Differential effects of low- and high-dose X-rays on N-ethyl-N-nitrosourea-induced mutagenesis in thymocytes of B6C3F1 gpt-delta mice

Carcinogenesis in humans is thought to result from exposure to numerous environmental factors. Little is known, however, about how these different factors work in combination to cause cancer. Because thymic lymphoma is a good model of research for combined exposure, we examined the occurrence of mutations in thymic DNA following exposure of B6C3F1 gpt-delta mice to both ionizing radiation and N-ethyl-N-nitrosourea (ENU). Mice were exposed weekly to whole body X-irradiation (0.2 or 1.0 Gy), ENU (200 ppm) in the drinking water, or X-irradiation followed by ENU treatment. Thereafter, genomic DNA was prepared from the thymus and the number and types of mutations in the reporter transgene gpt was determined. ENU exposure alone increased mutant frequency by 10-fold compared to untreated controls and over 80% of mutants had expanded clonally. X-irradiation alone, at either low or high dose, unexpectedly, reduced mutant frequency. Combined exposure to 0.2 Gy X-rays with ENU dramatically decreased mutant frequency, specifically G:C to A:T and A:T to T:A mutations, compared to ENU treatment alone. In contrast, 1.0 Gy X-rays enhanced mutant frequency by about 30-fold and appeared to accelerate clonal expansion of mutated cells. In conclusion, repeated irradiation with 0.2 Gy X-rays not only reduced background mutation levels, but also suppressed ENU-induced mutations and clonal expansion. In contrast, 1.0 Gy irradiation in combination with ENU accelerated clonal expansion of mutated cells. These results indicate that the mode of the combined mutagenic effect is dose dependent.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) co-localizes with activity-dependent neuroprotective protein (ADNP) in the mouse brains

The crustacean hyperglycemic hormone is the most abundant neuropeptide present in the eyestalk of Crustacea and its main role is to control the glucose level in the hemolymph. Our study was aimed at assessing the importance of C-terminal amidation for its biological activity. Two recombinant peptides were produced, Asl-rcHH-Gly with a free carboxyl terminus and Asl-rcHH-amide with an amidated C-terminus. Homologous bioassays performed on the astacid crayfish Astacus leptodactylus showed that the amidated peptide had a stronger hyperglycemic effect compared to the non-amidated peptide. To assess the relevance of amidation also in other decapods and how much the differences in the cHH amino acid sequence can affect the functionality of the peptides, we carried out heterologous bioassays on the cambarid Procambarus clarkii and palaemonid Palaemon elegans. The Asl-rcHH-amide elicited a good response in P. clarkii and in P. elegans. The injection of Asl-rcHH-Gly evoked a weak response in both species. These results prove the importance of C-terminal amidation for the biological activity of cHH in crayfish as well as the role of the peptide primary sequence for the species-specificity hormone-receptor recognition.     

Molecular assembly of Zn porphyrin complexes using synthetic light-harvesting model polypeptides

Synthetic single α-helix hydrophobic polypeptides, which have similar amino acid sequences to the hydrophobic core in the native light-harvesting 1-β polypeptide from Rhodobacter sphaeroides, formed Zn porphyrin complexes on a gold electrode, as well as in n-octyl-β-glucoside micelles: this process is dependent on the structure of the pigments and the polypeptides. Interestingly, an enhanced photoelectric current was observed when Zn mesoporphyrin monomer complexed with the synthetic light-harvesting model polypeptide in an α-helical configuration was assembled with a defined orientation onto the electrode. Analog of these light-harvesting model complexes are also useful in providing insights into the effect of polypeptide structure on the formation of light-harvesting complexes on and off electrodes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lipocalin 2-mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells

Lipocalin 2 (LCN2), a secreted protein of the lipocalin family, induces apoptosis in some types of cells and inhibits bacterial growth by sequestration of the iron-laden bacterial siderophore. We have recently reported that LCN2 inhibits the production of red blood cells in the mouse. Here we analyzed the role of LCN2 in human hematopoiesis. Expression of LCN2 was observed not only in mature cells such as those of the granulocyte/macrophage and erythroid lineages but also in hematopoietic stem/progenitor cells. We also examined expression of two candidate receptors for LCN2, brain type organic cation transporter (BOCT) and megalin, in various cell types. BOCT showed relatively high levels of expression in erythroid and hematopoietic stem/progenitor cells but lower levels in granulocyte/macrophage and T lymphoid cells. Megalin was expressed at high levels in T lymphoid and erythroid cells but at lower levels in granulocyte/macrophage lineage cells. LCN2 suppressed the growth of erythroid and monocyte/macrophage lineages in vitro, but did not have this effect on cells of other lineages. In addition, immature hematopoietic stem/progenitor cells were not sensitive to LCN2. These results demonstrate a lineage-specific role for LCN2 in human hematopoiesis that is reminiscent of its effects upon mouse hematopoiesis and strongly suggest an important in vivo function of LCN2 in the regulation of human hematopoiesis.     

Aberrant genetic control of invariant TCR-bearing NKT cell function in New Zealand mouse strains: possible involvement in systemic lupus erythematosus pathogenesis

Both suppressive and promoting roles of NKT cells have been reported in the pathogenesis of systemic lupus erythematosus (SLE). Herein, we found that although New Zealand mice have normal frequencies of NKT cells, their in vitro potential to produce IL-4 and IFN-gamma in response to alpha-galactosylceramide was remarkably impaired in New Zealand Black (NZB) mice prone to mild SLE, while production was highly up-regulated in nonautoimmune New Zealand White (NZW) mice and at intermediate levels in (NZB x NZW)F(1) mice, which are prone to severe SLE. Because this aberration is evident in young mice before disease onset, genetic mechanisms are thought to be involved. Genome-wide quantitative trait locus analysis and association studies revealed that a locus linked to D11Mit14 on chromosome 11 may be involved in the difference in cytokine-producing potential between NZB and NZW NKT cells. Additionally, (NZB x NZW)F(1) x NZB backcross progeny with the NZW genotype for D11Mit14 showed significantly increased frequencies of age-associated SLE phenotypes, such as high serum levels of IgG, IgG anti-DNA Abs, and lupus nephritis. In coculture studies, alpha-galactosylceramide-stimulated NKT cells from NZW and (NZB x NZW)F(1) mice, but not from NZB mice, showed significantly enhanced Ig synthesis by B cells. These findings suggest that the D11Mit14-linked NZW locus may contribute to the development of SLE in (NZB x NZW)F(1) mice through a mechanism that up-regulates NKT cell function. Thus, this NZW allele may be a candidate of the NZW modifiers that act to promote (NZB x NZW)F(1) disease.     

Possible involvement of phosphatidylinositol 3-kinase, but not protein kinase B or glycogen synthase kinase 3beta, in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica

It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3beta (GSK3beta) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3beta are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3beta, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3beta is a component of the PI3K signal transduction pathway.     

Atherosclerotic plaque imaging using phase-contrast X-ray computed tomography

Reliable, noninvasive imaging modalities to characterize plaque components are clinically desirable for detecting unstable coronary plaques, which cause acute coronary syndrome. Although recent clinical developments in computed tomography (CT) have enabled the visualization of luminal narrowing and calcified plaques in coronary arteries, the identification of noncalcified plaque components remains difficult. Phase-contrast X-ray CT imaging has great potentials to reveal the structures inside biological soft tissues, because its sensitivity to light elements is almost 1,000 times greater than that of absorption-contrast X-ray imaging. Moreover, a specific mass density of tissue can be estimated using phase-contrast X-ray CT. Ex vivo phase-contrast X-ray CT was performed using a synchrotron radiation source (SPring-8, Japan) to investigate atherosclerotic plaque components of apolipoprotein E-deficient mice. Samples were also histologically analyzed. Phase-contrast X-ray CT at a spatial resolution of 10-20 mum revealed atherosclerotic plaque components easily, and thin fibrous caps were detected. The specific mass densities of these plaque components were quantitatively estimated. The mass density of lipid area was significantly lower (1.011 +/- 0.001766 g/ml) than that of smooth muscle area or collagen area (1.057 +/- 0.001407 and 1.080 +/- 0.001794 g/ml, respectively). Moreover, the three-dimensional assessment of plaques could provide their anatomical information. Phase-contrast X-ray CT can estimate the tissue mass density of atherosclerotic plaques and detect lipid-rich areas. It can be a promising noninvasive technique for the investigation of plaque components and detection of unstable coronary plaques.     

Crystal structure of human D-amino acid oxidase: context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring

In the brain, the extensively studied FAD-dependent enzyme D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent activator of N-methyl-D-aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the pathophysiology of schizophrenia. Indeed, its potential clinical importance highlights the need for structural and functional analyses of human DAO. We recently succeeded in purifying human DAO, and found that it weakly binds FAD and shows a significant slower rate of flavin reduction compared with porcine DAO. However, the molecular basis for the different kinetic features remains unclear because the active site of human DAO was considered to be virtually identical to that of porcine DAO, as would be expected from the 85% sequence identity. To address this issue, we determined the crystal structure of human DAO in complex with a competitive inhibitor benzoate, at a resolution of 2.5 Angstrom. The overall dimeric structure of human DAO is similar to porcine DAO, and the catalytic residues are fully conserved at the re-face of the flavin ring. However, at the si-face of the flavin ring, despite the strict sequence identity, a hydrophobic stretch (residues 47-51, VAAGL) exists in a significantly different conformation compared with both of the independently determined porcine DAO-benzoate structures. This suggests that a context-dependent conformational variability of the hydrophobic stretch accounts for the low affinity for FAD as well as the slower rate of flavin reduction, thus highlighting the unique features of the human enzyme.