Kenkichi Sugimoto

Cloning and characterization of amphibian cold inducible RNA-binding protein

Gene expression of cold inducible RNA-binding protein (CIRP) was examined in the frog. In Xenopus laevis, expression of CIRP (XCIRP) was observed in both brain and liver at 24 degrees C. Circadian expression of XCIRP was observed in brain. Expression of XCIRP in brain was induced by cold treatment and gradually decreased to the control level at 24 degrees C, but no significant changes were observed in liver. Employing the sequence of murine CIRP, bullfrog (Rana catesbeiana) CIRP gene was cloned. The bullfrog CIRP gene, designated BFCIRP, was 706 bp in length and encoded a putative protein of 164 amino acid residues. The deduced protein contained one consensus sequence of RNA-binding domain (CS-RBD) and a glycine rich domain (GRD). The amino acid sequence of BFCIRP was 78.4% identical to XCIRP. Expression of BFCIRP in brain was stronger in winter than that in summer. These findings suggest that BFCIRP expression in brain may link to hibernation.

Age-related decrease in the number of hemopoietic stem cells and progenitors in senescence accelerated mice

Senescence accelerated mice (SAM-P) were used for the study of the possible aging of hemopoiesis. The number of peripheral leukocytes decreased significantly with age, whereas hematocrit showed only a slight decrease. Although the number of total nucleated cells in the bone marrow increased, the number of hemopoietic stem cells (CFU-S) as well as that of granulocyte-macrophage colony forming cells (GM-CFC) showed a decrease in old mice. A significant decrease in the number of GM-CFC was observed in the spleen of old SAM-P mice, whereas no decrease was found in the number of CFU-S. Such a profound reduction of the recruitment of GM-CFC from CFU-S in the spleen together with a reduction of bone marrow hemopoiesis may be responsible for the decrease in the number of peripheral leukocytes in the old mice. SAM-P mice could provide a good model for the study of the aging of hemopoietic system.

Expression of macro non-coding RNAs Meg8 and Irm in mouse embryonic development

Non-coding RNAs (ncRNAs) Meg8 and Irm were previously identified as alternatively splicing isoforms of Rian gene. Ascertaining ncRNAs spatiotemporal expression patterns is crucial for understanding the physiological roles of ncRNAs during tissue and organ development. In this study in mouse embryos, we focused on the developmental regulation expression of imprinted macro ncRNAs, Meg8 and Irm by using in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). The in situ hybridization results showed that Meg8 and Irm were expressed in the developing brain at embryonic day 10.5 (E10.5) and E11.5, while Irm expression signals were strikingly detected in the somite, where Meg8 expression signals were undetectable. By E15.5, they were expressed in brain, tongue, liver, lung and neuroendocrine tissues, while Irm displayed more restricted expression in tongue and skeletal muscle than Meg8. Furthermore, quantitative analysis confirmed that they were highly expressed in tongue and brain at E12.5, E15.5 and E18.5. These results indicated that Meg8 and Irm might be coordinately expressed and functionally correlated in diverse of organs. Notably, Irm was more closely associated with morphogenesis of skeletal muscle in contrast to Meg8 during embryonic development.

Cold stress and light signals induce the expression of cold-inducible RNA binding protein (cirp) in the brain and eye of the Japanese treefrog (Hyla japonica).

Hibernation is an important physiological animal behavior. However, the molecular mechanism by which hibernation is regulated remains unknown. The Japanese treefrog (Hyla japonica) usually hibernates in the winter. Since this treefrog is an ectothermic animal, its hibernation is thought to be linked to the environmental temperature. In murine cells, gene expression for the cold-inducible RNA binding protein (cirp) is induced simply by cold stress. Therefore, it was hypothesized that the treefrog would also have increased expression of cirp during the hibernation season. In this report, we describe the cloning of the treefrog cirp gene and a quantitative analysis of its expression with real-time PCR. Like its homologs, treefrog cirp was found to be expressed in response to a cold stress, and its transcript was detectable in the brain, eye and ovary. Furthermore, we found that light signals could also induce the expression of cirp, and the total amount of cirp expression in both the brain and eye was significantly higher in December than in July. These results suggest that the expression of cirp in this treefrog is physiologically induced by environmental factors, such as cold stress or light signals.

Induction of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Granulocyte Colony-Stimulating Factor (G-CSF) Expression in Bone Marrow and Fractionated Marrow Cell Populations by Interleukin 3 (IL-3): IL-3-Mediated Positive Feedback Mechanisms of Granulopoiesis

Interleukin 3 (IL-3) induces proliferation and differentiation of mast cell progenitors in vitro, whereas it induces granulocytosis in vivo. In this paper, a positive feedback mechanism of granulopoiesis was studied in order to elucidate the granulocytosis induced by IL-3 in mouse. IL-3 induced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in total bone marrow cells and a marrow adherent cell population. In fractionated marrow cell populations, a different expression pattern of induction of IL-3 stimulation was observed; GM-CSF was expressed in macrophages and fraction 1 (P < 1.061) and 2(1.061 < P 1.074) of bone marrow cells fractionated by equilibrium density centrifugation, G-CSF was expressed in macrophages and fraction 2 and 3 (1.074 < P < 1.097), and interleukin 6 (IL-6) in macrophages and fraction 1 to 3. These results indicate a hierarchical regulation of cytokine production and the existence of a positive feedback mechanism in granulopoiesis. IL-6, induced by IL-3, stimulates stem cells into cycle and induces stem cells to respond to IL-3. The stem cells differentiate to granulocyte-macrophage colony-forming cells by the combined effect of IL-3 and IL-6. IL-3 also induces GM- and G-CSF expression which in turn makes granulocyte-macrophage colony-forming cells differentiate to granulocytes. These factors organize a cytokine network in granulopoiesis.

Age-related decreases in the reconstituting ability of hemopoietic cells and the ability of hemopoietic microenvironment to support hemopoietic reconstitution in senescence accelerated (SAM-P) mice

The effect of the aging process on the hemopoietic system in senescence-accelerated (SAM-P) mice with respect to the reconstituting ability of hemopoietic cells and the ability of the microenvironment to support hemopoietic reconstitution was investigated by bone marrow transplantation (reconstitution assay). When the bone marrow cells, obtained from young or old mice, were transplanted to lethally irradiated young SAM-P mice no difference in the reconstituting pattern of femoral spleen colony-forming units (CFU-S), splenic CFU-S and splenic granulocyte macrophage colony-forming units (CFU-GM) was observed between the mice transplanted with young and old donor cells. However, the reconstitution of femoral CFU-GM in mice transplanted with old donor cells was delayed compared to that in mice transplanted with young donor cells. Moreover, the recovery of WBC in mice transplanted with old donor cells was noticeably delayed. When the bone marrow cells obtained from young mice were transplanted to young or old recipient mice, no difference in the reconstituting pattern of femoral CFU-S and CFU-GM as well as splenic CFU-S and CPU-GM was observed between young and old recipient mice. However, the recovery of WBC in old recipient mice was noticeably delayed. These data indicate that the functions of both the hemopoietic cells and the hemopoietic microenvironment deteriorated with age in SAM-P mice.

ESTABLISHMENT OF A SARCOMA CELL LINE; MS-K, EXPRESSING KI-RAS PROTO-ONCOGENE PRODUCT FROM MOUSE BONE MARROW STROMAL CELLS

A sarcoma cell line, MS-K, was established from a long-term culture of mouse bone marrow stromal cells. When inoculated into syngeneic 3HC/HeNS1c mice, the cells formed large necrosis-free tumors, but there were no apparent changes in hematological features or in general conditions of tumor-bearing mice. The tumor had a fibroblastic appearance, was well vasculated and differentiated into adipocytes at the peripheral region. Immunohistochemical studies revealed that the cells were positive for vimentin and S-100 protein, indicating that the cells were of lipoblast origin. A significant amount of fat-deposition was induced in the cytoplasm of the cells when MS-K cells were cultured in the presence of hydrocortisone and insulin. Antibody-staining for oncogene products showed that the cells were negative for c-fos but strongly positive for Ki-ras. MS-K cells did not adhere hemopoietic stem cells or support their proliferation, which contrasts with previously established MS-1, which is a hemopoietic-supportive and stem cell-adherent lipoblast cell line. These properties of MS-K and MS-1 should be useful in the identification of the surface structures for stem cell anchorage.

Hierarchical regulation of interleukin production : induction of interleukin 6(IL-6)production from bone marrow cells and marrow stromal cells by interleukin 3 (IL-3)

IL-3 stimulated the production of IL-6 from a bone marrow-adherent cell population, macrophages and from hemopoietic supportive stromal cell lines. It also induced IL-6 production from a stem cell-enriched population of bone marrow cells which did not produce IL-6 without stimulation. In contrast, stimulation with IL-6 of all the cell populations studied in the present experiments did not induce IL-3 production. These results indicate a hierarchical network in the regulation of interleukin production, and existence of a positive feedback mechanism; IL-3 induces IL-6 production which in turn stimulates stem cells into cycle and induces stem cells to respond to IL-3.

MiR-1188 at the imprinted Dlk1-Dio3 domain acts as a tumor suppressor in hepatoma cells

MiR-1188 at the imprinted Dlk1-Dio3 domain is a novel player in hepatocellular carcinoma. MiR-1188 expression was found to be decreased in hepatoma cells, and, when overexpressed, miR-1188 inhibited Bcl2 and Sp1 expression, suppressed cell proliferation and migration, promoted apoptosis in vitro, and inhibited tumor growth in vivo.

Induction of the Expression of SCF in Mouse by Lethal Irradiation

Abstract To clarify what kinds of cytokines are actually contributing to proliferation of hemopoietic stem cells in vivo after lethal irradiation, we have investigated the expression of some cytokines by RT-PCR method. Above all, expression of the SCF was increased significantly in the bone marrow cells soon after lethal irradiation in both the Sca-1 (+) bone marrow cells injected and non-injected mice. The day 6 serum from the lethally irradiated mice could support the proliferation of the Sca-1 (+) bone marrow cells, even though the serum from normal mice could not. The quantification analyses have revealed the increase of the amounts of IL-6 and flt3-ligand in their serum, but not significant increase of the amount of SCF. Precise PCR analysis has revealed that the cell surface associated form of SCF was significantly induced in the bone marrow after lethal irradiation. These data indicate that the cell surface form of SCF mainly promotes the proliferation of hemopoietic stem cells with some soluble cytokines under sever lack of hemopoietic stem cells in vivo caused by lethal irradiation and also suggest the importance of direct cell-to-cell interaction on proliferation of hematopoietic stem cells in vivo.