Long Bones Exhibit Adaptive Responses to Chronic Low-Dose-Rate Ionizing Radiation despite its Lifespan-Shortening and Carcinogenic Effects on C57BL/6 Mice.

Ionizing radiation (IR) is a well-known carcinogen. High-dose-rate (HDR) IR is known to damage long bones (in terms of mass and structure), but the relationships among dose rates (particularly low-dose-rate [LDR] IR), long-bone condition, cancer incidence, and lifespan shortening remain elusive. The aim of this study was to elucidate the effects of LDR-IR on long-bone condition by comparing the long-term consequences of HDR- and LDR-IR exposure in mice. We utilized micro-computed tomography (µCT) scans of the long bones at similar ages (mean 77-96 weeks) to compare mice receiving approximately equivalent total doses of internal LDR-IR or external HDR-IR starting at 4 weeks of age, and their respective control groups. The lifespan-shortening effects of LDR-IR and HDR-IR were similar in these mixed-sex cohorts. Notably, compared to HDR-IR mice, mice internally exposed to chronic LDR-IR with continuous hypohematopoiesis showed a significantly higher trabecular bone connective density [femur: 247% (p = 0.0042), tibia: 169% (p = 0.0005)] and midshaft cortical bone thickness/area (femur: 130% [p = 0.0079]/120% [p = 0.021], tibia: 148% [p = 0.0004]/129% [p = 0.002]). Consistent with this result, when comparing 26-32 weeks post-IR with 2-8 weeks post-IR, compared to HDR-IR-treated mice, LDR-IR-treated mice exhibited higher levels of an osteoblast marker (OPG; p = 0.67 for HDR-IR, p = 0.068 for LDR-IR) and lower levels of an osteoclast marker (CTX-I; p = 0.0079 for HDR-IR, p = 0.72 for LDR-IR) despite significantly higher levels of inflammatory markers (CCL2 and CXCL1; p = 0.36-0.8 for HDR-IR, p = 0.013-0.041 for LDR-IR). These results suggest that long bones under chronic LDR-IR stress may exhibit an adaptive response to stresses such as chronic inflammation associated with IR-induced lifespan shortening. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

STUDY OF BIOLOGICAL EFFECTS OF TRITIUM IN MICE.

The mutation, apoptosis and chromosomal aberration induced by tritiated water (HTO) in spleen T lymphocytes of mice were investigated and compared with those by acute or chronic 137Cs gamma irradiation. p53 wild-type (p53+/+) and null (p53-/-) mice were exposed to tritium (3H) beta rays via a single injection of HTO. 137Cs gamma irradiation was carried out at dose-rate of 0.86Gy min-1 (acute) and at a low dose-rate (0.71-0.09 mGy min-1) that mimicked internal exposure (gamma simulation-irradiation). Each dose of irradiation was 3Gy. When compared on the basis of the induced TCR variant fractions in p53-/- mice at 3Gy, 3H beta rays appeared to be more mutagenic than chronic gamma ray reference. On the other hand, both of the frequency chromosomal aberration was not different significantly between HTO injected and 137Cs gamma irradiated mice.

Comparison of the fertility of tumor suppressor gene-deficient C57BL/6 mouse strains reveals stable reproductive aging and novel pleiotropic gene.

Tumor suppressor genes are involved in maintaining genome integrity during reproduction (e.g., meiosis). Thus, deleterious alleles in tumor suppressor-deficient mice would exhibit higher mortality during the perinatal period. A recent aging model proposes that perinatal mortality and age-related deleterious changes might define lifespan. This study aimed to quantitatively understand the relationship between reproduction and lifespan using three established tumor suppressor gene (p53, APC, and RECQL4)-deficient mouse strains with the same C57BL/6 background. Transgenic mice delivered slightly reduced numbers of 1st pups than wild-type mice [ratio: 0.81-0.93 (p = 0.1-0.61)] during a similar delivery period, which suggest that the tumor suppressor gene-deficient mice undergo relatively stable reproduction. However, the transgenic 1st pups died within a few days after birth, resulting in a further reduction in litter size at 3 weeks after delivery compared with that of wild-type mice [ratio: 0.35-0.68 (p = 0.034-0.24)] without sex differences, although the lifespan was variable. Unexpectedly, the significance of reproductive reduction in transgenic mice was decreased at the 2nd or later delivery. Because mice are easily affected by environmental factors, our data underscore the importance of defining reproductive ability through experiments on aging-related reproduction that can reveal a trade-off between fecundity and aging and identify RECQL4 as a novel pleiotropic gene.

The first fatal case of Corynebacterium ulcerans infection in Japan.

Introduction.Corynebacterium ulcerans (C. ulcerans) is a zoonotic pathogen that occasionally causes diphtheria-like symptoms in humans. Cases of C. ulcerans infection have been increasing in recent years, and C. ulcerans has been recognized as an emerging pathogen. Case presentation. Here we report a case of asphyxia death due to pseudomembrane caused by diphtheria toxin (DT)-producing C. ulcerans. This is, to our knowledge, the first fatal case of C. ulcerans infection in Japan. A strain of C. ulcerans was obtained from the patient's pet cat and was confirmed to be identical to the patient's isolate by sequencing of the 16S rRNA gene and the DT gene, by pulsed-field gel electrophoresis (PFGE) and by ribotyping. In the same way, it was revealed that the isolate in this case belonged to the same molecular type as the C. ulcerans 0102 isolated from the first case in Japan in a distant prefecture 15 years earlier, in 2001. Conclusion. DT-producing C. ulcerans can be contracted from a companion animal and causes human death if the appropriate treatment is delayed. The finding indicates that this molecular type of virulent C. ulcerans is currently widespread in Japan.

Estimation of Biological Effects of Tritium.

Nuclear fusion technology is expected to create new energy in the future. However, nuclear fusion requires a large amount of tritium as a fuel, leading to concern about the exposure of radiation workers to tritium beta radiation. Furthermore, countermeasures for tritium-polluted water produced in decommissioning of the reactor at Fukushima Daiichi Nuclear Power Station may potentially cause health problems in radiation workers. Although, internal exposure to tritium at a low dose/low dose rate can be assumed, biological effect of tritium exposure is not negligible, because tritiated water (HTO) intake to the body via the mouth/inhalation/skin would lead to homogeneous distribution throughout the whole body. Furthermore, organically-bound tritium (OBT) stays in the body as parts of the molecules that comprise living organisms resulting in long-term exposure, and the chemical form of tritium should be considered. To evaluate the biological effect of tritium, the effect should be compared with that of other radiation types. Many studies have examined the relative biological effectiveness (RBE) of tritium. Hence, we report the RBE, which was obtained with radiation carcinogenesis classified as a stochastic effect, and serves as a reference for cancer risk. We also introduce the outline of the tritium experiment and the principle of a recently developed animal experimental system using transgenic mouse to detect the biological influence of radiation exposure at a low dose/low dose rate.

A novel ATM/TP53/p21-mediated checkpoint only activated by chronic γ-irradiation.

Different levels or types of DNA damage activate distinct signaling pathways that elicit various cellular responses, including cell-cycle arrest, DNA repair, senescence, and apoptosis. Whereas a range of DNA-damage responses have been characterized, mechanisms underlying subsequent cell-fate decision remain elusive. Here we exposed cultured cells and mice to different doses and dose rates of γ-irradiation, which revealed cell-type-specific sensitivities to chronic, but not acute, γ-irradiation. Among tested cell types, human fibroblasts were associated with the highest levels of growth inhibition in response to chronic γ-irradiation. In this context, fibroblasts exhibited a reversible G1 cell-cycle arrest or an irreversible senescence-like growth arrest, depending on the irradiation dose rate or the rate of DNA damage. Remarkably, when the same dose of γ-irradiation was delivered chronically or acutely, chronic delivery induced considerably more cellular senescence. A similar effect was observed with primary cells isolated from irradiated mice. We demonstrate a critical role for the ataxia telangiectasia mutated (ATM)/tumor protein p53 (TP53)/p21 pathway in regulating DNA-damage-associated cell fate. Indeed, blocking the ATM/TP53/p21 pathway deregulated DNA damage responses, leading to micronucleus formation in chronically irradiated cells. Together these results provide insights into the mechanisms governing cell-fate determination in response to different rates of DNA damage.

Involvement of reactive oxygen species in stimuli-induced shedding of heparin-binding epidermal growth factor-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes, such as wound healing, atherosclerosis and cancer proliferation. HB-EGF is synthesized as a membrane form (proHB-EGF), and is shedded at the cell surface to yield soluble HB-EGF, resulting in making it active. In this study, the involvement of reactive oxygen species (ROS) in stimuli-induced shedding of HB-EGF was investigated using monkey kidney Vero cells overexpressing HB-EGF (Vero-H cells). 12-O-tetradecanoylphorbol-13-acetate (TPA), lysophosphatidic acid (LPA) as a ligand for seventransmembrane G protein coupled receptors (GPCR) and sorbitol as stress induced shedding of HB-EGF mediated protein kinase C (PKC)-δ, mitogen-activated protein kinase (MAPK) and p38MAPK, respectively. These stimuli-induced sheddings of HB-EGF were inhibited by N-acetyl-L-cysteine (NAC), suggesting the involvement of ROS. As specific inhibitors of these protein kinases inhibited the shedding of HB-EGF, these signaling pathways seem to be independent, respectively. In contrast, γ-ray irradiation did not induce shedding although it did increase intracellular ROS levels. Taken together, these results suggest that the synergistic generation of ROS and the activation of protein kinase are required to promote stimuli-induced shedding of HB-EGF.

CxxC-ZF domain is needed for KDM2A to demethylate histone in rDNA promoter in response to starvation.

The transcription of ribosomal RNA genes (rDNA) is a rate-limiting step in ribosome biogenesis and changes profoundly in response to environmental conditions. Recently we reported that JmjC demethylase KDM2A reduces rDNA transcription on starvation, with accompanying demethylation of dimethylated Lys 36 of histone H3 (H3K36me2) in rDNA promoter. Here, we characterized the functions of two domains of KDM2A, JmjC and CxxC-ZF domains. After knockdown of endogenous KDM2A, KDM2A was exogenously expressed. The exogenous wild-type KDM2A demethylated H3K36me2 in the rDNA promoter on starvation and reduced rDNA transcription as endogenous KDM2A. The exogenous KDM2A with a mutation in the JmjC domain lost the demethylase activity and did not reduce rDNA transcription on starvation, showing that the demethylase activity of KDM2A itself is required for the control of rDNA transcription. The exogenous KDM2A with a mutation in the CxxC-ZF domain retained the demethylase activity but did not reduce rDNA transcription on starvation. It was found that the CxxC-ZF domain of KDM2A bound to the rDNA promoter with unmethylated CpG dinucleotides in vitro and in vivo. The exogenous KDM2A with the mutation in the CxxC-ZF domain failed to reduce H3K36me2 in the rDNA promoter on starvation. Further, it was suggested that KDM2A that bound to the rDNA promoter was activated on starvation. Our results demonstrate that KDM2A binds to the rDNA promoter with unmethylated CpG sequences via the CxxC-ZF domain, demethylates H3K36me2 in the rDNA promoter in response to starvation in a JmjC domain-dependent manner, and reduces rDNA transcription.

Ablation of Mina53 in mice reduces allergic response in the airways.

The mina53 (myc-induced nuclear antigen with a 53 kDa molecular mass; also known as mina) was identified as a direct transcriptional target of the oncoprotein Myc and encodes a conserved protein in vertebrates. While Mina53 is known to be associated with tumorigenesis, it is not clear what role Mina53 plays in non-neoplastic tissues. To directly address the roles of Mina53 in non-neoplastic tissues, we created mina53-deficient mice. Both male and female mina53-deficient mice reached adulthood and were fertile, suggesting that Mina53 is dispensable for the basic developmental processes. Since we found that Mina53 was expressed in cells responsible for immune responses, we investigated whether Mina53 was involved in immune responses. When mice were exposed intranasally to house dust mites as an allergen, the airway tract showed hyperresponsiveness to methacholine in wild-type mice but not in mina53-deficient mice. The mina53-deficient mice also showed a significantly reduced migration of immune cells, including eosinophils, into bronchoalveolar lavage fluid compared with wild-type mice. The levels of Th2 cytokines, IL-4 and IL-5, produced in response to house dust mites were lower in the mina53-deficient mice than in wild-type mice. The level of IFN-γ in bronchoalveolar lavage fluid was significantly decreased by exposure to house dust mites in wild-type mice but not in the mina53-deficient mice. These results suggest that Mina53 plays a role in the allergic response to inhaled allergens, possibly through controlling IL-4 production.

Bacterial hazards of sludge brought ashore by the tsunami after the great East Japan earthquake of 2011.

OBJECTIVES: The aim of this study was to identify bacteria in sludge brought by the 2011 tsunami in Japan to determine the necessary precautions for workers who handle the sludge. METHODS: Two sludge samples and one water sample were collected from each of two sites in Miyagi Prefecture in June 2011. We also obtained control samples from a paddy field and a dry beach in Fukuoka, Japan. The samples were subjected to physicochemical analyses, conventional cultivation methods, and molecular methods for bacterial flora analysis. The bacterial floras were analyzed using a clone library method employing fragments of the 16S ribosomal RNA gene (rDNA) amplified with universal primers. RESULTS: We detected 51-61 genera in sludge samples and 14 and 17 genera in water samples collected in the tsunami-affected areas. In sludge samples collected in the tsunami-affected areas, more genera belonged to Proteobacteria than to Bacteroidetes, but in water samples collected in these areas, more genera belonged to Bacteroidetes than to Proteobacteria. Non-O1, non-O139 V. cholerae (non-agglutinable vibrio) was found at approximately 10(4) cells/m/ near the coast of the tsunami affected area. Sulfate-reducing bacteria were detected in sludge collected from the paddy field, and a relatively high concentration of sulfate ions was found in the water sample (258 mg/l). CONCLUSIONS: Sludge brought by the tsunami contained some pathogens; therefore, frequent hand washing is recommended for workers who have direct contact with the sludge to minimize their risk of infection. Under the anaerobic conditions of paddy fields, hydrogen sulfide could be produced by sulfate-reducing bacteria metabolizing sulfate ions.

JmjC enzyme KDM2A is a regulator of rRNA transcription in response to starvation.

The rate-limiting step in ribosome biogenesis is the transcription of ribosomal RNA, which is controlled by environmental conditions. The JmjC enzyme KDM2A/JHDM1A/FbxL11 demethylates mono- and dimethylated Lys 36 of histone H3, but its function is unclear. Here, we show that KDM2A represses the transcription of ribosomal RNA. KDM2A was localized in nucleoli and bound to the ribosomal RNA gene promoter. Overexpression of KDM2A repressed the transcription of ribosomal RNA in a demethylase activity-dependent manner. When ribosomal RNA transcription was reduced under starvation, a cell-permeable succinate that inhibited the demethylase activity of KDM2A prevented the reduction of ribosomal RNA transcription. Starvation reduced the levels of mono- and dimethylated Lys 36 of histone H3 marks on the rDNA promoter, and treatment with the cell-permeable succinate suppressed the reduction of the marks during starvation. The knockdown of KDM2A increased mono- and dimethylated Lys 36 of histone H3 marks, and suppressed the reduction of ribosomal RNA transcription under starvation. These results show a novel mechanism by which KDM2A activity is stimulated by starvation to reduce ribosomal RNA transcription.

A comparison of the mutagenic and apoptotic effects of tritiated water and acute or chronic caesium-137 gamma exposure on spleen T lymphocytes on normal and p53-deficient mice.

PURPOSE: This study was carried out to compare the mutagenic effects on spleen T lymphocytes of mice exposed to tritiated water (HTO) and chronic or acute (137)Cs gamma irradiation. MATERIALS AND METHODS: p53 wild type (p53(+/+)) and p53 null type (p53(-/-)) mice were exposed to a total dose of 3 Gy of HTO, chronic (137)Cs and acute (137)Cs. RESULTS: In spontaneous T-cell receptor (TCR) variant fractions and fractions following exposure to HTO, chronic (137)Cs and acute (137)Cs, TCR variant fractions in p53(+/+) mice were 5.9 x 10(-4), 9.8 x 10(-4), 6.4 x 10(-4) and 20.1 x 10(-4), respectively. In contrast, those fractions were increased in p53(-/-) mice to 11.2 x 10(-4), 18.8 x 10(-4), 15.7 x 10(-4) and 31.3 x 10(-4), respectively. The frequency of apoptotic cells of the spleen 12 h after HTO injection increased to 5.0% in p53(+/+) mice, but did not increase at all in p53(-/-) mice. CONCLUSIONS: When compared on the basis of induced TCR variant fractions in p53(-/-) mice, HTO (7.6 x 10(-4)) was 1.7 times more mutagenic than chronic (137)Cs (4.5 x 10(-4)), but 2.6 times less mutagenic than acute (137)Cs gamma irradiation (20.1 x 10(-4)).

Expression of the TAF4b gene is induced by MYC through a non-canonical, but not canonical, E-box which contributes to its specific response to MYC.

Transcription factor binding sites are short DNA sequences that interact with transcription factors and the proper control of gene expression appears to require the mechanisms including the regulation through the genome context around the transcription factor binding sites. The MYC proteins are central regulators of cell growth. Many genes have been reported to be regulated by MYC through E-box sites. However, the characters of E-box that Myc selects to function are not clear and identification of additional genes controlled by MYC will provide information to completely understand the functions of MYC. Here we report that MYC directly induces TAF4b expression. We mapped the transcription start site and characterized functional promoter elements for MYC response in the TAF4b promoter. There are several E-box sequences near the transcription start site, including canonical (CACGTG) and non-canonical (CGCGTG) ones. We found that c-MYC induces TAF4b expression through one of the non-canonical E-box sites, which is in a highly conserved region of TAF4b promoters in mammals, suggesting the importance of the genome context around the target E-box. When the non-canonical E-box in the TAF4b promoter was mutated to a canonical one, MYC functioned on both E-boxes, while another E-box-binding transcription factor, USF, did so on only the canonical E-box. These results suggest that in addition to the context where the target E-box exists, a sequence within an E-box is involved in the mechanisms by which specific E-box sites are selected by Myc.

[Mechanism for activation of heparin-binding EGF-like growth factor induced by stimuli].

Heparin-binding epidermal growth factor-like growth factor(HB-EGF), which belongs to the EGF family, is a critical growth factor for a number of physiological and pathological processes, such as wound healing and cardiac hypertrophy. HB-EGF is synthesized as a membrane-anchored form(pro-HB-EGF), and pro-HB-EGF is cleaved at the cell surface to yield soluble HB-EGF by a mechanism called "ectodomain shedding". Soluble HB-EGF has mitogenic activity. Ectodomain shedding of proHB-EGF is induced by stimuli, including 12-O-tetradecanoylphorbol-13-acetate(TPA), a ligand for seven-transmembrane G protein-coupled receptors(GPCR), stress and inflammatory cytokine. Lysophosphatidic acid(LPA), a ligand for GPCR, stimulates the shedding of proHB-EGF, which constitutes a GPCR-mediated transactivation of the EGF receptor. Ras-Raf-MEK signal and the small GTPase Rac are essential for the LPA-induced shedding. On the other hand, protein kinase C and ADAM 9 protease are essential for the TPA-induced shedding. Furthermore, p38 MAPK is essential for the stress- and IL-1 beta-induced shedding. Finally there is a mechanism for activation of HB-EGF regulated by the environment in the living body.

c-myc induces autophagy in rat 3Y1 fibroblast cells.

The proto-oncogene c-myc is a multifunctional gene that regulates cell division, cell growth, and apoptosis. Here we report a new function of c-myc: induction of autophagy. Autophagy is a bulk degradation system for intracellular proteins. Autophagy proceeds with characteristic morphologies, which begins with the formation of a double-membrane structure called the autophagosome surrounding a portion of the cytoplasm, after which its outer membrane then fuses with the lysosomal membrane to become an autolysosome. Autophagosomes and autolysosomes are generally called autophagic vacuoles. When c-Myc protein was overexpressed in rat 3Y1 fibroblasts or when the chimeric protein c-MycER was activated by estrogen, the number of autophagic vacuoles in cells increased significantly. The formation of autophagic vacuoles induced by c-Myc was completely blocked by a specific inhibitor of autophagosome formation, 3-methyladenine. A c-Myc mutant lacking Myc Box II induced neither apoptosis nor oncogenic transformation, but still stimulated autophagy. An inhibitor of caspases suppressed apoptosis but not autophagy. These results suggest that the autophagy caused by c-myc is not due to the apoptosis or tumorigenesis induced by c-myc. Taken together, our results suggest that the induction of autophagy is a novel function of c-myc.

The stress- and inflammatory cytokine-induced ectodomain shedding of heparin-binding epidermal growth factor-like growth factor is mediated by p38 MAPK, distinct from the 12-O-tetradecanoylphorbol-13-acetate- and lysophosphatidic acid-induced signaling cascades.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes. HB-EGF is synthesized as a membrane-anchored form (pro-HB-EGF), and pro-HB-EGF is cleaved at the cell surface to yield soluble HB-EGF by a mechanism called "ectodomain shedding." We show here that the ectodomain shedding of pro-HB-EGF in Vero cells is induced by various stress-inducing stimuli, including UV light, osmotic pressure, hyperoxidation, and translation inhibitors. The pro-inflammatory cytokine interleukin-1beta also stimulated the ectodomain shedding of pro-HB-EGF. An inhibitor of p38 MAPK (SB203580) or the expression of a dominant-negative (dn) form of p38 MAPK inhibited the stress-induced ectodomain shedding of pro-HB-EGF, whereas an inhibitor of JNK (SP600125) or the expression of dnJNK1 did not. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and lysophosphatidic acid (LPA) are also potent inducers of pro-HB-EGF shedding in Vero cells. Stress-induced pro-HB-EGF shedding was not inhibited by the inhibitors of TPA- or LPA-induced pro-HB-EGF shedding or by dn forms of molecules involved in the TPA- or LPA-induced pro-HB-EGF shedding pathway. Reciprocally, SB203580 or dnp38 MAPK did not inhibit TPA- or LPA-induced pro-HB-EGF shedding. These results indicate that stress-induced pro-HB-EGF shedding is mediated by p38 MAPK and that the signaling pathway induced by stress is distinct from the TPA- or LPA-induced pro-HB-EGF shedding pathway.