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Biomedical subjects

A Miyajima

Publications and source records attributed to A Miyajima.

At least 19 recordsLinked to original sources
PubMed2003

Oncostatin M, a multifunctional cytokine.

Oncostatin M (OSM) is a multifunctional cytokine that belongs to the Interleukin (IL)-6 subfamily. Among the family members, OSM is most closely related to leukemia inhibitory factor (LIF) and it in fact utilizes the LIF receptor in addition to its specific receptor in the human. While OSM was originally recognized by its unique activity to inhibit the proliferation of tumor cells, accumulating evidence now indicates that OSM exhibits many unique biological activities in inflammation, hematopoiesis, and development. Here, we review the profile of OSM activities, receptors, and signal transduction.

Animals↗
PubMed2003

Enhanced in vitro maturation of subcultivated fetal human hepatocytes in three dimensional culture using poly-L-lactic acid scaffolds in the presence of oncostatin M.

Fetal human liver cell fractions, which contain large numbers of hepatocyte progenitors, have high proliferation potential in vitro. To create an engineered liver tissue equivalent of a clinically significant size, however, repeated subcultivation and functional maturation are necessary in vitro. A commercially available human fetal liver cell fraction that was cultivated for some time in vitro has been reported to lose liver specific functions almost completely. We therefore investigated the effects of oncostatin M (OSM) and hepatocyte growth factor (HGF) in long-term three-dimensional (3D) culture using macroporous poly-L-lactic acid (PLLA) scaffolds on the restoration of such liver-specific functions of the fraction. 3D culture using PLLA scaffolds with OSM remarkably enhanced the albumin production and cytochrome P450 1A1/2 capacity with the culture time. HGF alone had no preferable effect on these functions even in 3D culture. Alpha-fetoprotein production was consistently suppressed in the 3D culture compared with that in monolayers. This suppression was not observed in the same types of culture of hepatocarcinoma Hep G2 cells. Despite these favorable observations on the 3D culture with OSM, the final attained functional levels at the 5th week were still over ten-times lower than those of Hep G2 cells when standardized with a cellular DNA amount. Although further improvement is needed for the complete functional restoration and maturation in vitro, these results demonstrate that a combination of 3D culture using PLLA scaffolds and OSM offers promising culture conditions for in vitro maturation of human hepatocyte progenitors.

Albumins↗
PubMed2002

Enhanced in vitro maturation of fetal mouse liver cells with oncostatin M, nicotinamide, and dimethyl sulfoxide.

Although cells isolated from fetal liver are one of the major sources for liver tissue engineering, it is still very difficult to induce them to fully differentiate in vitro into mature hepatocytes. We therefore investigated the effects of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) on differentiation in terms of the expression of various liver-specific functions, because these factors have been reported to induce the emergence of possible hepatocyte progenitor cells (small hepatocytes) in adult rat hepatocyte culture or maturation of fetal mouse liver cells in culture. Fetal liver cells isolated from mouse embryos were cultured for 5 weeks in collagen-precoated plates. NA (10 mM) and DMSO (1%) remarkably enhanced the emergence of small hepatocytes, and OSM also synergistically enhanced the selective growth of small hepatocytes and inhibited the growth of blood cell populations. In the presence of these three factors, such small hepatocytes became dominant in culture, so that they covered almost 60-70% of confluence after week 2. In addition, some of them piled up over the small hepatocyte monolayer and displayed distinctively differentiated morphology, such as the emergence of binucleated cells, formation of tight gap junctions, and possible bile duct structures. Although OSM alone had very weak effects on hepatocyte functions, albumin secretion and cytochrome P450IA1/2 capacity were greatly enhanced when combined with NA or DMSO. This functional observation closely agreed with the emergence of small hepatocytes. In contrast, ammonium removal was strongly dependent on DMSO alone. DNA amount basis functions of fetal cells with three factors at week 5 were 1/7 for albumin secretion, 3 times higher for ammonium removal, and 1/10 for P450 capacity, compared with those of cultured adult mouse hepatocytes. These results show that inclusion of NA, DMSO, and OSM in the culture medium significantly enhances in vitro maturation of fetal liver cells when compared with conventional culture conditions.

Albumins↗
PubMed2001

Oncostatin M and hepatocyte growth factor induce hepatic maturation via distinct signaling pathways.

Liver development is regulated by soluble factors as well as cell-cell contacts. We previously reported that oncostatin M (OSM) induced hepatic maturation in a primary culture of embryonic day 14 liver cells. While OSM expression in the liver starts in mid gestation and decreases in postnatal stages, hepatocyte growth factor (HGF) is mainly expressed in the liver in the first few days after birth. In this study, we compared the effect of OSM and HGF on the differentiation of fetal hepatic cells in vitro. Like OSM, HGF in the presence of dexamethasone induced expression of glucose-6-phosphatase, tyrosine amino transferase and carbamoyl-phosphate synthase, and accumulation of glycogen in fetal hepatic cells, although to a lesser extent than OSM. Interestingly, while both OSM and HGF up-regulated production of albumin, secretion of albumin occurred only in response to OSM. In addition, although hepatic maturation induced by OSM depends on STAT3, HGF failed to activate STAT3 and HGF-induced differentiation was independent of STAT3. These results indicate that OSM and HGF induce hepatic maturation through different signaling pathways.

Animals↗
PubMed2001

Involvement of SGS1 in DNA damage-induced heteroallelic recombination that requires RAD52 in Saccharomyces cerevisiae.

The SGS1 gene of Saccharomyces cerevisiae is homologous to the genes that are mutated in Bloom's syndrome and Werner's syndrome in humans. Disruption of SGS1 results in high sensitivity to methyl methanesulfonate (MMS), poor sporulation, and a hyper-recombination phenotype including recombination between heteroalleles. In this study, we found that SGS1 forms part of the RAD52 epistasis group when cells are exposed to MMS. Exposure to DNA-damaging agents causes a striking, Rad52-dependent, increase in heteroallelic recombination in wild-type cells, but not in sgs1 disruptants. However, in the absence of DNA damage, the frequency of heteroallelic recombination in sgs1 disruptants was several-fold higher than in wild-type cells, as described previously. These results imply a function for Sgs1: it acts to suppress spontaneous heteroallelic recombination, and to promote DNA damage-induced heteroallelic recombination.

Alleles↗
PubMed2001

The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants.

The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome. Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles. SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ. A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region). In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end. These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe. In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants. In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes. Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes. Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.

Amino Acid Sequence↗
PubMed2001

Oncostatin M suppresses generation of lymphoid progenitors in fetal liver by inhibiting the hepatic microenvironment.

OBJECTIVE: Interaction between hematopoietic cells and stromal cells is important for regulation of hematopoiesis. Numerous soluble and membrane-bound factors directly regulating hematopoiesis have been documented, but little is known about how stromal cell activity is controlled. We previously reported that fetal hepatic cells in primary culture create the hematopoietic microenvironment and support expansion of blood cells from hematopoietic stem cells. In this study, we focused on lymphopoiesis reconstituted in our culture system and analyzed how stroma-mediated lymphopoiesis is regulated during embryonic development. MATERIALS AND METHODS: Subconfluent cultures of murine fetal hepatic cells were cocultured with hematopoietic stem cells derived from fetal liver in the presence of various cytokines. After 10 days of incubation, hematopoietic cells floating over the stromal layer were analyzed by various assays, including cell proliferation and FACS analysis. RESULTS: We found that oncostatin M, an inducer of hepatic development, strongly inhibited generation of B220(+) lymphocytic cells and colony-forming unit-interleukin-7 (CFU-IL-7) from hematopoietic stem cells in our coculture system. In contrast, oncostatin M did not directly inhibit proliferation of B cells in response to IL-7 and SCF in semisolid cultures. Analysis of antigen expression in lymphoid cells revealed that oncostatin M apparently did not arrest cells at a particular stage of B-cell development. CONCLUSIONS: The results suggest that oncostatin M inhibits lymphopoiesis by suppressing stromal activity of fetal hepatic cells to stimulate generation of CFU-IL-7 from their progenitors rather than by acting directly on lymphocytic cells.

Animals↗
PubMed2001

Incidentally discovered giant renal arteriovenous malformation.

A case is presented of giant renal arteriovenous malformation (AVM). A 61-year-old woman was admitted to the National Defense Medical College Hospital for further evaluation of a renal cyst. Doppler ultrasonography and magnetic resonance imaging revealed a giant renal AVM, although the patient had no history nor clinical sign suggesting an AVM. Under the diagnosis of a right renal AVM, the patient underwent AVM resection.

Arteriovenous Malformations↗
PubMed2001

Role of nitric oxide in renal tubular apoptosis of unilateral ureteral obstruction.

BACKGROUND: The obstructed kidney in unilateral ureteral obstruction (UUO) is characterized by renal atrophy and tissue loss, which is mediated by renal tubular apoptosis. We sought to determine whether NO is involved in renal tubular apoptosis in vitro and in vivo. METHODS: Rat renal tubular epithelial cells (NRK-52E) were subjected to mechanical stretch, and apoptosis and cell size were analyzed by flow cytometry. Furthermore, we studied UUO in mice lacking the gene for inducible nitric oxide synthase (iNOS-/-) and their wild-type littermates. Tubular apoptosis and proliferation were detected by immunostaining. NOS activity and NOS expression were assessed by a citrulline assay and Western blot, respectively. RESULTS: Stretching-induced apoptosis in NRK-52E, which was reduced when NO was increased; conversely, stretch-induced apoptosis was increased when a NOS inhibitor was added to the cells. Stretched cells are larger and more apoptotic than unstretched cells. In UUO, the obstructed kidney of iNOS-/- mice exhibited more apoptotic renal tubules than the wild-type mice through 14 days of UUO. The obstructed kidney of iNOS-/- mice at day 3 showed more proliferative tubules compared with wild type. The obstructed kidney of wild-type mice exhibited higher total NOS activity until day 7 after UUO compared with iNOS-/- mice. However, the obstructed kidney of day 14 wild-type mice exhibited significantly lower iNOS activity and protein compared with the day 0 kidney. CONCLUSION: These results suggest that mechanical stretch is related to renal tubular apoptosis and that NO plays a protective role in this system in UUO.

Animals↗
PubMed2001

Captopril restores transforming growth factor-beta type II receptor and sensitivity to transforming growth factor-beta in murine renal cell cancer cells.

PURPOSE: Captopril is known to inhibit the growth of renal cancer but the mechanism involved has been unclear. The current study elucidates the mechanism of captopril induced inhibition of the growth of the Renca mouse renal cancer cell line involving transforming growth factor-beta, which is known to be a growth inhibitory cytokine in epithelial cells and tissues. MATERIALS AND METHODS: Transforming growth factor-beta in conditioned medium was measured by bioassay. Levels of transforming growth factor-beta and transforming growth factor-beta type II receptor expression messenger RNA were determined by reverse transcriptase-polymerase chain reaction and flow cytometry. Cell viability was determined by bromodeoxyuridine (BrdU) incorporation and tetrazolium bromide assay. RESULTS: Captopril (0.01 to 1 mM.) showed no significant effect on transforming growth factor-beta synthesis or transforming growth factor-beta messenger RNA in Renca cells. On the other hand, 1 mM. captopril significantly inhibited Renca cell growth. Reverse transcriptase-polymerase chain reaction and flow cytometry showed that 1 mM. captopril up-regulated type II receptor expression. CONCLUSIONS: These findings suggest that captopril restores transforming growth factor-beta type II receptor expression and inhibits the growth of Renca cells by increasing their sensitivity to transforming growth factor-beta.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2001

Role of the microenvironment of the embryonic aorta-gonad-mesonephros region in hematopoiesis.

Although various cytokines, growth factors, and chemokines are known to regulate hematopoiesis, expansion of hematopoietic stem cells (HSCs) in vitro with the use of such agents has proved problematic. Stromal cells are major components of the microenvironment that surrounds hematopoietic cells and are thought to play an important role in hematopoiesis in vivo. Co-culture of HSCs with stromal cells promotes hematopoiesis and self-renewal of HSCs. Definitive hematopoietic cells first appear during mammalian embryonic development in the aorta-gonad-mesonephros (AGM) region, and it is therefore thought that the microenvironment of this region plays an important role in HSC ontogeny. We have adopted two approaches to studying the contribution of the AGM microenvironment to hematopoiesis. In the first approach, we have developed an in vitro culture system for mouse AGM explants. Hematopoiesis is enhanced in such cultures by the presence of the combination of stem cell factor (SCF), basic fibroblast growth factor, leukemia inhibitory factor, and oncostatin M (SFLO culture). However, transplantation assays revealed that HSCs capable of long-term reconstitution of the hematopoietic compartment of irradiated mice (LTR-HSCs) do not expand in AGM-SFLO cultures; rather, these cultures appear to provide a favorable microenvironment for hematogenic angioblasts that are precursors of both endothelial and hematopoietic cells. In our second approach, we have established various stromal cell lines from the mouse AGM region. The AGM-S3 cell line supports human and mouse primitive hematopoietic cells as well as mouse LTR-HSCs. Maintenance of LTR-HSCs is mediated by a mechanism other than SCF signaling through its receptor (c-Kit). These two in vitro approaches should prove useful for further elucidation of the mechanisms that underlie hematopoiesis and HSC self-renewal.

Animals↗
PubMed2001

Downregulation of Bim, a proapoptotic relative of Bcl-2, is a pivotal step in cytokine-initiated survival signaling in murine hematopoietic progenitors.

Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (betac chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-x(L) through activation of STAT5. The other pathway emanates from the distal region of the betac chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.

Animals↗
PubMed2001

Ammocidin, a new apoptosis inducer in Ras-dependent cells from Saccharothrix sp. I. Production, isolation and biological activity.

A new apoptosis inducer, ammocidin, was isolated from the culture broth of Saccharothrix sp. AJ9571. Ammocidin induced apoptotic cell death in Ras-dependent Ba/F3-V12 cells with an IC50 of 66 ng/ml. No cell death was observed in IL-3-dependent Ba/F3-V12 cells at less than 100 microg/ml of ammocidin. Ammocidin significantly reduced the phosphorylation level of MAPK and S6K that mediate the anti-apoptotic function of Ras.

Actinomycetales↗
PubMed2001

Tranilast ameliorates renal tubular damage in unilateral ureteral obstruction.

PURPOSE: We determined whether tranilast, the anti-allergic agent N-(3, 4-dimethoxyciannamoyl)-anthranilic acid, would diminish renal transforming growth factor-beta (TGF-beta) levels in unilateral ureteral obstruction and concomitantly affect renal tubular apoptosis and proliferation in that condition. MATERIALS AND METHODS: Tranilast (150 mg./kg.) was administered to rats 1 day before unilateral ureteral obstruction and each day thereafter. Kidneys were harvested day 14 after unilateral ureteral obstruction. Tissue TGF-beta was measured by bioassay using mink lung epithelial cells. Renal tubular proliferation and apoptosis were detected by immunostaining proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay, respectively. Fibrosis was assessed by measuring collagen deposition with trichrome stained slides. RESULTS: TGF-beta bioassay showed that obstructed kidneys in controls contained significantly higher mean TGF-beta plus or minus standard deviation than unobstructed kidneys in controls (73.7 +/- 13.6 versus 14.1 +/- 5.5 pg./mg. tissue) and tranilast significantly decreased tissue TGF-beta in obstructed kidneys (15.9 +/- 4.8 pg./mg. tissue). The terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay demonstrated that obstructed kidneys in controls had significantly more mean tubular apoptosis than the unobstructed counterparts (36.6 +/- 6.7 versus 5.8 +/- 5.5 nuclei per high power field) and tranilast significantly decreased mean renal tubular apoptosis in obstructed kidneys (16.2 +/- 1.7 nuclei per high power field). In addition, immunostaining proliferating cell nuclear antigen showed that obstructed kidneys in controls had significantly more mean renal tubular proliferation than unobstructed kidneys (20.7 +/- 3.4 versus 6.2 +/- 2.1 per high power field) and tranilast significantly increased proliferating renal tubules in obstructed and unobstructed kidneys (26.5 +/- 8.3 and 14.5 +/- 3.4 per high power field, respectively). Control obstructed kidneys exhibited significantly more fibrosis, which was also blunted by tranilast. CONCLUSIONS: Tranilast significantly decreases tissue TGF-beta, resulting in a reduction in tubular apoptosis and an increase in tubular proliferation. This finding suggests that tranilast is a promising agent for preventing renal tubular damage in unilateral ureteral obstruction.

Animals↗
PubMed2001

Does cyclooxygenase-2 inhibitor prevent renal tissue damage in unilateral ureteral obstruction?

PURPOSE: We determined whether the cyclooxygenase-2 inhibitor etodolac affects renal tubular damage and interstitial fibrosis in unilateral ureteral obstruction. MATERIALS AND METHODS: Etodolac (10 mg./kg.) was administered to rats 1 day before unilateral ureteral obstruction and every day thereafter. Kidneys were harvested at day 14 after unilateral ureteral obstruction. Tissue transforming growth factor-beta and prostaglandin E2 were measured by bioassay using mink lung epithelial cells and enzyme linked immunosorbent-sandwich assay. Renal tubular proliferation and apoptosis were detected by immunostaining with proliferating cellular nuclear antigen and by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling, respectively. Cyclooxygenase-2 expression was detected by immunohistochemistry. Fibrosis was assessed by measuring collagen deposition in trichrome stained slides. RESULTS: Bioassay showed that in the control group obstructed kidneys contained significantly higher mean transforming growth factor-beta1 than unobstructed kidneys (79.1 +/- 8.3 versus 33.6 +/- 4.2 ng./gm. tissue) and etodolac significantly decrease the mean value in obstructed kidneys (46.2 +/- 10.0 ng./gm. tissue). Assay demonstrated that obstructed control kidneys had significantly more mean tubular apoptosis than their unobstructed counterparts (26.6 +/- 5.4 versus 2.2 +/- 1.4 nuclei per high power field) and etodolac significantly decreased mean renal tubular apoptosis in the obstructed kidneys (16.2 +/- 1.9 nuclei per high power field). In addition, immunostaining with proliferating cellular nuclear antigen showed that obstructed kidneys in the control group had significantly more mean renal tubular proliferation than unobstructed kidneys (9.8 +/- 3.4 versus 3.9 +/- 0.1 per high power field) and etodolac significantly increased mean proliferating renal tubule in the obstructed kidneys (24.9 +/- 4.3 per high power field). Control obstructed kidneys had significantly more fibrosis and prostaglandin E2 production, which were also significantly blunted by etodolac. CONCLUSIONS: The cyclooxygenase-2 inhibitor etodolac significantly reduces tissue transforming growth factor-beta, resulting in decreased tubular damage and interstitial fibrosis. This finding suggests that etodolac is a promising agent for preventing renal tissue damage in unilateral ureteral obstruction.

Animals↗
PubMed2000

Interleukin-6 decreases estrogen production and messenger ribonucleic acid expression encoding aromatase during in vitro cytodifferentiation of rat granulosa cell.

To elucidate the influence of interleukin-6 (IL-6) on the maturation of rat granulosa cell (GC), we have established a differentiation model of rat GC in vitro and examined the expression of IL-6 and its receptors, and its possible actions during GC maturation. Bioactive IL-6 was detectable in the conditioned media of GC at approximately 2.5 ng/ml/24 h per 5x10(5) cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that messenger RNAs encoding components of functional IL-6 receptor, namely both ligand-binding subunit (IL-6R) and gp130, were expressed in GC. Treatment of GC with IL-6 for 72 h during the process of in vitro GC maturation dose-dependently inhibited the accumulation of estradiol-17beta (E(2)) and the expression of cytochrome P450 aromatase (P450arom). IL-6 did not change nitric oxide (NO) production and inducible NO synthase expression, implying that IL-6-induced suppression on E(2) levels is dissociated with NO expression. Further, GC which had been incubated with neutralizing anti-IL-6 antibody showed a distinct increase in the levels of P450arom mRNA. These results suggest that IL-6 may attenuate E(2) production partially by inhibiting the expression of aromatase mRNA as an intraovarian regulator for suppressing GC maturation.

Animals↗
PubMed2000

Thrombopoietin and interleukin-2 induce association of CRK with STAT5.

Crk (Crk I and II) proteins and closely related CrkL are adapters which are commonly involved in various signaling processes in various cells, and these proteins share many ligands. Whether they have redundant or distinct physiologic roles is unclear. By coprecipitation and far Western blotting analysis, we demonstrate that Crk (I/II) binds to tyrosine phosphorylated STAT5 in cells stimulated by cytokines such as thrombopoietin (TPO) and interleukin-2 (IL-2). The association did not require nuclear elements and can be observed in primary cells as this was also demonstrated in TPO-stimulated platelets. Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that CrkL (a close relative of Crk) antiserum, but not Crk antiserum, supershifted the STAT5-DNA complex by an electrophoretic mobility shift assay, suggesting that CrkL, but not Crk, is the major component of the complex. Thus, Crk and CrkL may have distinct roles in the regulation of STAT5.

Base Sequence↗