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A Miyajima

Publications and source records attributed to A Miyajima.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cell density-dependent regulation of hepatic development by a gp130-independent pathway.

We previously demonstrated that oncostatin M (OSM) promotes hepatic development in concert with glucocorticoid. The livers from mice deficient for gp130, a signaling subunit of the OSM receptor, displayed reduced expression of hepatic differentiation marker and defective glycogenic function. However, these phenotypes were not completely abolished in gp130(-/-) mice, suggesting that there is an alternative pathway regulating hepatic development in vivo. To test this possibility, we cultured gp130(-/-) fetal hepatic cells and investigated a signal that induces hepatic differentiation. When hepatocytes were forced to interact with each other by inoculating cells at high densities, hepatic differentiation was induced even in the absence of gp130. Moreover, cells stimulated with OSM and/or cultured at a high density possess many other metabolic functions. These observations suggest that fetal hepatic cells acquire multiple characteristics of differentiated hepatocytes in response to the signals generated by cell-cell contacts as well as by OSM.

Ammonia↗
PubMed2000

Receptor subunit-specific action of oncostatin M in hepatic cells and its modulation by leukemia inhibitory factor.

The related cytokines, interleukin-6 (IL-6), oncostatin M (OSM), and leukemia inhibitory factor (LIF) direct the formation of specific heteromeric receptor complexes to achieve signaling. Each complex includes the common signal-transducing subunit gp130. OSM and LIF also recruit the signaling competent, but structurally distinct OSMRbeta and LIFRalpha subunits, respectively. To test the hypothesis that the particularly prominent cell regulation by OSM is due to signals contributed by OSMRbeta, we introduced stable expression of human or mouse OSMRbeta in rat hepatoma cells which have endogenous receptors for IL-6 and LIF, but not OSM. Both mouse and human OSM engaged gp130 with their respective OSMRbeta subunits, but only human OSM also acted through LIFR. Signaling by OSMRbeta-containing receptors was characterized by highest activation of STAT5 and ERK, recruitment of the insulin receptor substrate and Jun-N-terminal kinase pathways, and induction of a characteristic pattern of acute phase proteins. Since LIF together with LIFRalpha appear to form a more stable complex with gp130 than OSM with gp130 and OSMRbeta, co-activation of LIFR and OSMR resulted in a predominant LIF-like response. These results suggest that signaling by IL-6 cytokines is not identical, and that a hierarchical order of cytokine receptor action exists in which LIFR ranks as dominant member.

Acute-Phase Proteins↗
PubMed2000

Constitutive activation of STAT5 by a point mutation in the SH2 domain.

We previously identified a constitutively active form of STAT (signal transducer and activator of transcription) 5A by polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening, which had two point mutations in the DNA-binding and transcriptional activation domains, and was designated STAT5A1*6. STAT5A1*6 showed markedly elevated DNA binding and transactivation activities with stable tyrosine phosphorylation and nuclear accumulation, and conferred autonomous cell growth on interleukin 3-dependent Ba/F3 cells. We now report another constitutively active mutant, STAT5A-N642H which has a single point mutation (N642H) in its SH2 domain, identified using the same strategy as that used to identify STAT5A1*6. STAT5A-N642H showed identical properties to those of STAT5A1*6 both biochemically and biologically. Interestingly the mutation in STAT5A-N642H resulted in restoration of the conserved critical histidine which is involved in the binding of phosphotyrosine in the majority of SH2-containing proteins. Introduction of an additional mutation (Y694F) to STAT5A-N642H, which disrupted critical tyrosine 694 required for dimerization of STAT5, abolished all the activities manifested by the mutant STAT5A-N642H, which indicates that dimerization is required for the activity of STAT5A-N642H as was the case for the wild-type STAT5A. The present findings also show that different mutations rendered STAT5A constitutively active, through a common mechanism, which is similar to that of physiological activation.

3T3 Cells↗
PubMed2000

Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom's syndrome gene.

The SGS1 of Saccharomyces cerevisiae is a homologue of the Bloom's syndrome and Werner's syndrome genes. The sgs1 disruptants show hyperrecombination, higher sensitivity to methyl methanesulfonate and hydroxyurea, and poor sporulation. In this study, we found that sister chromatid exchange was increased in sgs1 disruptants. We made mutated SGS1 genes coding a protein proved to lack DNA helicase activity (sgs1-hd), having equivalent missense mutations found in Bloom's syndrome patients (sgs1-BS1, sgs1-BS2). None of the mutated genes could suppress the higher sensitivity to methyl methanesulfonate and hydroxyurea and the increased frequency of interchromosomal recombination and sister chromatid exchange of sgs1 disruptants. On the other hand, all of the mutant genes were able to complement the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wild-type SGS1.

Alleles↗
PubMed2000

The AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic aorta-gonad-mesonephros region.

We examined the role of the AML1 transcription factor in the development of hematopoiesis in the paraaortic splanchnopleural (P-Sp) and the aorta-gonad-mesonephros (AGM) regions of mouse embryos. The activity of colony-forming units of colonies from the P-Sp/AGM region was reduced severalfold by heterozygous disruption of the AML1 gene, indicating that AML1 functioned in a dosage-dependent manner to generate hematopoietic progenitors. In addition, no hematopoietic progenitor activity was detected in the P-Sp/AGM region of embryos with an AML1 null mutation. Similar results were obtained when a dispersed culture was first prepared from the P-Sp/AGM region before assay of the activity of the colony-forming units. In a culture of cells with the AML1(+/+) genotype, both hematopoietic and endothelial-like cell types emerged, but in a culture of cells with the AML1(-/-) genotype, only endothelial-like cells emerged. Interestingly, introduction of AML1 cDNA into the P-Sp/AGM culture with the AML1(-/-) genotype partially restored the production of hematopoietic cells. This restoration was observed for cultures prepared from 9.5-day postcoitum (dpc) embryos but not for cultures prepared from 11.5-dpc embryos. Therefore, the population of endothelial-like cells capable of growing in the AML1(-/-) culture would appear to contain inert but nonetheless competent hematogenic precursor cells up until at least the 9.5-dpc period. All these results support the notion that the AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic P-Sp/AGM region.

Animals↗
PubMed2000

Expression of interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 in the murine cerebellum and neuropathological effect of leukemia inhibitory factor on cerebellar Purkinje cells.

Expression of glycoprotein 130 and the related receptors, including interleukin-6 receptor and leukemia inhibitory factor receptor, was examined in the murine cerebellum at the protein level. Western blot analysis revealed that interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 were expressed in the murine cerebellum. Immunoreactivities for interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 were strongly localized on the cell body of Purkinje cells, indicating that both interleukin-6 and leukemia inhibitory factor could act directly on Purkinje cells in murine adult mice. The expressions of interleukin-6 receptor, leukemia inhibitory factor receptor and glycoprotein 130 were observed on the cell membranes of Purkinje cells by immunoelectron microscopy. Immunoreactivity for the interleukin-6 receptor was also detected in the cytoplasm of Purkinje cells. Injection of a murine hemopoietic cell line, FDC-P1 cells, transfected with the complementary DNA encoding the leukemia inhibitory factor led to a reduction in calbindin-positive dendrites of the Purkinje cells.The present results suggest that the leukemia inhibitory factor affects cerebellar functions through Purkinje cells.

Animals↗
PubMed2000

Role of Oncostatin M in hematopoiesis and liver development.

Definitive hematopoietic stem cells (HSCs) first appear in the aorta/gonad/mesonephros (AGM) region and migrate to the fetal liver where they massively produce hematopoietic cells before establishing hematopoiesis in the bone marrow at a perinatal stage. In the AGM region, Oncostatin M (OSM) enhances the development of both hematopoietic and endothelial cells by possibly stimulating their common precursors, so-called hemangioblasts. During development of HSCs in the AGM region, the liver primodium is formed at the foregut and accepts HSCs. While fetal hepatic cells function as hematopoietic microenvironment for expansion of hematopoietic cells during mid to late gestation, they do not possess most of the metabolic functions of adult liver. Along with the expansion of hematopoietic cells in fetal liver, OSM is produced by hematopoietic cells and induces differentiation of fetal hepatic cells, conferring various metabolic activities of adult liver. Matured hepatic cells then lose the ability to support hematopoiesis. Thus, OSM appears to coordinate the development of liver and hematopoiesis in the fetus.

Animals↗
PubMed2000

Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction.

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-beta (TGF-beta), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-beta (1D11) in UUO. METHODS: Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-beta levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). RESULTS: Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-beta; 1D11 (10 microg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-beta as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-beta of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. CONCLUSION: The present study strongly suggests that an antibody to TGF-beta is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.

Animals↗
PubMed2000

Retroviral gene transfer of signaling molecules into murine fetal hepatocytes defines distinct roles for the STAT3 and ras pathways during hepatic development.

We recently demonstrated that oncostatin M (OSM) in the presence of glucocorticoid promotes development of fetal hepatic cells in a primary culture system. Our results also suggested that OSM transduces differentiation signals through gp130, a common subunit of the interleukin (IL)-6 family cytokine receptors. However, an essential downstream pathway required for hepatic development remains unknown. To address this issue, we expressed signal molecules by a retroviral expression vector in primary fetal hepatic cells and investigated a signaling pathway essential for OSM-mediated hepatic development. Expression of a dominant-negative form of STAT3 (DeltaSTAT3), but not DeltaSTAT5, suppressed differentiated phenotypes of hepatocytes induced by OSM. On the other hand, dominant-negative forms of Ras (RasN17) and SHP-2(C463A) rather augmented the expression of hepatic differentiation markers, suggesting that the Ras pathway negatively regulates hepatic development. Consistently, expression of a constitutively activated form of Ras (RasV12) inhibited cellular responses to OSM. Our results indicate that STAT3 is an essential signaling component for OSM-induced hepatic development, while activation of Ras appears to negatively regulate this process. Thus, retrovirus-mediated gene transfer is an effective means to analyze function of a gene in primary fetal hepatic culture.

Animals↗
PubMed2000

Metabolic activation of o-phenylphenol to a major cytotoxic metabolite, phenylhydroquinone: role of human CYP1A2 and rat CYP2C11/CYP2E1.

1. The in vitro metabolic activation of o-phenylphenol has been evaluated as yielding a toxic metabolite, 2,5-dihydroxybiphenyl (phenylhydroquinone), by p-hydroxylation in liver microsomes of rat and human. The involvement of rat CYP2C11, CYP2E1 and human CYP1A2 in the p-hydroxylation of o-phenylphenol is suggested. 2. 2,3- and phenylhydroquinone, which induced DNA single-strand scission in the presence of 1 microM CuCl2, were the most cytotoxic chemicals examined to cultured mammalian cell lines among o-phenylphenol, m-phenylphenol, p-phenylphenol, 2,2'-, 4,4'-, 2,3- and phenylhydroquinone. 3. Rat and human liver microsomes catalysed the formation of phenylhydroquinone, but not 2,3-dihydroxybiphenyl, using o-phenylphenol as a substrate. A higher rate of metabolic activation of o-phenylphenol was observed with livers of the male than the female rats by 5.6- and 2.6-fold respectively. 4. Inhibitory antibodies against the male-specific CYP2C11 inhibited hepatic o-phenylphenol p-hydroxylation in the male F344 and Sprague-Dawley rat by > 70%. Liver microsomes from the isoniazid-treated rats produced 1.8- and 3-fold induction of o-phenylphenol p-hydroxylation and chlorzoxazone 6-hydroxylation (a CYP2E1-dependent activity) respectively. 5. Human CYP1A2, expressed by baculovirus-mediated cDNA expression systems, exhibited a remarkably higher capacity for o-phenylphenol p-hydroxylation at concentrations of 5 (> 5-fold), 50 (> 2-fold) and 500 microM (> 2-fold) than CYP2A, CYP2B, CYP2Cs, CYP2D6, CYP2E1 and CYP3A4 on the basis of pmol P450. 6. Among various CYP inhibitors tested here, 7,8-benzoflavone and furafylline, typical human CYP1A2 inhibitors, inhibited the microsomal p-hydroxylation of o-phenylphenol in human livers most potently by 70 and 50% respectively. 7. The results thus indicate the involvement of rat CYP2C11/CYP2E1 and human CYP1A2 in the hepatic p-hydroxylation of o-phenylphenol.

Animals↗
PubMed2000

Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions.

The SGS1 gene of Saccharomyces cerevisiae is a homologue for the Bloom's syndrome and Werner's syndrome genes. The disruption of the SGS1 gene resulted in very poor sporulation, and the majority of the cells were arrested at the mononucleated stage. The recombination frequency measured by a return-to-growth assay was reduced considerably in sgs1 disruptants. However, double-strand break formation, which is a key event in the initiation of meiotic DNA recombination, occurred; crossover and noncrossover products were observed in the disruptants, although the amounts of these products were slightly decreased compared with those in wild-type cells. The spores produced by sgs1 disruptants showed relatively high viability. The sgs1 spo13 double disruptants sporulated poorly, like the sgs1 disruptants, but spore viability was reduced much more than with either sgs1 or spo13 single disruptants. Disruption of the RED1 or RAD17 gene partially alleviated the poor-sporulation phenotype of sgs1 disruptants, indicating that portions of the population of sgs1 disruptants are blocked by the meiotic checkpoint. The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants.

Adenosine Triphosphatases↗
PubMed2000

Inverted papilloma arising in a juvenile.

A 15-year-old man presented with painless, gross hematuria. Excretory pyelography showed a filling defect in the bladder and ultrasonography revealed a solitary bladder tumor. Cystoscopy showed a solitary, papillary tumor on the bladder neck. Transurethral resection was then performed and histological examination showed an inverted papilloma. In addition, the expression of proliferative cellular nuclear antigen and p53 in the surgical specimen were 37.1 and 0%, respectively. Since an inverted papilloma arising during the first two decades of life is quite rare, we herein report the above case and review previous reports.

Adolescent↗
PubMed2000

Different domains of Sgs1 are required for mitotic and meiotic functions.

The SGS1 of Saccharomyces cerevisiae is a homologue for human Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome causative genes. Disruptants of SGS1 show high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea, and hyper recombination phenotypes including interchromosomal homologous recombination in mitotic growth. In addition, sgs1 disruptants show poor sporulation and a reduced level of meiotic recombination as assayed by return-to-growth. We examined domains of Sgs1 required for mitotic and meiotic functions of Sgs1 by transfecting variously mutated SGS1 into sgs1 disruptants. The N-terminal 1-401 amino acid region was required for complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations of sgs1 disruptants in mitotic growth and for complementation of poor sporulation and of reduced meiotic recombination. Although the N-terminal 1-125 amino acid region was absolutely required for the complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations in mitotic growth, it was dispensable for the meiotic functions. In contrast, the highly acidic region (400-596 amino acid) was dispensable for the mitotic functions but a deletion of this region affected the meiotic functions. The C-terminal 1271-1350 amino acid region containing a HRDC (helicase and RNaseD C-terminal) domain was dispensable for the mitotic and meiotic functions. Although DNA helicase activity of Sgs1 was not required for Sgs1 to complement the meiotic functions, a deletion of helicase motifs III-IV (842-1046 amino acid) abolished the complementing activity of Sgs1, indicating that a structurally intact helicase domain is necessary for Sgs1 to fulfill its meiotic functions.

Alleles↗
PubMed2000

Constitutively active STAT5A and STAT5B in vitro and in vivo: mutation of STAT5 is not a frequent cause of leukemogenesis.

We recently identified several constitutively active forms of signal transducers and activators of transcription 5 (STAT5) using polymerase chain reaction-driven random mutagenesis followed by retrovirus-mediated expression screening. All constitutively active STAT5 showed constitutive phosphorylation on their tyrosine residues and induced factor-independent growth in a mouse interleukin-3-dependent cell line, Ba/F3. Sequence analysis of these active STAT5 revealed two important mutations: S710F and N642H. The N642H mutation localized in the SH2 domain was able to induce autonomous growth of Ba/F3 cells by itself, whereas S710F in the effector domain was able to induce autonomous growth of Ba/F3 cells in concert with a second mutation including H298R and E150G. Recently, constitutive activation of STAT5 has been reported in patients' leukemic cells and is implicated in leukemogenesis. We attempted to clarify whether leukemic cells harbored activating mutations primarily in STAT5 proteins, and analyzed the sequence of STAT5 derived from 49 leukemic patients. No mutations were found, however, in the regions surrounding S710 and N642 of STAT5A and corresponding residues of STAT5B. We also cloned full-length cDNAs for STAT5s from three patients whose leukemic cells exhibited constitutive tyrosine phosphorylation of the STAT5 protein and expressed the derived STAT5 proteins in Ba/F3 cells. However, none of these clones exhibited constitutive tyrosine phosphorylation or gave rise to FI proliferation of Ba/F3 cells. These results indicate that constitutive activation of STAT5 is a secondary event in most leukemias.

Animals↗
PubMed2000

Interaction of nitric oxide and transforming growth factor-beta1 induced by angiotensin II and mechanical stretch in rat renal tubular epithelial cells.

PURPOSE: Changes in intrarenal pressure accompanying unilateral ureteral obstruction can result in tubular mechanical stretch and mediator release from renal tubules. Therefore, we examined the synthesis of nitric oxide and transforming growth factor-beta (TGF-beta), and their interaction in rat renal epithelial cells (NRK-52E) exposed to either angiotensin II or mechanical stretch. MATERIALS AND METHODS: NRK-52E were exposed to either angiotensin II or mechanical stretch. Nitrite and TGF-beta in the supernatant were assessed by the Greiss reaction and bioassay, respectively. The level of cell hypertrophy and intracellular TGF-beta protein was determined by flow cytometry. TGF-beta messenger RNA and inducible nitric oxide synthase protein were detected by reverse transcriptase polymerase chain reaction and Western blot, respectively. RESULTS: Angiotensin II stimulated TGF-beta1 and nitric oxide. The nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NAME) or angiotensin II type I receptor blocker, losartan, inhibited nitric oxide and TGF-beta1 induced by angiotensin II. Flow cytometry showed that either L-NAME or losartan inhibited angiotensin II induced cell hypertrophy. TGF-beta1 inhibited iNOS protein and nitric oxide, whereas an anti-TGF-beta antibody enhanced iNOS. Mechanical stretch induced TGF-beta, inducible NOS protein and nitric oxide. However, TGF-beta expression was not affected by L-arginine or L-NAME when cells were exposed to mechanical stretch. CONCLUSIONS: These results demonstrate that nitric oxide is an intermediate in angiotensin II stimulated TGF-beta1 expression but not in stretch induced TGF-beta expression, and that TGF-beta1 is a negative regulator of nitric oxide in rat renal epithelial cells. The complex interaction of these cytokines may be a target for intervention in the fibrotic and apoptotic processes in the obstructed kidney.

Angiotensin II↗
PubMed1999

A novel class of Na+ and Ca2+ channel dual blockers with highly potent anti-ischemic effects.

A series of novel arylpiperidines (4a-d) which have highly potent blocking effects for both neuronal Na+ and T-type Ca2+ channels with extremely low affinity for dopamine D2 receptors were synthesized. Among these compounds, 1-(2-hydroxy-3-phenoxy)propyl-4-(4-phenoxyphenyl)-piperidine hydrochloride (4c; SUN N5030) exhibited remarkable neuroprotective activity in a transient middle cerebral artery occlusion (MCAO) model.

Animals↗