Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.     

Nodal/Bozozok-independent induction of the dorsal organizer by zebrafish cell lines

Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades—Bozozok/Dharma (Boz) and Nodal signaling—act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cellsthemselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers incells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activatedfollowing implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.     

Glutathione is a physiologic reservoir of neuronal glutamate

Glutamate, the principal excitatory neurotransmitter of the brain, participates in a multitude of physiologic and pathologic processes, including learning and memory. Glutathione, a tripeptide composed of the amino acids glutamate, cysteine, and glycine, serves important cofactor roles in antioxidant defense and drug detoxification, but glutathione deficits occur in multiple neuropsychiatric disorders. Glutathione synthesis and metabolism are governed by a cycle of enzymes, the γ-glutamyl cycle, which can achieve intracellular glutathione concentrations of 1–10 mM. Because of the considerable quantity of brain glutathione and its rapid turnover, we hypothesized that glutathione may serve as a reservoir of neural glutamate. We quantified glutamate in HT22 hippocampal neurons, PC12 cells and primary cortical neurons after treatment with molecular inhibitors targeting three different enzymes of the glutathione metabolic cycle. Inhibiting 5-oxoprolinase and γ-glutamyl transferase, enzymes that liberate glutamate from glutathione, leads to decreases in glutamate. In contrast, inhibition of γ-glutamyl cysteine ligase, which uses glutamate to synthesize glutathione, results in substantial glutamate accumulation. Increased glutamate levels following inhibition of glutathione synthesis temporally precede later effects upon oxidative stress.     

Effects of the veterinary antiparasitic drug eprinomectin on dung beetles (Coleoptera: Scarabaeidae), the non-pest fly Neomyia cornicina and pest fly Haematobia irritans (Diptera: Muscidae) in Japan

Effects of the antiparasitic drug eprinomectin were studied in the laboratory and field experiments in Hokkaido, Japan, by pour-on administrations (500 μg/kg) on the pest fly Haematobia irritans (Linnaeus), nontarget coprophagous fly Neomyia cornicina (Fabricius), and the dung beetles Caccobius jessoensis Harold and Liatongus minutus (Motschulsky). Eprinomectin excreted into cattle dung was highest at 3 days post-treatment in both experiments, then it declined rapidly at 7 days and was not detected on or after day 14 post-treatment. In laboratory experiments, pupation and emergence rates of H. irritans and N. cornicina were hampered from 1 to 14 days post-treatment. There were no significant differences in the numbers of brood balls constructed by C. jessoensis in dung from treated and control cattle. Adult emergence rates of C. jessoensis were significantly reduced on days 1 and 3 post-treatment in dung from treated cattle. There were no significant differences in the numbers of brood balls constructed by L. minutus in dung from treated and control cattle, but survival rates of larvae were significantly reduced on days 1 and 3 post-treatment. In field experiments, the numbers of brood balls by L. minutus recovered from beneath dung pats were significantly larger in number in dung from treated cattle, suggesting that adult beetles are attracted to dung pats from treated cattle. Survival rates of larvae in these brood balls of L. minutus were significantly reduced 1 day post-treatment in dung pats from treated cattle, and equivalent levels to the control were restored 7 days post-treatment. The results are discussed in relation to the effects of endectocides on nontarget insects in grazing pastures.     

NUDT15 polymorphism and NT5C2 and PRPS1 mutations influence thiopurine sensitivity in acute lymphoblastic leukaemia cells

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B-cell precursor-ALL (BCP-ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA-incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7-day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP-ALL and 23 T-ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.     

Individual traits and environmental factors influencing sleep timing: a study of 225 Japanese couples

Behavioral and physiological processes, such as sleep-wakefulness, thermoregulation, and hormone secretion, exhibit 24-h rhythms in most organisms. These biological rhythms are driven by the circadian clock system and are entrained by the external environment, which in the case of humans includes social time schedules. Couples might be ideal experimental subjects to discriminate between individual traits and environmental factors, as they share lifestyle habits but not genetic backgrounds. In this study, sleep timing was compared between married Japanese couples (n?=?225) who had lived together for 1 yr or more (mean 17 yrs). Additionally, the authors evaluated the influence of individual traits and environmental factors on an individual's sleep timing per each couple. The results reveal that the sleep timings of a couple are mainly associated with the chronotypes of the husband and wife, whereas the sleep timings are significantly influenced by certain environmental factors. The findings suggest that chronotype remains one of the major determinants of an individual's sleep onset and wake times. Understanding an individual's chronotype may help improve the quality of life issues surrounding sleep.     

Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid

We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.     

A highly sensitive enzyme-linked immunosorbent assay for quantification of adipocytokines secreted by mouse adipocytes

The mouse preadipocyte 3T3-L1 line is the most useful cell line for the study of adipocytes. Adipocytes secrete adipocytokines, and abnormal adipocytokine production can cause the metabolic syndrome. Although it is important to understand the characteristics of mouse adipocytokine secretion, it is difficult to quantify these products because they are produced in low concentrations. Here, we developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for detecting the concentrations of mouse adipocytokines, such as TNFα and leptin. In this method, the antigen was sandwiched by using goat- and rabbit-derived polyclonal antibodies, and the fluorescence intensity produced in the reaction with 4-methylumbelliferyl-β-galacto-sidase pyranosidase (MUG) was measured. TNFα and leptin could be measured at concentrations as low as approximately 1 pg/ml. By using our ELISA method, we also measured the concentrations of TNFα and leptin in mouse 3T3-L1 preadipocytes and adipocytes. The differentiation of preadipocytes into adipocytes enhanced TNFα production and secretion and reduced the leptin production. The amount of TNFα produced in the adipocytes was 3.0 ng/mg-protein; this amount was considerably higher than that of leptin.