Genome-wide analysis of low-dose irradiated male Drosophila melanogaster with extended longevity.

Ionizing radiation generates oxidative stress, which is thought to be a major cause of aging. Although living organisms are constantly exposed to low levels of radiation, most studies examining the effect of radiation have focused on accelerated aging and diminished life span that result from high-dose radiation. On the other hand, several studies have suggested that low-dose radiation enhances the longevity of Drosophila melanogaster. Therefore, investigation of the biological effects of low-dose radiation could contribute to a more comprehensive understanding of the aging process. In this study, microarray and quantitative real time-PCR were used to measure genome-wide changes in transcript levels in low-dose irradiated fruit flies that showed enhanced longevity. In response to radiation, approximately 13% of the genome exhibited changes in gene expression, and a number of aging-related genes were significantly regulated. These data were compared with quantitative trait loci affecting life-span to identify candidate genes involved in enhanced longevity induced by low-dose radiation. This genome-wide survey revealed novel information about changes in transcript levels in low-dose irradiated flies and identified 39 new candidate genes for molecular markers of extended longevity induced by ionizing radiation. In addition, this study also suggests a mechanism by which low-dose radiation extends longevity.

Alteration of cytokine profiles in mice exposed to chronic low-dose ionizing radiation.

While a high-dose of ionizing radiation is generally harmful and causes damage to living organisms, a low-dose of radiation has been shown to be beneficial in a variety of animal models. To understand the basis for the effect of low-dose radiation in vivo, we examined the cellular and immunological changes evoked in mice exposed to low-dose radiation at very low (0.7mGy/h) and low (3.95mGy/h) dose rate for the total dose of 0.2 and 2Gy, respectively. Mice exposed to low-dose radiation, either at very low- or low-dose rate, demonstrated normal range of body weight and complete blood counts. Likewise, the number and percentage of peripheral lymphocyte populations, CD4(+) T, CD8(+) T, B, or NK cells, stayed unchanged following irradiation. Nonetheless, the sera from these mice exhibited elevated levels of IL-3, IL-4, leptin, MCP-1, MCP-5, MIP-1alpha, thrombopoietin, and VEGF along with slight reduction of IL-12p70, IL-13, IL-17, and IFN-gamma. This pattern of cytokine release suggests the stimulation of innate immunity facilitating myeloid differentiation and activation while suppressing pro-inflammatory responses and promoting differentiation of naïve T cells into T-helper 2, not T-helper 1, types. Collectively, our data highlight the subtle changes of cytokine milieu by chronic low-dose gamma-radiation, which may be associated with the functional benefits observed in various experimental models.

Risk factors for bone mineral density at the calcaneus in 40-59 year-old male workers: a cross-sectional study in Korea.

BACKGROUND: Few epidemiologic studies have attempted to investigate the prevalence and risk factors for osteopenia and osteoporosis in middle-aged Asian men. We performed this study to determine the prevalence and risk factors of osteopenia and osteoporosis in this population. METHODS: This cross-sectional study was conducted from March to July, 2004. The subjects were 2,073 males aged from 40 to 59 years in the KHNP (Korea Hydro & Nuclear Power) workplace-based cohort. Bone mineral density (BMD) was measured by peripheral, dual-energy, X-ray absorptiometry (DXA) at the calcaneus. Anthropometric and lifestyle factors were investigated using a standard, self-reported questionnaire. RESULTS: BMD was 0.60 +/- 0.09 g/cm2 (mean +/- standard deviation) and was negatively correlated with age (r = -0.18, P < 0.001), but positively correlated with waist-to-hip ratio (WHR; r = 0.15, P < 0.001), body fat (r = 0.10, P < 0.001), BMI (r = 0.35, P < 0.001), height (r = 0.26, P < 0.001), and weight (r = 0.43, P < 0.001). In multiple linear regression analysis, the independent determinants associated with BMD were increasing age (coefficient = -0.002, P < 0.001), physical activity (< or = 2/week vs. > or = 3/week; coefficient = 0.017, P < 0.001), WHR (coefficient = -0.796, P < 0.001), body mass index (BMI; coefficient = 0.023, P < 0.001) and smoking status (never vs. ever; coefficient = -0.018, P < 0.001). CONCLUSION: We suggest that BMD of the calcaneus is correlated negatively with exposure to smoke and increased WHR, but positively with regular exercise and increased BMI.

Specific proteolysis of the A-kinase-anchoring protein 149 at the Asp582 residue by caspases during apoptosis.

A-kinase-anchoring protein 149 (AKAP149) is a member of a structurally diverse, though functionally similar anchoring protein family and is localized to the outer membrane of mitochondria and in the endoplasmic reticulum-nuclear envelope network. AKAP149 plays an important role in controlling the subcellular localization and temporal specificity of protein phosphorylation and mRNA metabolism by tethering kinases and phosphatases, such as protein kinase A and type I protein phosphatase, through its N-terminal protein-binding motifs and mRNAs via its C-terminal RNA-binding motifs. It is well recognized that caspases play a central role in transducing and amplifying the intracellular death signal and that apoptosis is executed as a consequence of caspase-mediated cleavage of multiple cellular substrates. The identification of novel death substrates and elucidation of the consequences of their proteolytic cleavages by caspases are therefore crucial for our understanding of cell death and other biological processes. Herein, we demonstrated that AKAP149 is a direct substrate of active caspase-3, -8 -and -10 in vitro and in vivo. 35S-labeled full-length AKAP149 was completely cleaved in vitro by active caspase-3, -8 and -10 into two fragments of approximately 105 and 45 kDa, while caspase-2 cleaved it partially and caspase-1 did not cleave it at all. AKAP149 was also cleaved by caspases during Fas- and staurosporine-induced apoptosis in Jurkat T and HeLa cells, which were blocked by specific inhibitors of caspase-3 and -8. The specific cleavage site for these caspases was mapped in vitro and in vivo to Asp582 at AKAP149, which is located between the protein kinase A regulatory subunit anchoring and KH RNA-binding domains. In addition, HeLa cells transiently overexpressing AKAP149 D582E mutant were resistant to staurosporine-induced HeLa cell apoptosis. Taken together, these data suggest that AKAP149 activity may be deregulated by caspase-dependent proteolysis during apoptotic cell death and may provide useful information for elucidating the apoptosis signaling pathways in detail.

Genome-wide screening and identification of novel proteolytic cleavage targets of caspase-8 and -10 in vitro.

Apoptosis executed by the mammalian caspase family plays a fundamental role in cellular homeostasis. Deregulation of this process is associated with several human diseases. The multimerization of ligand-induced death receptors results in the recruitment of the death inducing signaling complex and autocatalytic activation of initiator caspases, including caspase-8 and -10. However, it is still unclear how initiator caspases trigger and control the early apoptotic signaling pathways, partly because the downstream proteolytic cleavage targets of the initiator caspases are not completely known. Although it is known that a number of proteins are cleaved by various members of the caspase family, the identification of specific cleavage substrates of the initiator caspases 8 and 10, has been hindered by a lack of systematic and broadly applicable strategies for substrate identification. In the present study we constructed a mouse cDNA library and used it to perform a systematic, genome-wide screen for novel in vitro substrates of caspase-8 and -10. From this, we successfully identified six putative caspase substrates, including five novel proteins (ABCF1, AKAP1, CPE, DOPEY1 and GOPC1) that may be targeted specifically by the initiator caspases 8 and 10 during the early stages of apoptosis. These findings may provide useful information for elucidating the apoptotic signaling pathways downstream of the death receptors.

Comprehensive analysis of time- and dose-dependent patterns of gene expression in a human mesenchymal stem cell line exposed to low-dose ionizing radiation.

We focused on the transcriptional responses induced by low and very low doses of ionizing radiation with time effect. Regardless of their importance only a few limited studies have been done. Here we applied a large-scale gene transcript profile to elucidate the genes and biological pathways. Immortalized human mesenchymal stem cells were irradiated with 0.01, 0.05, 0.2 and 1 Gy of gamma radiation and total RNA was extracted from each cell line at 1, 4, 12 and 48 h after exposure. The essential transcriptional responses were identified according to dose and time. A total of 6,016 genes showed altered expression patterns at more than one time point or dose level among the investigated 10,800 genes. Genes that showed dose-dependent expression responses were involved in signal transduction, regulation of transcription, proteolysis, peptidolysis and metabolism. Those that showed time-dependent responses were divided into two distinct groups: the up-and-down group was associated with 'cellular defense mechanisms' such as apoptosis, cell adhesion, stress response and immune response and the down-and-up group with 'fundamental cellular processes' such as DNA replication, mitosis, RNA splicing, DNA repair and translation initiation. Genes showing both dose-and time-dependent responses exhibited a mixture of both features. A highly non-linear relationship between the IR dose and the transcriptional relative response was obtained from the dose-dependent group. The time-dependent group also exhibited a non-linear relationship as the complex effect group did. Some of the early-reactive-phase (1-4 h) genes showed a differential expression response to 0.01, 0.05 and 0.2 Gy but were unresponsive to 1 Gy. Some of the late-recovery-phase (12-48 h) genes showed a differential expression to 1 Gy but were relatively unresponsive to other doses. We further characterized the gene expression patterns that could be implicated in the molecular mechanism of the cellular responses to low and very low-dose irradiation.

Incidence and risk factors of insulin resistance syndrome in 20-59 year-old Korean male workers.

We investigated the incidence of insulin resistance syndrome (IRS) according to the criteria of diagnoses suggested by the American College of Endocrinology/ American Association of Clinical Endocrinologists and the risk factors associated with the development of IRS. Among 2,048 subjects without a history of/or drug treatment for hypertension, diabetes, dyslipidemia with normal findings at baseline, 1,578 subjects aged 20-59 yr were followed prospectively for 2 yr. The incidence of IRS was 6.9 per 100 persons/year. The relative risk (RR) due to age was 1.03 (95% CI: 1.00-1.05) with every one-year increase in age. The RR associated with an abnormal waist-hip ratio group (> or =0.9) was increased by 1.74 (95% CI: 1.17-2.58) compared to the normal group (<0.9); RR associated with abnormal alanine transferase was increased (> or =35 IU/L) by 1.70 (95% CI: 1.20-2.41) compared to the normal group (<35 IU/L); and the RR associated with abnormal lowdensity lipoprotein (LDL) cholesterol was increased (> or =160 mg/L) by 1.70 (95% CI: 1.19-2.44) compared to the normal LDL cholesterol (<160 mg/L). Lastly, the RR of current smokers was increased by 1.63 (95% CI: 1.09-2.42) compared to that of non-smokers. It is necessary to develop methods of prevention and therapeutic approach to manage the integrated risk factors as opposed to individual factors.

Low-dose of ionizing radiation enhances cell proliferation via transient ERK1/2 and p38 activation in normal human lung fibroblasts.

This study shows the human cellular responses and the mechanism of low-dose ionizing radiation in CCD 18 Lu cells, which are derived from normal human lung fibroblasts. Cell proliferation and viability assay were measured for the cells following gamma-irradiation using trypan blue, BrdU incorporation, and Wst-1 assay. We also examined genotoxicity using a micronuclei formation assay. The activation of the MAPKs pathway was determined by Western blot analysis, and the siRNA system was used to inhibit the expression of ERK1/2 and p38. We found that 0.05 Gy of ionizing radiation stimulated cell proliferation and did not change Micronuclei frequencies. In addition, 0.05 Gy of ionizing radiation activated ERK1/2 and p38, but did not activate JNK1/2 in cells. A specific ERK1/2 inhibitor, U0126, decreased the phosphorylation of ERK1/2 proteins induced by 0.05 Gy of ionizing radiation, and a similar suppressive effect was observed with a p38 inhibitor, PD169316. Suppression of ERK1/2 and p38 phosphorylation with these inhibitors decreased cell proliferation, which was stimulated by 0.05 Gy of ionizing radiation. Furthermore, downregulation of ERK1/2 and p38 expression using siRNA blocked the cell proliferation that had been increased by 0.05 Gy of ionizing radiation. These results suggest that 0.05 Gy of ionizing radiation enhances cell proliferation through the activation of ERK1/2 and p38 in normal human lung fibroblasts.

Low-dose radiation stimulates the proliferation of normal human lung fibroblasts via a transient activation of Raf and Akt.

The biological effects of low-dose radiation have been investigated and debated for more than a century, but its cellular effects and regulatory mechanisms remain poorly understood. This study shows the human cellular responses to low-dose radiation in CCD-18 Lu cells, which are derived from normal human lung fibroblasts. We examined a colony-forming assay for cell survival by ionizing radiation. Live cell counting and cell cycle analysis were measured for cell proliferation and cell cycle progression following low-dose irradiation. We examined Raf and Akt phosphorylation to determine the proliferation mechanism resulting from low-dose radiation. We also observed that p53 and p21 were related to cell cycle response. We found that 0.05 Gy of ionizing radiation enhanced cell proliferation and did not change the progression of the cell cycle. In addition, 0.05 Gy of ionizing radiation transiently activated Raf and Akt, but did not change phospho-p53, p53 and p21 in CCD-18 Lu cells. However, 2 Gy of ionizing radiation induced cell cycle arrest, phosphorylation of p53, and expression of p53 and p21. The phosphorylation of Raf and Akt proteins induced by 0.05 Gy of ionizing radiation was abolished by pre-treatment with an EGFR inhibitor, AG1478, or a PI3k inhibitor, LY294002. Cell proliferation stimulated by 0.05 Gy of ionizing radiation was blocked by the suppression of Raf and Akt phosphorylation with these inhibitors. These results suggest that 0.05 Gy of ionizing radiation stimulates cell proliferation through the transient activation of Raf and Akt in CCD-18 Lu cells.

Potential of dark-striped field mice, Apodemus agrarius coreae, for use as a biological radiation dosimeter for human environments.

This study examined the possibility of using striped field mice as a biological dosimeter or indicator for surveillance of the ecological effects of boundary radiation emitted by nuclear power plants. For this study, the external morphological characteristics and isoenzymic electrophoretypes of Korean domestic dark-striped field mice were studied after they were captured, controlled for reproduction, and their exact species were identified. In terms of morphological external characteristics, the dark-brown coat, dark back stripe, head-to-tail length, tail length, and ear length matched the taxonomical characteristics of dark-striped field mice. In terms of isoenzymic electrophoretypes, the analyses on I-lactate dehydrogenase, aspartate aminotransferase, and malate dehydrogenase revealed that one species of dark-striped field mice, called Apodemus agrarius coreae, was scattered throughout a wide range of habitats. On the other hand, after irradiating the A. a. coreae (0, 0.5, 1, 3, 5, and 7 gray [Gy]) to analyze their survival rate and frequency of micronuclei in peripheral polychromatic erythrocytes, their LD50/30 was approximately 5 Gy. Also, the mice that contained 1 or 3 Gy gained weight compared with those that contained 0.5 Gy. Moreover, those with 0.5 Gy and higher showed an increase in white blood cells and platelets as well as in sodium and creatinine. However, decreased concentrations of alkaline phosphatase, alanine animotransferase, calcium, phosphorus, and globulin were observed in the A. a. coreae after irradiation. The results of the study reveal that wild A. a. coreae mice have high potential as a biological monitoring system to determine radiation effects in human environments such as those within the vicinity of nuclear power plants.

[The incidence and risk factors of hypertension that developed in a male-workers' cohort for 3 years].

OBJECTIVES: Cardiovascular disease is one of the main causes of death and morbidity in Korea. In this study, the prevalence and incidence of developing hypertension in a male-workers' cohort were investigated during 3-years follow-up with a view to find the risk factors that affected the development of hypertension. METHODS: Among the 5,374 people who participated in a routine health check up, 3,852 people with normal blood pressure and who had no history of hypertension were prospectively followed up for 3 years. The classification of hypertension was based on the JNC7 report (the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure). Life style factors and underlying diseases that were related to the risk factors of hypertension were collected by using a self-report questionnaires via the internet. RESULTS: The prevalence of hypertension was 28.3% (1,520/5,374) at the first screening (2001). It was found that the incidence in 2004 of hypertension for the follow-up subjects (3,711) who had normal blood pressure in 2001 was 7.6 per 100 person-year. Multiple logistic regression analysis of the variables related to the risk factors of hypertension was carried out. The relative risks were 1.037 (95% CI=1.022-1.053) as the age increased 1 year and 1.039 (95% CI=1.023-1.055) as the body mass index increased 1 kg/m2. The relative risk for the prehypertensive group was 2.501(95% CI=1.986-3.149) compared to the normotensive group. These results showed that age, body mass index and the baseline blood pressure were significantly related to the incidence of hypertension. CONCLUSIONS: The incidence of hypertension was 7.6 per 100 person-year during follow-up. It was concluded that the risk factors for developing hypertension in the short-term were age, BMI, and prehypertension; Especially, this showed that it is necessary for prehypertensives to manage their body weight and blood pressure to prevent hypertension in middle-age by modifying their life style.

Radioprotective effects of various cytokines in peripheral blood mononuclear cells and C3H mice.

The purpose of this study was to investigate the radioprotective effect of HGFs (GM-CSF, IL-3 and SCF) in irradiated human peripheral blood mononuclear cells (PBMCs) in vitro, and the survival effect of lethally irradiated C3H mice in vivo. The irradiation of human PBMCs using a (137)Cs irradiator showed a dose-dependent inhibition of cell growth up to a dose of 5 Gy. This cell growth inhibition induced apoptosis, which was associated with the down-regulation of Bcl-2, up-regulation of Bax, depolarization of mitochondrial transmembrane potential (Delta psi m), and caspase-3 and -9 activation. Following gamma-irradiation at 2 Gy, IL-3 (10 ng/ml) alone or combined with SCF (50 ng/ml) reduced the apoptotic portion of human PBMCs by 15 and 20% of the cell population, respectively, showing no activation of caspase-3 compared to the control group. To examine the in vivo effect of gamma-irradiation and cytokines, we investigated the survival rate and recovery of peripheral blood cells in C3H mice. C3H mice subjected to total body irradiation (TBI) at a dose of 7 Gy (lethal dose 83% at 30 days) showed time-dependent decreases in RBC, WBC and platelet counts, with the nadir occurring at 12 to 15 days. However, treatment with recombinant murine (rm) SCF (2 microg/day s.c.), rmIL-3 (2 microg/day s.c.), or rmG-CSF (2.5 microg/day s.c.) 24 h before and after irradiation did not promote hematologic recovery or survival in the lethally irradiated C3H mice. These findings indicate that the combined treatment of IL-3 and SCF prevents the apoptosis induced in PBMCs by gamma-irradiation in vitro, but it does not afford any in vivo radioprotective effect in lethally irradiated C3H mice.

Nuclear factor-kappaB activation correlates with better prognosis and Akt activation in human gastric cancer.

PURPOSE: Because the biological significance of constitutive nuclear factor-kappaB (NF-kappaB) activation in human gastric cancer is unclear, we undertook this study to clarify the regulatory mechanism of NF-kappaB activation and its clinical significance. EXPERIMENTAL DESIGN: Immunohistochemistry for NF-kappaB/RelA was done on 290 human gastric carcinoma specimens placed on tissue array slides. The correlations between NF-kappaB activation and clinicopathologic features, prognosis, Akt activation, tumor suppressor gene expression, or Bcl-2 expression were analyzed. We also did luciferase reporter assay, Western blot analysis, and reverse transcription-PCR using the SNU-216 human gastric cancer cell line transduced with retroviral vectors containing constitutively active Akt or the NF-kappaB repressor mutant of IkappaBalpha. RESULTS: Nuclear expression of RelA was found in 18% of the gastric carcinomas and was higher in early-stage pathologic tumor-node-metastasis (P = 0.019). A negative correlation was observed between NF-kappaB activation and lymphatic invasion (P = 0.034) and a positive correlation between NF-kappaB activation and overall survival rate of gastric cancer patients (P = 0.0228). In addition, NF-kappaB activation was positively correlated with pAkt (P = 0.047), p16 (P = 0.004), adenomatous polyposis coli (P < 0.001), Smad4 (P = 0.002), and kangai 1 (P < 0.001) expression. An in vitro study showed that NF-kappaB activity in gastric cancer cells is controlled by and controls Akt. CONCLUSIONS: NF-kappaB activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis and Akt activation. These findings suggest that NF-kappaB activation is a valuable prognostic variable in gastric carcinoma.

Gamma-irradiation and doxorubicin treatment of normal human cells cause cell cycle arrest via different pathways.

Ionizing radiation and doxorubicin both produce oxidative damage and double-strand breaks in DNA. Double-strand breaks and oxidative damage are highly toxic and cause cell cycle arrest, provoking DNA repair and apoptosis in cancer cell lines. To investigate the response of normal human cells to agents causing oxidative damage, we monitored alterations in gene expression in F65 normal human fibroblasts. Treatment with g-irradiation and doxorubicin altered the expression of 23 and 68 known genes, respectively, with no genes in common. Both agents altered the expression of genes involved in cell cycle arrest, and arrested the treated cells in G2/M phase 12 h after treatment. 24 h after g-irradiation, the percentage of G1 cells increased, whereas after doxorubicin treatment the percentage of G2/M cells remained constant for 24 h. Our results suggest that F65 cells respond differently to g-irradiation- and doxorubicin-induced DNA damage, probably using entirely different biochemical pathways.

Histopathological changes of the liver after praziquantel treatment in Clonorchis sinensis infected rabbits.

The rabbits were infected with Clnorchis sinensis and were treated with praziquantel at the dose of 50 mg/kg x 2 x 2 days afer 1, 2, 4, 8 weeks and 7 months from the infection. Their livers were observed histopathologically 1, 4 and 12 weeks after treatment. The findings are summarized as below: The changes of the liver in control rabbits were relatively mild until 2 weeks after infection. However, widening and thickening of bile ducts, proliferation of biliary epithelium and periductal fibrosis were moderate after 4 weeks from infection and those changes were severe after 8 weeks and 7months. Goblet cell metaplasia was found after 8 weeks from infection. The mild changes of 2-week infection group were completely recovered by 4 weeks after the treatment. In the groups of 4 or more weeks after infection, the changes of bile ducts became milder in the degree after the treatment, but were still found 12 weeks after the treatment. As the infection duration was passed, more severe changes were observed after the treatment. In this context, it is concluded that the liver changes of acute clonorchiasis in the early two weeks are reversible by treatment while chronic biliary epithelial changes are irreversible. Therefore, early treatment should be recommended as possible to minimize the remaining histopathological changes of liver in clonorchiasis.