Solid cancer incidence in atomic bomb survivors: 1958-1998

This is the second general report on radiation effects on the incidence of solid cancers (cancers other than malignancies of the blood or blood-forming organs) among members of the Life Span Study (LSS) cohort of Hiroshima and Nagasaki atomic bomb survivors. The analyses were based on 17,448 first primary cancers (including non-melanoma skin cancer) diagnosed from 1958 through 1998 among 105,427 cohort members with individual dose estimates who were alive and not known to have had cancer prior to 1958. Radiation-associated relative risks and excess rates were considered for all solid cancers as a group, for 19 specific cancer sites or groups of sites, and for five histology groups. Poisson regression methods were used to investigate the magnitude of the radiation-associated risks, the shape of the dose response, how these risks vary with gender, age at exposure, and attained age, and the evidence for inter-site variation in the levels and patterns of the excess risk. For all solid cancers as a group, it was estimated that about 850 (about 11%) of the cases among cohort members with colon doses in excess of 0.005 Gy were associated with atomic bomb radiation exposure. The data were consistent with a linear dose response over the 0- to 2-Gy range, while there was some flattening of the dose response at higher doses. Furthermore, there is a statistically significant dose response when analyses were limited to cohort members with doses of 0.15 Gy or less. The excess risks for all solid cancers as a group and many individual sites exhibit significant variation with gender, attained age, and age at exposure. It was estimated that, at age 70 after exposure at age 30, solid cancer rates increase by about 35% per Gy (90% CI 28%; 43%) for men and 58% per Gy (43%; 69%) for women. For all solid cancers as a group, the excess relative risk (ERR per Gy) decreases by about 17% per decade increase in age at exposure (90% CI 7%; 25%) after allowing for attained-age effects, while the ERR decreased in proportion to attained age to the power 1.65 (90% CI 2.1; 1.2) after allowing for age at exposure. Despite the decline in the ERR with attained age, excess absolute rates appeared to increase throughout the study period, providing further evidence that radiation-associated increases in cancer rates persist throughout life regardless of age at exposure. For all solid cancers as a group, women had somewhat higher excess absolute rates than men (F:M ratio 1.4; 90% CI 1.1; 1.8), but this difference disappears when the analysis was restricted to non-gender-specific cancers. Significant radiation-associated increases in risk were seen for most sites, including oral cavity, esophagus, stomach, colon, liver, lung, non-melanoma skin, breast, ovary, bladder, nervous system and thyroid. Although there was no indication of a statistically significant dose response for cancers of the pancreas, prostate and kidney, the excess relative risks for these sites were also consistent with that for all solid cancers as a group. Dose-response estimates for cancers of the rectum, gallbladder and uterus were not statistically significant, and there were suggestions that the risks for these sites may be lower than those for all solid cancers combined. However, there was emerging evidence from the present data that exposure as a child may increase risks of cancer of the body of the uterus. Elevated risks were seen for all of the five broadly classified histological groups considered, including squamous cell carcinoma, adenocarcinoma, other epithelial cancers, sarcomas and other non-epithelial cancers. Although the data were limited, there was a significant radiation-associated increase in the risk of cancer occurring in adolescence and young adulthood. In view of the persisting increase in solid cancer risks, the LSS should continue to provide important new information on radiation exposure and solid cancer risks for at least another 15 to 20 years.     

Regulation of glial development by cystatin C

Cystatin C (CysC) is an endogenous cysteine proteases inhibitor produced by mature astrocytes in the adult brain. Previously we isolated CysC as a factor activating the glial fibrillary acidic protein (GFAP) promoter, and showed that CysC is expressed in astrocyte progenitors during development. Here we show that protease inhibitor activity increased daily in conditioned medium, and that this activity was mainly a result of CysC released from primary cultured cells. Human CysC added to the culture medium of primary brain cells increased the number of GFAP-positive and nestin-positive cells. Human CysC also increased the number of neurospheres formed from embryonic brain, and thus it increases the number of neural stem/precursor cells in a manner similar to glycosylated rat CysC. The addition of a neutralizing antibody, on the other hand, greatly decreased the number of GFAP and glutamate aspartate transporter (GLAST)-positive astrocytes. This decrease was reversed by the addition of CysC but not by another cysteine protease inhibitor. Thus, the promotion of astrocyte development by CysC appears to be independent of its protease inhibitor activity. The antibody increased the number of oligodendrocytes and their precursors. Therefore, CysC modifies glial development in addition to its activity against neural stem/precursor cells.     

Critical role of the Fc receptor gamma-chain on APCs in the development of allergen-induced airway hyperresponsiveness and inflammation

The FcR common gamma-chain (FcRgamma) is an essential component of the receptors FcepsilonRI, FcgammaRI, and FcgammaRIII, which are expressed on many inflammatory cell types. The role of these receptors in the initiation or maintenance of allergic inflammation has not been well defined. FcRgamma-deficient (FcRgamma(-/-)) and control (wild-type (WT)) mice were sensitized and subsequently challenged with OVA. Following sensitization and challenge to OVA, FcRgamma-deficient (FcRgamma(-/-)) mice developed comparable levels of IgE and IgG1 as WT mice. However, numbers of eosinophils, levels of IL-5, IL-13, and eotaxin in bronchoalveolar lavage fluid, and mononuclear cell (MNC) proliferative responses to OVA were significantly reduced, as was airway hyperresponsiveness (AHR) to inhaled methacholine. Reconstitution of FcRgamma(-/-) mice with whole spleen MNC from WT mice before sensitization restored development of AHR and the numbers of eosinophils in bronchoalveolar lavage fluid; reconstitution after sensitization but before OVA challenge only partially restored these responses. These responses were also restored when FcRgamma(-/-) mice received T cell-depleted MNC, T and B cell-depleted MNC, or bone marrow-derived dendritic cells before sensitization from FcR(+/+) or FcgammaRIII-deficient but not FcRgamma(-/-) mice. The expression levels of FcgammaRIV on bone marrow-derived dendritic cells from FcR(+/+) mice were found to be low. These results demonstrate that expression of FcRgamma, most likely FcgammaRI, on APCs is important during the sensitization phase for the development of allergic airway inflammation and AHR.     

Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping

Somatic mutations of the fibroblast growth factor receptor 3 (FGFR3) gene were detected by peptide nucleic acid (PNA)-mediated real-time PCR clamping. Mutation was detected in negative control containing only wild-type DNA due to a misincorporation of dNTPs to PNA binding sites when the amount of template DNA was decreased to 1 ng. Thus, the amount of template DNA was critical determinant of the assay sensitivity in PNA-mediated PCR clamping. Assay conditions were optimized to detect FGFR3 mutations in exons 7, 10, and 15, at a concentration of more than 1% mutated DNA using 50 ng of genomic DNA as the template. Mutations were detected in 12 of 13 (92.3%) tumor tissues and 11 of 13 (84.6%) urine samples from patients with superficial bladder cancer, while no mutations were detected in tissues and/or urine samples from patients with muscle-invasive bladder cancer or chronic cystitis.     

Demonstration and characterization of manganese superoxide dismutase of Providencia alcalifaciens

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and play a role in the pathogenesis of certain invasive bacteria. In this study, we reported for the first time here that Providencia alcalifaciens, a member of the family Enterobacteriaceae, produces a superoxide dismutase (SOD) as a major protein in culture supernatants. This protein was purified by a series of column chromatographic separations. The N-terminal amino acid sequence of the protein was determined to be highly homologous to manganese superoxide dismutase of Escherichia coli or Salmonella reported. The gene (sodA) encoding for SOD of P. alcalifaciens was cloned and sequenced. The sodA-encoded protein has a molecular weight of about 23.5 kDa, and the DNA sequence of P. alcalifaciens sodA gene (627 bp) has about 83% identity to the E. coli SOD gene. We constructed a sodA deletion mutant and its complemented strain of P. alcalifaciens. In J774, a macrophage cell line, the sodA deletion mutant was more susceptible to killing by macrophages than the wildtype strain and its complemented strain. When we injected the mutant strain, its complemented strain and wildtype strain intraperitoneally into DDY strain mice, we found that the sodA deletion mutant proved significantly less virulent while the complemented strain recovered the virulence to the same level of wildtype strain of P. alcalifaciens. These results suggested that manganese superoxide dismutase plays an important role in intracellular survival of P. alcalifaciens.     

The effects of spacer sequences on silencing efficiency of plant RNAi vectors

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.     

Identification and characterization of VopT, a novel ADP-ribosyltransferase effector protein secreted via the Vibrio parahaemolyticus type III secretion system 2

Vibrio parahaemolyticus strain RIMD2210633 has two sets of genes encoding two separate type III secretion systems (T3SSs), called T3SS1 and T3SS2. T3SS2 has a role in enterotoxicity and is present only in Kanagawa phenomenon-positive strains, which are pathogenic to humans. Accordingly, T3SS2 is considered to be closely related to V. parahaemolyticus human pathogenicity. Despite this, the biological actions of T3SS2 and the identity of the effector protein(s) secreted by this system have not been well understood. Here we report that T3SS2 induces a cytotoxic effect in Caco-2 and HCT-8 cells. Moreover, it was revealed that VPA1327 (vopT), a gene encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. The VopT shows approximately 45% and 44% identity with the ADP-ribosyltransferase (ADPRT) domain of ExoT and ExoS, respectively, which are two T3SS-secreted effectors of Pseudomonas aeruginosa. T3SS2 was found to be necessary not only for the secretion, but also for the translocation of the VopT into host cells. We also demonstrate that VopT ADP-ribosylates Ras, a member of the low-molecular-weight G (LMWG) proteins both in vivo and in vitro. These results indicate that VopT is a novel ADPRT effector secreted via V. parahaemolyticus T3SS.     

Simultaneous Enantioseparation of Aldohexoses and Aldopentoses Derivatized With L‐Tryptophanamide by Reversed Phase HPLC Using Butylboronic Acid as a Complexation Reagent of Monosaccharides

Three aldohexoses, glucose, galactose, and mannose, and three aldopentoses, arabinose, xylose, and ribose, were derivatized with L‐tryptophanamide (L‐TrpNH₂) under alkaline conditions. Using a basic mobile phase (pH 9.2), the three aldohexoses or the three aldopentoses were simultaneously enantioseparated, respectively, but all the six monosaccharides could not be simultaneously enantioseparated. A large amount of nonreacted L‐TrpNH₂ was detected after the derivatized monosaccharides. In order to widen the separation window, a large portion of nonreacted L‐TrpNH₂ could be eliminated by liquid–liquid extraction with ethylacetate, and elution order of the derivatized monosaccharides and nonreacted L‐TrpNH₂ was found to be reversed using a neutral mobile phase. All of the six monosaccharides were simultaneously enantioseparated by reversed phase high‐performance liquid chromatography (HPLC) using InertSustainSwift C18 column (4.6 mm i.d. × 150 mm) and a mobile phase containing 180 mM phosphate buffer (pH 7.6), 1.5 mM butylboronic acid, and 5% acetonitrile at 40 °C. Nomenclature of D and L for monosaccharides is based on the configurations of the asymmetric C4 center for aldopentoses and C5 center for aldohexoses. It was found that the enantiomer elution order of these six monosaccharides and fucose in the proposed method conformed to be the absolute configuration of the C2 center. Chirality 27:417–421, 2015. © 2015 Wiley Periodicals, Inc.     

The FBXL10/KDM2B Scaffolding Protein Associates with Novel Polycomb Repressive Complex-1 to Regulate Adipogenesis

Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in a diet-induced model of obesity. Interestingly, inhibition of adipogenesis does not require the JmjC demethylase domain of FBXL10, but it does require the F-box and leucine-rich repeat domains, which we show recruit a noncanonical polycomb repressive complex 1 (PRC1) containing RING1B, SKP1, PCGF1, and BCOR. Knockdown of either RING1B or SKP1 prevented FBXL10-mediated repression of 3T3-L1 preadipocyte differentiation indicating that PRC1 formation mediates the inhibitory effect of FBXL10 on adipogenesis. Using ChIP-seq, we show that FBXL10 recruits RING1B to key specific genomic loci surrounding the key cell cycle and the adipogenic genes Cdk1, Uhrf1, Pparg1, and Pparg2 to repress adipogenesis. These results suggest that FBXL10 represses adipogenesis by targeting a noncanonical PRC1 complex to repress key genes (e.g. Pparg) that control conversion of pluripotent cells into the adipogenic lineage.