Mutagenicity of bisphenol A and its suppression by interferon-α in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1×10⁻⁷ to 1×10⁻⁵ M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1×10⁻⁸ M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua^R) phenotypic mutation was also found in cells treated with 1×10⁻⁷ and 1×10⁻⁵ M of bisphenol A. The induction of K-ras codon 12 mutations and Oua^R mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-α prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1×10⁻⁶ M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Membrane Translocation of Mannitol in Escherichia coli Without Phosphorylation

Galactosyl-mannitol can be transported into cells of Escherichia coli by beta-galactoside permease and can be hydrolyzed rapidly to mannitol and galactose by beta-galactosidase. When a mutant strain lacking enzyme I of the phosphoenolpyruvate phosphotransferase system and constitutive in the lactose system was presented with galactosyl-mannitol in which the mannitol moiety was labeled with (3)H, the liberated mannitol remained inside the cell if the Enzyme II complex of the phosphoenolpyruvate phosphotransferase system for mannitol was uninduced. It is postualted that one of the enzyme II proteins can still catalyze translocation of mannitol across the cell membrane even when phsophorylation is not possible.     

Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis

Mitochondrial DNA (mtDNA) analysis with Hae III, Hind. III and Msp I was performed in 45 Exophiala jeanselmei strains (30 Phialophora jeanselmei and 15 Phialophora gougerotii strains) and 31 Exophiala dermatitidis strains. The results were as follows, 1) P. jeanselmei and P. gougerotii are identical, 2) E. jeanselmei is classified into 18 types based on restriction profiles, 3) two strains of E. jeanselmei CBS 577.76 and CBS 578.76 are identified as E. dermatitidis, 4) E. dermatitidis has no intraspecific variation and is definitely distinct from E. jeanselmei, 5) E. jeanselmei is suggested to be a complex organism because of extensive mtDNA polymorphism.     

DNA Sequencing Using Ultra Small Amounts of Reagents and Template

One of the key points in the genome project is finding waysto reduce the running cost in DNA sequencing. One way is touse a highly-sensitive fluorescent DNA sequencer, where onlytrace amounts of template DNA and reagents are needed. An experimentalprotocol optimized for the trace amounts of DNA analysis wasestablished by using the hybridization reaction rate coefficientof primers on template DNA, which was estimated to be 7.5x10⁵M^–1 sec^–1 at 37°C. One femtomole of templateDNA with 0.001 unit of modified T7 DNA polymerase (SequenaseVer. 2.0) and also 0.45 fmol of M13 template DNA with 0.01 unitof Taq DNA polymerase were enough to sequence DNA of up to 400bases.     

Fluoride‐sensitivity of growth and acid production of oral Actinomyces: comparison with oral Streptococcus

Actinomyces are predominant oral bacteria; however, their cariogenic potential in terms of acid production and fluoride sensitivity has not been elucidated in detail and compared with that of other caries‐associated oral bacteria, such as Streptococcus. Therefore, this study aimed to elucidate and compare the acid production and growth of Actinomyces and Streptococcus in the presence of bicarbonate and fluoride to mimic conditions in the oral cavity. Acid production from glucose was measured by pH‐stat at pH 5.5 and 7.0 under anaerobic conditions. Growth rate was assessed by optical density in anaerobic culture. Although Actinomyces produced acid at a lower rate than did Streptococcus, their acid production was more tolerant of fluoride (ID_{acid production} 50 = 110–170 ppm at pH 7.0 and 10–13 ppm at pH 5.5) than that of Streptococcus (ID_{acid production} 50 = 36–53 ppm at pH 7.0 and 6.3–6.5 ppm at pH 5.5). Bicarbonate increased acid production by Actinomyces with prominent succinate production and enhanced their fluoride tolerance (ID_{acid production} 50 = 220–320 ppm at pH 7.0 and 33–52 ppm at pH 5.5). Bicarbonate had no effect on these variables in Streptococcus. In addition, although the growth rate of Actinomyces was lower than that of Streptococcus, Actinomyces growth was more tolerant of fluoride (ID_growth 50 = 130–160 ppm) than was that of Streptococcus (ID_growth 50 = 27–36 ppm). These results indicate that oral Actinomyces are more tolerant of fluoride than oral Streptococcus, and bicarbonate enhances the fluoride tolerance of oral Actinomyces. Because of the limited number of species tested here, further study is needed to generalize these findings to the genus level.     

Precise localization of the human gene encoding cell adhesion kinase β (CAKβ/PYK2) to chromosome 8 at p21.1 by fluorescence in situ hybridization

Cell adhesion kinase β (CAKβ) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily with large N- and C-domains in addition to the central kinase domain. The cDNA of the human CAKβ has been cloned and used as a probe for the assignment of this gene by fluorescence in situ hybridization. CAKβ is sublocalized on chromosome 8p21.1, a locus frequently involved in allelic losses in colorectal cancers and prostate carcinomas. Received: 14 February 1996 / Revised: 29 April 1996     

The distribution of geraniin and mallotusinic acid in the order geraniales

The distribution of geraniin and mallotusinic acid in Geraniaceae, Euphorbiaceae and other families in the Geraniales were investigated by HPLC. Geraniin composes the main part of the tannin in all of the investigated species of Geranium, but not in species of Pelargonium. The species of Geraniaceae lack mallotusinic acid. Geraniin was also detected in most of the species in the subfamily Euphorbioideae of Euphorbiaceae, although the amount was generally smaller than in Geranium, except for some woody species. Several species of Euphorbioideae contained mallotusinic acid. Characteristic of the subfamily Phyllanthoideae is the absence of mallotusinic acid and poor distribution of geraniin. Geraniin and mallotusinic acid were not detected in most of the species of other families in the Geraniales, except in Erythroxylum coca. Hyperin was found to be the main flavonol glycoside in most species of Geranium.     

Different biosynthetic pathways of the pyrimidine moiety of thiamin in procaryotes and eucaryotes

[¹⁴C]Formate is incorporated into the C-2 of the pyrimidine moiety of thiamin by Escherichia coli and Salmonella typhimurium. In Saccharomyces cerevisiae, it is incorporated into C-4. Radioactive carbons of [1-¹⁴C]glycine and [2-¹⁴C]glycine are incorporated by S. typhimurium into the C-4 and C-6 of the pyrimidine, respectively, but not by S. cerevisiae. These facts suggest that procaryotes and eucaryotes have different biosynthetic pathways for pyrimidine. In this study, the procaryotes tested incorporated [¹⁴C]formate into the C-2 and the eucaryotes incorporated it into the C-4 of the pyrimidine.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.