129I/127I and Δ14C records in a modern coral from Rowley Shoals off northwestern Australia reflect the 20th-century human nuclear activities and ocean/atmosphere circulations.

Radionuclides produced by 20th-century human nuclear activities from 1945 (e.g., atmospheric nuclear explosions and nuclear-fuel reprocessing) made significant impacts on earth's surface environments. Long-lived shallow-water corals living in tropical/subtropical seas incorporate the anthropogenically-produced radionuclides, including 129I and 14C, into their skeletons, and provide time series records of the impacts of nuclear activities. Here, we present 129I/127I and Δ14C time series records of an annually-banded modern coral skeleton from Rowley Shoals, off the northwestern coast of Australia, in the far eastern Indian Ocean. The 129I/127I and Δ14C records, covering the period 1930s-1990s, exhibit distinct increases caused by the nuclear activities, and their increasing profiles are clearly different from each other. The first distinct 129I/127I increase occurs from 1955 to 1959, followed by a decrease in 1960-1963. The increase is probably due to US atmospheric nuclear explosions in Bikini and Eniwetok Atolls in 1954, 1956 and 1958. The 129I produced in those nuclear tests would be transported by the North Equatorial Current, a portion of which passes through the Indonesian Throughflow and then reaches Rowley Shoals. This initial increase from 1955 is, however, absent in the Δ14C record, which shows a distinct increase from 1959 and its peak around the mid-1970s, followed by a gradual decrease. This absence and the 4-year-delayed Δ14C increase are likely due to dilution of explosion-produced 14C with natural carbon (by seawater mixing and air-sea gas exchange) being much more intense than that of explosion-produced 129I with natural iodine (by the same processes), suggesting that the 129I/127I ratio is a more conservative anthropogenic tracer in surface ocean waters, as compared to Δ14C. The second 129I/127I increase is contemporaneous with a rapid Δ14C increase during 1964-1967, followed by a rapid 129I/127I decrease in 1968-1969; the increases can be ascribed to very large atmospheric nuclear explosions conducted in the former Soviet Union in 1961-1962. The third 129I/127I increase appears between 1969/1970 and 1992, which can be attributed to airborne 129I released from nuclear-fuel reprocessing facilities in Europe, the former Soviet Union and the US. The coral 129I/127I and Δ14C time series records, combined with previous studies, enhance our understanding of the behavior of anthropogenic 129I and 14C in the global ocean and atmosphere.

Identification of osteoblast stimulating factor 5 as a negative regulator in the B-lymphopoietic niche.

Recent studies have revealed the crucial role of the niche which supports B-lymphocyte differentiation from hematopoietic stem cells. In this study, we aimed to identify a novel regulator of B lymphopoiesis secreted in the specific niche using the signal sequence trap method. Among the identified proteins from MS5 stromal cells, expression of pleiotrophin, placental proliferin 2, and osteoblast stimulating factor 5 (OSF-5) was dominantly high in several stromal cell lines. We found that OSF-5 suppressed early B lymphopoiesis in transgenic mice producing the target protein. The number of pre-B and immature B cells was reduced by more than half compared with control in the transgenic mice. In vitro studies showed that a secreted variant of OSF-5 inhibited the proliferation and colony formation of pre-B cells, whereas cell-intrinsic form had no influence on B lymphopoiesis. The main components of the B-lymphopoietic niche, osteoblasts in mice and mesenchymal cells in humans, are primary producers of OSF-5. These results define a novel mechanism of B lymphopoiesis in bone marrow. In the specific niche, B-lymphocyte differentiation is fine-tuned by negative regulators as well as supportive factors.

Signal-transducing adaptor protein-2 regulates macrophage migration into inflammatory sites during dextran sodium sulfate induced colitis.

Signal-transducing adaptor protein-2 (STAP-2) was cloned as a c-fms/M-CSF receptor interacting protein. STAP-2 is an adaptor protein carrying pleckstrin homology and Src homology 2 like domains, as well as a YXXQ motif. STAP-2 has been indicated to have an ability to bind and modulate a variety of signaling and transcriptional molecules. Especially, our previous in vitro studies showed that STAP-2 is crucial for immune and/or inflammatory responses. Here, we have investigated the role of STAP-2 in intestinal inflammation in vivo. The disruption of STAP-2 attenuates dextran sodium sulfate induced colitis via inhibition of macrophage recruitment. To study whether hematopoietic or epithelial cell derived STAP-2 is required for this phenomenon, we generated BM chimeric mice. STAP-2-deficient macrophages impair the ability of CXCL12-induced migration. Intriguingly, STAP-2 also regulates production of proinflammatory chemokines and cytokines such as CXCL1 and TNF-α from intestinal epithelial cells. Therefore, STAP-2 has a potential to regulate plural molecular events during pathological inflammatory responses. Furthermore, our findings not only indicate that STAP-2 is important in regulating intestinal inflammation, but also provide new insights toward the development of novel therapeutic approaches.

The anti-apoptotic gene Anamorsin is essential for both autonomous and extrinsic regulation of murine fetal liver hematopoiesis.

Anamorsin (AM) is an antiapoptotic molecule that confers factor-independent survival on hematopoietic cells. AM-deficient (AM(-/-)) mice are embryonic lethal because of a defect in definitive hematopoiesis; however, the significance of AM in embryonic hematopoiesis remains unknown. This study characterized the hematopoietic defects in AM(-/-) fetal livers. The AM(-/-) fetal liver displayed significantly reduced numbers of c-Kit(+)Sca-1(+)Lin(-) (KSL) cells. An in vitro colony-forming unit assay showed that fetal liver cells isolated from AM(-/-) embryos gave rise to fewer colonies in all cell types. The reconstitution activity in AM(-/-) hematopoietic stem cells (HSCs) was markedly reduced in all lineages. Furthermore, the limiting dilution assay revealed that the number of fetal liver HSCs was reduced because of AM deficiency. Retrovirus-mediated AM expression rescued the defective hematopoietic colony-forming activities of AM(-/-) KSL cells. We also investigated the effects of AM deficiency on fetal liver stromal cells, which support hematopoiesis. Interestingly, primary stromal cell cultures from wild type fetal liver supported the growth of AM(-/-) KSL cells, but stromal cultures from AM(-/-) fetal liver provided little support of wild type KSL cell growth. These results demonstrated that AM was essential for both autonomous and extrinsic regulation of fetal liver hematopoiesis. This study provided new insight into the molecular regulation of hematopoiesis.

The endothelial antigen ESAM monitors hematopoietic stem cell status between quiescence and self-renewal.

Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAM(hi) HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAM(hi) HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.

Signal-transducing adaptor protein-2 modulates Fas-mediated T cell apoptosis by interacting with caspase-8.

We found that an adaptor protein, signal-transducing adaptor protein (STAP)-2, is a new member of the Fas-death-inducing signaling complex and participates in activation-induced cell death in T cells. STAP-2 enhanced Fas-mediated apoptosis and caspase-8 aggregation and activation in Jurkat T cells. Importantly, STAP-2 directly interacted with caspase-8 and Fas, resulting in enhanced interactions between caspase-8 and FADD in the Fas-death-inducing signaling complex. Moreover, STAP-2 protein has a consensus caspase-8 cleavage sequence, VEAD, in its C-terminal domain, and processing of STAP-2 by caspase-8 was crucial for Fas-induced apoptosis. Physiologic roles of STAP-2 were confirmed by observations that STAP-2-deficient mice displayed impaired activation-induced cell death and superantigen-induced T cell depletion. Therefore, STAP-2 is a novel participant in the regulation of T cell apoptosis after stimulation.

NAD-dependent histone deacetylase, SIRT1, plays essential roles in the maintenance of hematopoietic stem cells.

Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-Kit(high)Sca-1(+)Lineage(-) (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.

Predictability of the response to tyrosine kinase inhibitors via in vitro analysis of Bcr-Abl phosphorylation.

It would be of great value to predict the efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of individual CML patients. We propose an immunoblot system for detecting the phosphorylation of Crkl, a major target of Bcr-Abl, in blood samples after in vitro incubation with TKIs. When the remaining phosphorylated Crkl after treatment with imatinib was evaluated as the "residual index (RI)", high values were found in accordance with imatinib resistance. Moreover, RI reflected the outcome of imatinib- as well as second generation TKIs with a high sensitivity and specificity. Therefore, this system should be useful in the selection of TKIs.

Identification of functional domains and novel binding partners of STIM proteins.

With a signal trap method, we previously identified stromal interaction molecule (STIM: originally named as SIM) as a protein, which has a signal peptide in 1996. However, recent works have accumulated evidences that STIM1 and STIM2 reside in endoplasmic reticulum (ER) and that both mainly sense ER Ca(2+) depletion, which plays an essential role in store operated calcium entry. In the present study, we extensively analyzed the domain functions and associated molecules of STIMs. A STIM1 mutant lacking the coiled-coil domains was massively expressed on the cell surface while mutants with the coiled-coil domains localized in ER. In addition, STIM1 mutants with the coiled-coil domains showed a longer half-life of proteins than those without them. These results are likely to indicate that the coiled-coil domains of STIM1 are essential for its ER-retention and its stability. Furthermore, we tried to comprehensively identify STIM1-associated molecules with mass spectrometry analysis of co-immunoprecipitated proteins for STIM1. This screening clarified that both STIM1 and STIM2 have a capacity to bind to a chaperone, calnexin as well as two protein-transporters, exportin1 and transportin1. Of importance, our result that glycosylation on STIM1 was not required for the association between STIM1 and calnexin seems to indicate that calnexin might function on STIM1 beyond a chaperone protein. Further information concerning regulatory mechanisms for STIM proteins including the data shown here will provide a model of Ca(2+) control as well as a useful strategy to develop therapeutic drugs for intracellular Ca(2+)-related diseases including inflammation and allergy.

Regulation of soluble epoxide hydrolase (sEH) in mice with diabetes: high glucose suppresses sEH expression.

Soluble epoxide hydrolase (sEH) is a xenobiotic-metabolizing enzyme that metabolizes epoxides to produce vicinal diols. Diabetes is a common pathological condition which effects drug metabolism. This study, investigates changes in the levels of sEH in mice with diabetes induced by streptozotocin (STZ). Diabetes reduced the amount of sEH protein in the liver and insulin restored the level of protein. The kidneys are a target of diabetes. Diabetes significantly decreased levels of sEH protein and also mRNA. The distribution of sEH in the kidney was studied with immunostaining. There was distinct staining in the proximal tubules but not in the glomerulus or other regions. Diabetes is characterized by high glucose concentrations that lead to increased production of reactive oxygen species (ROS). High glucose suppressed sEH mRNA and protein expression in Hep3B cells. NADPH oxidase is the main source of ROS generation in high glucose condition. The NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPIC), inhibited decrease in sEH expression at high glucose and hydrogen peroxide suppressed sEH expression. These findings indicate that diabetes reduces sEH expression by inducing ROS and may have important effects on the metabolism of xenobiotics and endogenous substrates of sEH.