CD41+ extracellular vesicles produced by avian thrombocytes contain microRNAs.

Avians have thrombocytes in their blood circulation rather than mammalian platelets. However, many details of thrombocyte characteristics have not been determined. Here, chicken thrombocytes were isolated, and extracellular vesicle (EV) production was investigated. The thrombocyte-specific markers cd41 and cd61 were expressed in the yolk sac at 24 h. According to the embryonic developmental stage, the cd41-expressing tissues changed from the yolk sac to the bone marrow and spleen. Accordingly, the bone marrow and spleen were the main tissues producing thrombocytes in adult chickens. Avian thrombocytes were separated from adult spleen cells through a combination of discontinuous density gradient centrifugation, phagocytic cell removal, and fluorescence-activated cell sorting. Isolated thrombocytes produced CD41+ EVs (CD41+ EVs), and the CD41+ EVs also expressed CD9. Microarray analysis revealed that CD41+ EVs contain many microRNAs. Macrophage lines (RAW264.7) phagocytosed CD41+ EVs, and their phagocytosis and migration activity were suppressed. Microarray analysis also revealed that EVs altered gene expression in macrophages. These data indicated that the CD41+ EV was a carrier of microRNAs produced from thrombocytes and affected the cell characteristics of the received cells. Therefore, the CD41+ EVs of avians worked as a communication tool.

Amphibian thrombocyte-derived extracellular vesicles, including microRNAs, induce angiogenesis-related genes in endothelial cells.

Thrombocytes circulate in the blood of nonmammalian vertebrates and are involved in hemostasis; however, many detailed characteristics of thrombocytes remain unclear. Recently, we established an amphibian thrombocyte cell line. Here, we report the finding that thrombocytes produce integrin alpha IIb (CD41)-positive extracellular vesicles (EVs), which include microRNAs (miRs). Flow cytometric analysis showed the expression of CD41+ and phosphatidylserine on the surface of EVs. Nanotracking analysis showed that these CD41+ EVs were approximately 100 nm in diameter. As CD41+ EVs were also observed from African clawed frogs, the production of CD41+ EVs might be common to amphibians. Microarray analysis showed that the CD41+ EVs contain many kinds of miRs. These CD41+ EVs were phagocytosed by endothelial cells and macrophages. qPCR analysis showed that many angiogenesis-related genes were up-regulated in CD41+ EV-treated endothelial cells. Over-expression of some miRs in the CD41+ EVs increased the proliferation of endothelial cells. These results indicated that thrombocytes produced CD41+ EVs, including miRs, that were received by endothelial cells to induce the expression of angiogenesis-related genes. These results indicated that the CD41+ EVs produced from thrombocytes act as signaling molecules to repair damaged blood vessels.

Analysing the evolutional and functional differentiation of four types of Daphnia magna cryptochrome in Drosophila circadian clock.

Cryptochrome (CRY) plays an important role in the input of circadian clocks in various species, but gene copies in each species are evolutionarily divergent. Type I CRYs function as a photoreceptor molecule in the central clock, whereas type II CRYs directly regulate the transcriptional activity of clock proteins. Functions of other types of animal CRYs in the molecular clock remain unknown. The water flea Daphnia magna contains four Cry genes. However, it is still difficult to analyse these four genes. In this study, we took advantage of powerful genetic resources available from Drosophila to investigate evolutionary and functional differentiation of CRY proteins between the two species. We report differences in subcellular localisation of each D. magna CRY protein when expressed in the Drosophila clock neuron. Circadian rhythm behavioural experiments revealed that D. magna CRYs are not functionally conserved in the Drosophila molecular clock. These findings provide a new perspective on the evolutionary conservation of CRY, as functions of the four D. magna CRY proteins have diverse subcellular localisation levels. Furthermore, molecular clocks of D. magna have been evolutionarily differentiated from those of Drosophila. This study highlights the extensive functional diversity existing among species in their complement of Cry genes.

MicroRNA-342 inhibits tumor growth via targeting chemokine CXCL12 involved in macrophages recruitment/activation.

MicroRNAs (miRNAs) play important roles in initiation, development, progression and metastasis of tumors. MiR-342 has been reported as a tumor suppressor or an onco-miRNA based on functions or expression changes in various types of cancers. However, the biological roles and underlying molecular mechanisms of miR-342 in tumorigenesis remain largely unknown. Here, we found that miR-342 was expressed significantly less in a murine MS-K tumor cell line that showed riched blood vessels. Over-expression of miR-342 in MS-K cells inhibited cell proliferation, colony formation, reduced frequency of S phase population in vitro and suppressed tumor growth in vivo. Moreover, increasing miR-342 impeded blood vessels formation and accumulation of macrophages (CD11b+ ) in tumors. By bioinformatic analysis and dual-luciferase reporter assays, chemokine CXCL12 was identified as a direct target of miR-342. Restored Cxcl12 expression in MS-K-miR-342 cells could rescue cell proliferation in vitro. In MS-K-miR-342 tumor-infiltrated macrophages, expression of proangiogenic genes (Vegf-A and Thbs1) and M2-subtype macrophage markers (Cd163, Dectin1 and Ym1) was significantly down-regulated compared with controls. Moreover, lower level of Cxcl12 and its receptor Cxcr4 was observed in the macrophages of MS-K-miR-342 tumors, and MS-K-miR-342 derived miR-342, but not endogenous miR-342, might contribute to Cxcl12 suppression in TAM. These results suggest that miR-342 is involved in MS-K tumor growth as a tumor suppressor by targeting chemokine CXCL12.

Aquaporin-1, a New Maternally Expressed Gene, Regulates Placental Development in the Mouse.

Imprinted genes play an important role in placental and embryonic development. Abnormalities in their regulation can result in placental and embryonic dysplasia, leading to congenital diseases. The imprinting state, expression, and function of aquaporin-1 (Aqp1) were explored in knockout mice by imprinting analysis, real-time PCR, and immunohistochemistry. In the present study, Aqp1 was identified as a new, imprinted, and placenta-specific maternally expressed gene in the mouse. Compared with wild-type Aqp1(+/+) mice, there was significant placental and embryonic overgrowth in Aqp1(-/+) (loss of maternal allele) and Aqp1(-/-) mice, but not in Aqp1(+/-) (loss of paternal allele) mice at Embryonic Day (E) 12.5-E18.5. In addition, the masses of Postnatal Day 0 (P0) embryos (Aqp1(-/-) and Aqp1(-/+)) were highest among the four types. In Aqp1(-/+) and Aqp1(-/-) mice, phenotypic analysis indicated that the number and branching of blood vessels, as well as the labyrinth area, increased significantly in placentae of E12.5-E18.5 mice. Moreover, there were abnormalities in the placental junctional zone and the labyrinthine zone at E15.5. Quantitative analysis showed that Aqp1 expression decreased significantly in the placentae of Aqp1(-/+) and Aqp1(-/-) mice at E15.5, and that the AQP1 protein expression signals were detected weakly in the decidual and spongioblast layers. Our results demonstrate that Aqp1 is maternally expressed in the placenta, and that its deficiency resulted in placental abnormalities in the mouse. Aqp1 may have a specific inhibitory role in mouse placental development. These results provide new insights for the treatment of diseases relating to placental and embryonic development.

CCL11-induced eosinophils inhibit the formation of blood vessels and cause tumor necrosis.

We previously demonstrated that IL-18 and CCL11 were highly expressed in an NFSA tumor cell line that showed limited angiogenesis and severe necrosis. However, IL-18 was not responsible for the immune cell accumulation and necrosis. Here, we attempted to clarify the relevance of CCL11 in angiogenesis and tumor formation. We established CCL11-overexpressing MS-K cell clones (MS-K-CCL11) to assess the role of CCL11 in immune cell accumulation and angiogenesis. The MS-K-CCL11 cells did not form tumors in mice. MS-K-CCL11-conditioned medium (CM) and recombinant CCL11 induced macrophage and eosinophil differentiation from bone marrow cells. The MS-K-CCL11-CM effectively recruited the differentiated eosinophils. Furthermore, the eosinophils damaged the MS-K, NFSA and endothelial cells in a dose-dependent manner. Administration of an antagonist of CCR3, a CCL11 receptor, to NFSA tumor-bearing mice restored the blood vessel formation and blocked the eosinophil infiltration into the NFSA tumors. Furthermore, other CCL11-overexpressing LM8 clones were established, and their tumor formation ability was reduced compared to the parental LM8 cells, accompanied by increased eosinophil infiltration, blockade of angiogenesis and necrosis. These results indicate that CCL11 was responsible for the limited angiogenesis and necrosis by inducing and attracting eosinophils in the tumors.

Inhibition of blood vessel formation in tumors by IL-18-polarized M1 macrophages.

We previously showed that interleukin (IL)-18 produced by NFSA cells induced the M1 type of macrophages in NFSA tumors, caused the destruction of endothelial cells in vitro and may have resulted in the necrosis of NFSA tumors by enhancing macrophage phagocytosis and cytotoxicity. However, the effect of IL-18 on blood vessel formation in vivo has not been elucidated. MS-K cells do not express il-18, and they form tumors with well-developed blood vessels. Here, we established IL-18-over-expressing MS-K cell clones (MS-K-IL-18) to address the roles of IL-18 in angiogenesis. The over-expression of IL-18 inhibited the proliferation rate of the MS-K-IL-18 cells in vitro and blood vessel formation in the MS-K-IL-18 tumors. Interestingly, CD14-positive cells from the MS-K-IL-18 tumor had up-regulated expression of the M1-type macrophage marker il-6 and down-regulated expression of interferon (ifn)-γ. Furthermore, FACS analysis showed more accumulation of CD11b+/CD80+ M1 macrophages in the MS-K-IL-18 tumors than in the parental MS-K tumor. Moreover, an in vitro coculture assay showed that MS-K-IL-18-conditioned medium (CM) stimulated macrophages to induce the apoptosis of endothelial cells. Cumulatively, our data showed that IL-18 inhibited tumor blood vessel formation in vivo.

The atypical IκB protein IκB(NS) is important for Toll-like receptor-induced interleukin-10 production in B cells.

Although a major function of B cells is to mediate humoral immunity by producing antigen-specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). However, the mechanism by which IL-10 is induced in B cells has not been fully elucidated. Here, we report that IκBNS , an inducible nuclear IκB protein, is important for Toll-like receptor (TLR)-mediated IL-10 production in B cells. Studies using IκB(NS) knockout mice revealed that the number of IL-10-producing B cells is reduced in IκB(NS)(-/-) spleens and that the TLR-mediated induction of cytoplasmic IL-10-positive cells and IL-10 secretion in B cells are impaired in the absence of IκB(NS). The impairment of IL-10 production by a lack of IκB(NS) was not observed in TLR-triggered macrophages or T-cell-receptor-stimulated CD4(+) CD25(+) T cells. In addition, IκB(NS)-deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL-10(+) CD138(+) plasmablasts. These results suggest that IκB(NS) is selectively required for IL-10 production in B cells responding to TLR signals, so defining an additional role for IκB(NS) in the control of the B-cell-mediated immune responses.

Establishment of a sticky, large, oval-shaped thrombocyte cell line from tree frog as an ancestor of mammalian megakaryocytes.

Maintenance of blood vessels is important for homeostasis. Many types of cells and cytokines are involved in angiogenesis and blood vessel repair. In mammals, platelets, which are produced from megakaryocytes, play a major role in hemostasis. Other vertebrates have no platelets in their bloodstream. In these animals, thrombocytes aggregate to form a thrombus. Therefore, I established a frog hematopoietic cell line to elucidate the mechanism of hematopoiesis in this species. The frog-derived thrombocytic cell line was established from a long-term bone marrow culture of Hyla japonica and was designated as a frog-derived unique hematopoietic non-adherent (FUHEN) cell line. The FUHEN cells had unique characteristics in that they proliferated in suspension culture without adherence to the culture flask, and the shapes of the FUHEN cells changed drastically to become very large ovals with growth. These cells reached more than 40 µm in length and had multi-lobed nuclei. The FUHEN cells expressed CD41, a specific surface marker of thrombocytes. These results indicated that the FUHEN cells were thrombocytes. Deprivation of divalent ions quickly induced adherence of the cells to the petri dish. This characteristic may be important for hemostasis. Furthermore, some of the FUHEN cells survived at 16 °C for 1 month and re-established proliferation when the cells were moved to 28 °C. Taken together, this new thrombocytic frog cell line, as an ancestor of mammalian megakaryocytes, could provide useful material to study the functions of thrombocytes and the hemostasis mechanism of amphibians.

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

The aberrant expression of microRNAs (miRNAs) has frequently been reported in cancer studies; miRNAs play roles in development, progression, metastasis, and prognosis. Recent studies indicate that the miRNAs within the Dlk1-Dio3 genomic region are involved in the development of liver cancer, but the role of miR-1188 in hepatocellular carcinoma (HCC) and the pathway by which it exerts its function remain largely unknown. Here we demonstrate that miR-1188 is significantly down-regulated in mouse hepatoma cells compared with normal liver tissues. Enhanced miR-1188 suppresses cell proliferation, migration, and invasion in vitro and inhibits the tumor growth of HCC cells in vivo. Moreover, overexpressed miR-1188 promotes apoptosis, enhances caspase-3 activity, and also up-regulates the expression of Bax and p53. MiR-1188 directly targets and negatively regulates Bcl-2 and Sp1. Silencing of Bcl-2 and Sp1 exactly copies the proapoptotic and anti-invasive effects of miR-1188, respectively. The expression of apoptosis- and invasion-related genes, such as Vegfa, Fgfr1, and Rprd1b, decreases after enhancement of miR-1188, as determined by gene expression profiling analysis. Taken together, our results highlight an important role for miR-1188 as a tumor suppressor in hepatoma cells and imply its potential role in cancer therapy.

Role of FGF10 on tumorigenesis by MS-K.

Murine MS-K and NFSA cell lines formed tumor after inoculation into mouse and both cell lines expressed high level of vascular endothelial growth factor-A (vegf-A) and produced same level of VEGF-A. However, poor blood vessel formation, and necrosis was significantly observed in NFSA-tumor, contrary to well-developed blood vessel formation in MS-K tumor. The microarray analysis showed high expression of fibroblast growth factor-10 (fgf-10) in MS-K than NFSA. In this report, the role of fgf-10 on tumor growth was studied. MS-K enhanced more proliferation of endothelial cells by direct co-culture than NFSA, and rFGF10 supported the proliferation of HUVEC in combination with VEGF-A. fgf-10-knocked down MS-K, MS-K (fgf-10-KD), proliferated slower in vitro and the tumorigenicity of them was also slower than control. The blood vessel formation in these MS-K (fgf-10-KD) clones was reduced compared with the MS-K (normal). qPCR analysis showed the suppression of vegf-A, vegf-C and fgfr-1-expression in the MS-K (fgf-10-KD) clones. Taken together, these results indicated that FGF10, which was produced from tumor cells, was essential for the proliferation of tumor cell itself and also supports proliferation of endothelial cells. Thus, FGF10 plays an important role for tumor growth by both paracrine and autocrine manner.

Enhancement of phagocytosis and cytotoxicity in macrophages by tumor-derived IL-18 stimulation.

Inoculation of mice with the murine NFSA cell line caused the formation of large tumors with necrotic tumor cores. FACS analysis revealed accumulations of CD11b(+) cells in the tumors. Microarray analysis indicated that the NFSA cells expressed a high level of the pro-inflammatory factor interleukin-18 (il-18), which is known to play a critical role in macrophages. However, little is known about the physiological function of IL-18-stimulated macrophages. Here, we provide direct evidence that IL-18 enhances the phagocytosis of RAW264 cells and peritoneal macrophages, accompanied by the increased expression of tumor necrosis factor (tnf-α), interleukin-6 (il-6) and inducible nitric oxide synthase (Nos2). IL-18-stimulated RAW264 cells showed an enhanced cytotoxicity to endothelial F-2 cells via direct cell-to-cell interaction and the secretion of soluble mediators. Taken together, our results demonstrate that tumor-derived IL-18 plays an important role in the phagocytosis of macrophages and that IL-18-stimulated macrophages may damage tumor endothelial cells.

Dynamic expression pattern of Pde4d and its relationship with CpG methylation in the promoter during mouse embryo development.

Mouse Pde4d is located on chromosome 13 and serves many functions in important physiological processes involving cyclic adenosine monophosphate. In this study, imprinting analysis indicated that Pde4d exhibits a dynamic and specific allelic expression pattern during embryo development. This showed paternal-origin sex bias in embryonic day 9.5 (E9.5) whole embryos and placenta, and biallelic expression in the major embryonic organs and placenta at E15.5. In situ hybridization determined the spatiotemporal expression pattern of Pde4d in mouse embryos from the mid- to late-embryonic stages. This demonstrated that Pde4d was widely expressed in the neural tissues, including the forebrain, midbrain, hindbrain, and neural tube, at the mid-embryonic stage. By the late-embryonic stage, Pde4d was extensively detected throughout the developing organism, including in the liver, brain, lung, kidney, and tongue. In addition, methylation analyses indicated that tissue-specific CpG methylation of the Pde4d promoter was correlated with Pde4d mRNA expression in major E15.5 tissues. Furthermore, stage-specific CpG methylation of the Pde4d promoter was associated with gene expression in the liver at three developmental stages. Our results suggest that Pde4d might serve specific biological functions in regulating the development process of the mouse embryo, and that CpG methylation of the Pde4d promoter may play an important role in regulating Pde4d at a transcriptional level.

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.

Effect of vegf gene knockdown on growth of the murine sarcoma cell line MS-K.

The murine sarcoma cell line MS-K was previously established as a Ki-ras-positive cell line. Inoculation of this cell line under the flank of C3H/HeN mice results in the growth of large tumors with well-developed blood vessels within day 30 of transplantation without any metastasis because MS-K cells produce vascular endothelial growth factor (VEGF). To elucidate the role of VEGF in tumor formation in vivo, stable vegf-knockdown-MS-K clones were obtained using plasmid-based knockdown vectors. Interestingly, tumorigenesis was completely suppressed in a vegf-A-knockdown-MS-K clone [designated MS-K (A-KD)]. Proliferation and colony formation capacity of the MS-K (A-KD) cells in a semi-solid medium under low serum conditions was significantly lower than that of control MS-K (SCR) cells; however, the expression of vegf-receptor 1 (vegf-r-1) was not changed. Addition of the recombinant VEGF-A(165) partially restored the colony formation capacity of MS-K (A-KD) cells and caused the phosphorylation of VEGF-r-1 (Flt-1) in MS-K (Normal) cells. Furthermore, tumorigenicity of the vegf-r-1-knockdown-MS-K clone [designated MS-K (R1-KD)] had obviously delayed or strongly suppressed compared with the MS-K (Normal). These results indicate that Vascular endothelial growth factor-A, produced from MS-K, acts as a growth factor for MS-K cells itself and supports tumor formation in vivo by inducing the blood vessel formation.

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.

HB-1, an acute myeloid leukemic cell line with the capability of infiltrating into the brain in CBA/N mice.

An acute myeloid leukemic HB-1 cell line was cloned and established from the spleen cells of irradiated CBA/N mice. Acute myeloma leukemia-like syndrome would be induced in normal CBA/N mice after intravenous injection of HB-1 cells, and the death of mouse happened within about two weeks. In general, leukemic cells transplanted into the mice would infiltrate into the hematopoietic organs, lungs, kidneys and liver. An interesting observation in our study was that HB-1 cells were present not only in the lung, kidney, and liver but also in the cerebrum and cerebellum. It was beyond our expectation that the leukemic cells could go through the blood-brain barrier in most circumstances. On the basis of the observation, we expect that HB-1 cells could be used as a very useful model to elucidate the mechanism of infiltrating the blood-brain barrier for certain type of cells.

Murine serum obtained from bone marrow-transplanted mice promotes the proliferation of hematopoietic stem cells by co-culture with MS-5 murine stromal cells.

To examine whether serum obtained from bone marrow-transplanted mice can selectively expand hematopoietic stem cells (HSCs) among whole bone marrow cells in vitro, whole bone marrow cells were cultured with or without MS-5 murine stromal cells in the presence of serum obtained from transplanted mice on day 3 (day 3 serum) or serum from normal mice for 7 days. When whole bone marrow cells and MS-5 cells were co-cultured in day 3 serum for 7 days, the c-kit-positive, Sca-1-positive, lineage marker-negative cells (KSL cells) expanded approximately 25 times; however, when they were co-cultured in normal serum for 7 days, the KSL cells expanded approximately 1.3 times. Direct contact between the whole bone marrow cells and MS-5 cells was essential for expansion of KSL cells in the co-culture, and it upregulated the expression of some cytokines in MS-5. Above all, the day 3 serum specifically upregulated the expression of SCF, SDF-1 alpha, G-CSF, IL-11 and IL-6 in MS-5. The level of testosterone in the day 3 serum was higher than normal serum and the addition of the testosterone in the culture expanded the KSL cells among whole bone marrow cells on MS-5 cells and also upregulated the expression of SDF-1 alpha, IL-11 and IL-6 in MS-5. These data indicates that the serum of bone marrow-transplanted mice contains a factor(s) that induced changes in the expression levels of various cytokines in MS-5 stromal cells and enabled the MS-5 cells to expand the KSL cells among whole bone marrow cells.

Cloning of hibernation-related genes of bullfrog (Rana catesbeiana) by cDNA subtraction.

Seven genes specifically expressed during hibernation in the bullfrog (Rana catesbeiana) were cloned from a subtracted cDNA library constructed from livers of winter bullfrogs. Those genes were fibrinogen alpha-subunit, fibrinogen gamma-subunit, complement component C3, alpha-1-microglobulin/bikunin precursor (AMBP), transferrin, apoferritin middle subunit and one novel gene. Northern hybridization has indicated that these seven genes were specifically induced or enhanced in winter. Above all, expression of the novel gene was specifically induced in winter in liver, though the expression of that was neither induced in bullfrog nor Xenopus laevis by cold treatment. The novel gene, which was designated as rc-hirp (Rana catesbeiana hibernation-related protein), encoded 420 base pairs length and a putative protein of 139 amino acid residues. Annual analyses of the expression of these genes have suggested that the seven winter-specific genes are playing an important role in hibernation processes.

Identification of negative regulator of interleukin-3 (NIL-3) in bone marrow.

We have reported that an inhibitor of interleukin-3 (NIL-3) is produced from murine bone marrow cells in response to excess stimulation of interleukin-3. In this report, we attempted the purification of the NIL-3 activity from bone marrow culture supernatant in the presence of interleukin-3. The purified NIL-3 activity was a protein with relative molecular weight of 54.5 kDa (SDS-PAGE), which inhibited the growth of IL-3 dependent DA-1 cell growth in a dose dependent manner. The N-terminal amino acid sequence of purified NIL-3 activity was determined to be homologous to beta-2 glycoprotein I (apolipoprotein H: APO-H). The gene expression of APO-H was detected by nested-PCR in STIL-3 C5-CM stimulated total bone marrow cells and STIL-3 C5-CM stimulated bone marrow fraction 2 (Fr. 2) which has been reported as a hematopoietic stem cell rich fraction. These observations indicate the possibility that the APO-H is the NIL-3 which was produced from bone marrow cells in response to excess IL-3 stimuli.