasporal inclusions ofBacillus thuringiensis strain PG-14 (serotype 8a∶8b)

A simple method using an antibody-mediated affinity chromatography was developed for rapid and specific purification of the 25-kilodalton protein from alkali-solubilized and silkworm (Bombyx mori) larval gut juice-digested parasporal inclusions of theBacillus thuringiensis strain PG-14 (serotype 8a8b). Affinity-purified 25-kilodalton protein was highly hemolytic to red blood cells (RBCs) of two avian (chicken and goose) and six mammalian (horse, mouse, cow, rabbit, guinea pig, and sheep) species. The concentration of the 25-kolodalton protein required for 100% hemolysis was in the range of 2–16 g/ml, and an apparent RBC species-dependent variation was observed in hemolytic activity of this protein. Of the RBCs tested, chicken and house RBCs were the most susceptible to hemolysis by this protein; sheep RBCs wre 4–8 times less susceptible than the others.     

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5 and 3 ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) _n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) _n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.Paper 3082 of the Forest Research Laboratory, Oregon State University