Toyo Okui

Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells

We investigated the lethal, UV killing-potentiating and repair-inhibiting effects of trivalent arsenic trioxide (As2O3) and pentavalent sodium arsenate (Na2HAsO4) in normal human and xeroderma pigmentosum (XP) fibroblasts. The presence of As2O3 for 24 h after UV irradiation inhibited the thymine dimer excision from the DNA of normal and XP variant cells and thus the subsequent unscheduled DNA synthesis (UDS): excision inhibitions were partial, 30-40%, at a physiological dose of 1 microgram/ml and 100% at a supralethal dose of 5 micrograms/ml. Correspondingly, As2O3 also potentiated the lethal effect of UV on excision-proficient normal and XP variant cells in a concentration-dependent manner, but not on excision-defective XP group A cells. Na2HAsO4 (As5+) was approximately an order of magnitude less effective in preventing all the above repair events than As2O3 (As3+) which is highly affinic to SH-containing proteins. The above results provide the first evidence that arsenic inhibits the excision of pyrimidine dimers. Partially repair-suppressing small doses of As2O3 (0.5 microgram/ml) and Na2HAsO4 (5 micrograms/ml) enhanced co-mutagenically the UV induction of 6-thioguanine-resistant mutations of V79 Chinese hamster cells. Thus, such a repair inhibition may be one of the basic mechanisms for the co-mutagenicity and presumably co-carcinogenicity of arsenic. XP group A and variant strains showed a unique higher sensitivity to As2O3 and Na2HAsO4 killing by a yet unidentified mechanism.

Radiation hypersensitivity of LEC strain rats controlled by a single autosomal recessive gene

LEC strain rats (LEC rats), which are known to develop hereditarily spontaneous fulminant hepatitis 4-5 months after birth, were highly sensitive to whole-body X-irradiation when compared to WKAH strain rats. The radiosensitivity of F1 hybrids of LEC and WKAH rats was similar to that of WKAH rats and significantly lower than that of LEC rats. Segregation data of backcross hybrids (F1 x LEC and LEC x F1) suggested that the hypersensitivity of LEC rats to whole-body irradiation is controlled by a single autosomal recessive gene. The radiosensitivity of fibroblasts from LEC rats was higher than that of fibroblasts from WKAH rats. The repair process of DNA double-strand breaks in LEC cells was slower than that in WKAH cells. LEC rats could provide a useful animal model to assist in understanding the mechanism of radiation-induced DNA damage and repair.

Gelatin-containing diphtheria–tetanus–pertussis (DTP) vaccine causes sensitization to gelatin in the recipients

Gelatin-specific T cell response was performed to determine whether a series of vaccinations with gelatin-containing DTP is a primary sensitization process in gelatin allergy. Thirty-seven recipients with gelatin-containing DTP who developed adverse reactions after vaccination and eight recipients of DTP without gelatin who also developed adverse reactions were studied. In addition, 10 subjects receiving gelatin-containing vaccine and 10 subjects inoculated with non-gelatin vaccine who did not show any adverse reactions were also investigated. All subjects inoculated with gelatin-containing DTP vaccine showed positive T cell responses against gelatin, however, occurrence of adverse reactions did not correlate with T cell responses. We conclude that DTP vaccine containing gelatin induces sensitization to gelatin in the recipients, but the mechanism of local reactions remains unknown.

A strong association between HLA-DR9 and gelatin allergy in the Japanese population

The frequency of HLA class I and II phenotypes was determined among 23 patients with positive gelatin IgE, eight of whom developed anaphylaxis, 18 patients who did not have gelatin IgE but who experienced non-immediate reactions after exposure to gelatin. HLA-DR9, which is unique to Orientals, was present in 56.5% of the gelatin IgE positive patients, as compared to control population frequency of 24% (P < 0.002). In the non-immediate reaction group, who did not generate IgE, phenotype distribution resembled controls. HLA-DR9 positive individuals have a relative risk of 4.1 for developing gelatin allergy with positive IgE.

Hypertonic Treatment Inhibits Radiation-Induced Nuclear Translocation of the Ku Proteins G22p1 (Ku70) and Xrcc5 (Ku80) in Rat Fibroblasts

Abstract Endoh, D., Okui, T., Kon, Y. and Hayashi, M. Hypertonic Treatment Inhibits Radiation-Induced Nuclear Translocation of the Ku Proteins G22p1 (Ku70) and Xrcc5 (Ku80) in Rat Fibroblasts. The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on the subcellular localization of the Ku proteins G22p1 (also known as Ku70) and Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivity were examined using confocal laser microscopy and immunoblotting. Although these proteins were observed mainly in the nuclei of human fibroblasts, approximately 80% of the intensities of immunofluorescence from both G22p1 and Xrcc5 was observed in the cytoplasm of rat fibroblasts. When the rat cells were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation. No significant differences were observed between the total intensities of the cellular fluorescence from the proteins in unirradiated and irradiated rat fibroblasts. The results showed that the proteins were translocated from the cytoplasm to the nucleus in the rat cells after X irradiation. The nuclear translocation of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair process of potentially lethal damage (PLD). When the rat cells were treated with 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited. The surviving fraction was approximately 60% of that of irradiated cells that were not treated with 0.5 M NaCl. The surviving fraction increased with incubation time in the growth medium before treatment with NaCl. The proportions of the intensities of fluorescence from G22p1 in the nuclei of X-irradiated cells also increased from 20% to 50% with increasing interval between X irradiation and treatment with NaCl. These results suggest that nuclear translocation of G22p1 and Xrcc5 is important for the fast repair process of PLD in rat cells.

Sensitive and rapid detection of norovirus using duplex TaqMan reverse transcription-polymerase chain reaction

Conventional reverse transcription‐polymerase chain reaction (RT‐PCR) to detect norovirus (NV) is a complex of multi‐step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the final diagnosis. A duplex TaqMan RT‐PCR was developed to detect and classify genogroup (G) I and GII of NV. The primers and TaqMan probes for this assay were selected from the region of open reading frame (ORF) 1‐ORF2 junction. A total of 796 stool specimens from 103 outbreaks of gastroenteritis, and a series of 46 stool specimens containing most NV genotypes was used for this study. For these specimens from 103 outbreaks, NV was detected and classified by the duplex TaqMan RT‐PCR in 536 of the 541 specimens tested previously to be positive using the conventional RT‐PCR. Two hundred fifty‐one of the 255 specimens that were negative by the conventional RT‐PCR were also negative by the TaqMan RT‐PCR. No false positive result was observed for other enteric RNA viruses such as rotavirus and sapovirus. This is the first report on the development of a duplex TaqMan RT‐PCR end‐point assay for detection and differentiation of GI and GII of NV strains simultaneously followed by genotyping. These results suggest the practical application of this duplex TaqMan RT‐PCR is useful for the detection of NV in clinical specimens. J. Med. Virol. 80:913–920, 2008.

Varicella-Zoster Virus—Specific Cellular Immunity in Subjects Given Acyclovir after Household Chickenpox Exposure

The time course of primary cell-mediated immune responses to varicella-zoster virus (VZV) among persons receiving acyclovir prophylaxis after exposure to chickenpox has not been well defined. Fifteen children who had household exposure to varicella received prophylactic acyclovir (40 mg/kg/day for 7-14 days after exposure) and were studied for development of both antibody and cell-mediated immunity (CMI) to VZV. Twelve developed antibodies and/or CMI; 10 had no symptoms and 2 manifested mild varicella. Two were already immune to varicella and had booster immune responses. One was not infected and subsequently developed full-blown varicella. Although acyclovir given after exposure to VZV is highly effective and does not appear to attenuate the immune response, it remains necessary to confirm whether, in the absence of clinical varicella, persons acquire specific immunity.

Hypoxia and etanidazole alter radiation-induced apoptosis in HL60 cells but not in MOLT-4 cells

Purpose : To examine how hypoxia influences ionizing irradiation-induced apoptosis in cultured mammalian cells and how a hypoxic cell radiosensitizer sensitizes apoptosis under hypoxic conditions. Materials and methods : Two cell lines derived from human lymphocytes, HL60 and MOLT-4, were exposed to 15 Gy X-rays under aerobic and hypoxic conditions. Etanidazole was used as a hypoxic cell radiosensitizer. The apoptotic morphological changes of nuclei and the induction of ladder-like DNA fragmentation were assessed by fluorescence microscopy and agarose gel electrophoresis, respectively. Results : In HL60 cells, apototic cell death and the activation of caspases 8, 9 and 3 were less induced under the hypoxic conditions than under the aerobic ones. Treatment of hypoxic cells with etanidazole enhanced X-ray-induced apoptosis and caspase activation. However, in MOLT-4 cells, neither hypoxia nor etanidazole influenced X-ray-induced apoptosis and caspase activation. In both cell lines, the frequency of X-ray-induced DNA double-strand breaks (DSB) under hypoxia was significantly smaller than that in aerobic conditions. Treatment of hypoxic cells with etanidazole enhanced them. Conclusion : These results suggested that X-ray-induced apoptosis in HL60 cells was initiated by DNA DSB and the treatment of hypoxic cells with etanidazole sensitized them through the enhancement of DSB induction, whereas X-ray-induced apoptosis in MOLT-4 cells occurred through damage other than to DNA.

Visualization of yellowish-orange luminescence from cuprous metallothioneins in liver of Long-Evans Cinnamon rat

We describe the first use of an emission probe, based on the cuprous thiolate chromophore, for direct microscopical observation of cuprous metallothioneins located in liver of 15-week-old (just before spontaneous hepatitis) Long-Evans Cinnamon rats. The rats show remarkable accumulations of copper and cuprous metallothioneins. In the mildly fixed liver, we visualized the same yellowish-orange luminescence as the specific emission from cuprous metallothioneins, following excitation in 330-385 nm region. In liver from Long-Evans Agouti rat, a counter part of Long-Evans Cinnamon rat, no similar luminescence was found. So, it was thought that cuprous metallothioneins accumulated in the Long-Evans Cinnamon rat liver might emit the yellowish-orange light. To verify this presumption, we tentatively defined three histochemical criteria, quenching tests by oxidation, protonation and mercury treatment, based on the coordination chemical characteristics of metallothioneins. The emission completely satisfied these criteria. Furthermore, the reliability of these criteria was supported by immunocytochemical and biochemical results. Consequently, all results sufficiently indicate that the yellowish-orange luminescence in the Long-Evans Cinnamon rat liver is the emission from cuprous metallothioneins.

Clonal structure of Shiga toxin (Stx)-producing and β-D-glucuronidase-positive Escherichia coli O157:H7 strains isolated from outbreaks and sporadic cases in Hokkaido, Japan

A total of 22 clonal phenotypic variants of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 was isolated from six different locations in Hokkaido, Japan. These isolates were negative for sorbitol fermentation but positive for beta-D-glucuronidase (GUD+). They carried eaeA, EHEC-hlyA, pas and etpD genes like typical E. coli O157:H7 and, in addition, st1 and stx2 genes. However, they were shown to lack katP and espP genes that are present in typical STEC O157:H7. All these atypical GUD+ STEC O157:H7 isolates had very similar antimicrobial susceptibilities. Pulsed-field gel electrophoresis analysis with XbaI, SfiI, SwaI, SpeI and NotI indicated that they were identical or closely related to one another. From their phenotypic and genotypic features, these GUD+ STEC O157:H7 isolates may represent a distinct clone among STEC O157.