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T Suda

Publications and source records attributed to T Suda.

At least 289 records · Page 16Linked to original sources
PubMed1997

Activation of cytosolic phospholipase A2 by platelet-derived growth factor is essential for cyclooxygenase-2-dependent prostaglandin E2 synthesis in mouse osteoblasts cultured with interleukin-1.

The synthesis of prostaglandins (PGs) is regulated by the arachidonic acid release by phospholipase A2 (PLA2) and its conversion to PGs by cyclooxygenase (COX). In the present study, we examined the regulation of PG synthesis by interleukin (IL)-1alpha in primary mouse osteoblastic cells isolated from mouse calvaria. Although IL-1alpha greatly enhanced cox-2 mRNA expression and its protein levels, PGE2 was not produced until 24 h. When arachidonic acid was added to osteoblastic cells precultured with IL-1alpha for 24 h, PGE2 was produced within 10 min. Of several growth factors tested, platelet-derived growth factor (PDGF) specifically initiated the rapid synthesis of PGE2, which was markedly suppressed by a selective inhibitor of cox-2 (NS-398). In mouse osteoblastic cells, cytosolic PLA2 (cPLA2) mRNA and its protein were constitutively expressed and increased approximately 2-fold by IL-1alpha, but secretory PLA2 mRNA was not detected. PDGF rapidly stimulated PLA2 activity, which was blocked completely by a cPLA2 inhibitor (arachidonyltrifluoromethyl ketone). The PDGF-induced cPLA2 activation was accompanied by phosphorylation of its protein. These results indicate that cox-2 induction by IL-1alpha is not sufficient, but cPLA2 activation by PDGF is crucial for IL-1alpha-induced PGE2 synthesis in mouse osteoblasts.

Animals↗
PubMed1997

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Animals↗
PubMed1997

Functional characterization of the promoter for the gene encoding murine CD34.

Since CD34 expression is restricted to the hematopoietic stem cells and decreases in differentiating cells, the analysis of the CD34 promoter is of interest to understand regulation of gene expression in stem cells. To characterize the cis-acting elements which control murine CD34 (mCD34) gene expression, we sought to clone, sequence, and functionally analyze the mCD34 promoter. An 80% decrease in promoter activity was obtained when sequences between -119 bp and -59 bp upstream of the transcriptional start site were deleted. We identified several DNA-protein complexes which correspond to functional segments defined by the linker-scanning mutants. These findings indicated the presence of the important regulatory element between bp -119 and -100, GGTTAAAAGTGAAGTAGGAA. Furthermore, from the result of functional promoter analysis in the DNase I hypersensitive site (HS) located within 5 kb upstream of the mCD34 gene, the presence of the enhancer region in the NcoI/PstI fragment of 5' upstream, -2.8 kb to -1.9 kb, has been identified. These data will provide useful information on the gene transfer using the CD34 promoter and enhancer.

Animals↗
PubMed1997

Bone morphogenetic protein-2 inhibits terminal differentiation of myogenic cells by suppressing the transcriptional activity of MyoD and myogenin.

Bone morphogenetic protein (BMP) is a family of cytokines that induce ectopic bone formation when implanted into muscular tissues. We reported that BMP-2 inhibits the terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells (Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A., and Suda, T. (1994) J. Cell Biol. 127, 1755-1766). In the present study, we examined the molecular mechanism of the inhibitory effect of BMP-2 on terminal differentiation of myogenic cells. When either MyoD or myogenin cDNA was introduced into C3H10T1/2 (10T1/2) cells with a muscle-specific CAT reporter containing four copies of the right E-box of muscle creatine kinase (MCK) enhancer, the CAT activity was dose-dependently suppressed by BMP-2. Furthermore, BMP-2 inhibited the terminal differentiation of these subclonal 10T1/2 cells that stably expressed MyoD or myogenin into mature myotubes that expressed myosin heavy chain and troponin T. The differentiation of a subclone of the MyoD-transfected NIH3T3 cells into mature muscle cells was also inhibited by BMP-2. BMP-2 induced alkaline phosphatase activity in 10T1/2-derived, but not in NIH3T3-derived MyoD-transfected cells. These cells constitutively expressed exogenous MyoD and myogenin, which were localized exclusively in the nuclei irrespective of the presence and the absence of BMP-2. However, these cells failed to express the mRNAs of endogenous myogenic factors and MCK when cultured with BMP-2. In the electrophoresis mobility shift assay using nuclear extracts of the myogenic cells, MyoD and myogenin bound to the right E-box in the enhancer region of the MCK gene even in the presence of BMP-2. These results suggest that BMP-2 inhibits the terminal differentiation of myogenic cells by suppressing the transcriptional activity of the myogenic factors.

3T3 Cells↗
PubMed1997

Lack of vacuolar proton ATPase association with the cytoskeleton in osteoclasts of osteosclerotic (oc/oc) mice.

We examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. An immunoelectron microscopic analysis revealed that in the osteoclasts of these mice, no ruffled borders formed, and that vacuolar H+-ATPase (V-ATPase) was present throughout the cytoplasm but not on the apical membranes. The activity of V-ATPase in oc/oc mice was similar to that in normal mice. In normal spleen cell-derived osteoclast-like cells (OCLs), immunoreactivity for V-ATPase was detected in association with Triton X-100-insoluble cellular structure, but not in oc/oc spleen cell-derived OCLs. Moreover, in renal proximal convoluted tubules of oc/oc mice, the basal striation did not form. These results suggest that osteosclerosis in oc/oc mice is possibly due to the dissociation of V-ATPase and cytoskeleton in osteoclasts.

Adenosine Triphosphatases↗
PubMed1997

Phosphatidylinositol-3 kinase is involved in ruffled border formation in osteoclasts.

Phosphatidylinositol (PI)-3 kinase has been implicated in several aspects of intracellular membrane trafficking, although the detailed mechanism is yet to be established. We previously reported that wortmannin (WT), a selective inhibitor of PI-3 kinase, inhibited the bone-resorbing activity of osteoclasts (Nakamura et al., 1995, FEBS Lett., 361:79-84). In this study, we examined how PI-3 kinase was involved in membrane trafficking in osteoclasts which are primary bone-resorbing cells. Osteoclasts exhibit a highly polarized cytoplasmic organization, the ruffled border. Ruffled borders are formed by numerous deep membrane invaginations, on which vacuolar H(+)-ATPase (V-ATPase) is localized in a high density. Immunoelectron microscopic analyses revealed that PI-3 kinase was specifically present along ruffled border membranes and the limiting membranes of associated intracellular vacuoles in rat authentic osteoclasts. WT and LY294002, another inhibitor of PI-3 kinase, caused the accumulation of numerous acidic vacuoles which were stained with acridine orange in murine osteoclast-like multinucleated cells formed in vitro. An electron microscopic examination showed that these vacuoles contained V-ATPase along their limiting membranes and appeared to be derived from the Golgi apparatus as ruffled border precursors. A time course study revealed that WT-induced vacuoles began to accumulate in the region close to the apical membrane and were finally distributed throughout the cytoplasm. Removal of WT from the culture medium resulted in the disappearance of vacuoles in the cytoplasm, leading to the formation of ruffled borders. During the culture period, some vacuoles were observed to fuse with the ruffled border membrane. A pit formation assay on dentine slices also showed that the pit-forming activity of osteoclast-like cells was recovered by the removal of WT from the assay. These results suggest that PI-3 kinase plays an important role in ruffled border formation in osteoclasts, probably in the fusion of membrane vacuoles with the plasma membrane.

Androstadienes↗
PubMed1997

Can pancreatic steatorrhea be diagnosed without chemical analysis?

CONCLUSION: Visual observation of feces, considering fecal output, is considered to be an excellent method of detection of steatorrhea when judged by experienced doctors. METHODS: Feces from 192 patients with untreated chronic pancreatitis or under pancreatic enzyme therapy were investigated. Feces were collected for three consecutive days and homogenized with water. Fecal samples were freeze-dried and analyzed for fatty acids by gas chromatography (GLC). The quantity of fat was calculated from the amount of fatty acid to obtain daily fecal fat excretion. Comparison of GLC method with van de Kamer method gave a significant (p < 0.01) positive correlation with correlation coefficient of 0.916 (n = 38). Steatorrhea was defined as fecal fat excretion exceeding 5 g/d. Mild steatorrhea was defined as 5-10 g/d, and severe steatorrhea as more than 10 g/d. RESULTS: Three visual identification items were used to consider fecal output exceeding 200 g/d: fecal fat concentration exceeding 4%, appearance, and odor. The results were compared with the results from GLC method. Detection of steatorrhea by means of visual properties was the most accurate, and correlation coefficient was 0.843 (p < 0.01) by Spearman's rank correlation test. This detection method was also significantly effective for differentiation of normal stool from mild and severe steatorrhea. The sensitivity and specificity were 89.3 and 91.1%, respectively, indicating a favorable result.

Celiac Disease↗
PubMed1997

Reduction of oxysterol levels up-regulates HMG-CoA reductase activity in rat liver.

Cholesterol regulates hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity by feedback inhibition. It has been suggested that oxidized derivatives of cholesterol (oxysterols) play an important role, as an intracellular mediator, in the feedback inhibition of cholesterol biosynthesis. We, therefore, investigated the role of intracellular oxysterols in the regulation of HMG-CoA reductase activity. Rats were fed with food (control), cholesterol, clofibrate as a potentiator of the microsomal monooxygenase cytochrome P-450 enzyme system, ketoconazole as a strong inhibitor of the system, or butylated hydroxytoluene (BHT) as an antioxidant. We analyzed and compared hepatic microsomal oxysterol levels among the groups. The results of this study indicated that the oxysterol level, especially 7beta-hydroxycholesterol and 7-ketocholestrol, in the liver was lowered by the administration of ketoconazole and BHT, and HMG-CoA reductase activity was increased in response to these agents. However, there was no change in the HMG-CoA reductase activity, after the administration of clofibrate. We conclude that reduced levels of oxysterol may release the inhibitory effect on the HMG-CoA reductase enzyme and lead to up-regulation of the enzyme.

Animals↗
PubMed1997

Why do defects in the Fas-Fas ligand system cause autoimmunity?

We have previously isolated genes that encode Fas and Fas ligand, a receptor-ligand pair that mediates an apoptotic signal. We also have demonstrated that lpr and gld mice, well-known animal models of autoimmune disease are loss-of-function mutants of the Fas and Fas ligand genes, respectively. Patients with autoimmune lymphoproliferative disorders have been found to bear mutations of the Fas gene. These findings indicate that the Fas-Fas ligand system plays an important role in the maintenance of self-tolerance among both humans and mice. During T-cell development, mouse T cells initially express Fas in the thymus and maintain their expression thereafter. Peripheral B cells usually express Fas at much lower levels than do T cells, but various stimuli enhance Fas expression on B cells. In contrast, among the lymphocyte subsets, only activated T cells and natural killer cells express readily detectable levels of Fas ligand. Reactivation of previously activated T cells through T-cell receptors induces apoptosis. This phenomenon (activation-induced cell death) is mediated by means of the Fas-Fas ligand interaction. We recently discovered that peripheral naive T cells in mice are susceptible to Fas ligand but not to agonistic anti-Fas antibodies. To our surprise, engagement of T-cell receptors on naive T cells was shown to induce Fas ligand resistance. On the basis of these findings and other reports, we discuss how the breakdown of self-tolerance occurs as the result of defects in the Fas-Fas ligand system.

Animals↗
PubMed1997

Tiludronate inhibits protein tyrosine phosphatase activity in osteoclasts.

Signaling pathways mediated by tyrosine phosphorylation and dephosphorylation have been reported to be involved in the regulation of cytoskeletal organization in osteoclasts, the principal cells responsible for bone resorption. We examined the effects of tiludronate [(4-chlorophenyl)thiomethylene bisphosphonate] on the cytoskeleton and the balance of phosphotyrosine levels in osteoclast-like multinucleated cells (OCLs) formed in cocultures of mouse osteoblastic cells and bone marrow cells. When OCLs were placed on plastic dishes in the presence of 10% fetal bovine serum, they formed a ringed structure of F-actin dots (actin ring) within 2 h. Tiludronate did not inhibit the process of actin ring formation, but it disrupted preformed actin rings in a time- and a dose-dependent manner. Western blot analysis using an antiphosphotyrosine antibody revealed that tyrosine phosphorylation of certain proteins in OCLs was stimulated by tiludronate added to the purified OCLs. Tyrosine kinase activity of the p60c-src immunoprecipitated from cell lysates of the purified OCLs was not affected by tiludronate directly added to the kinase assay. OCL lysates stimulated dephosphorylation of tyrosine-phosphorylated substrates such as phosphoneuroprotein 14 and epidermal growth factor receptors. Like sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, tiludronate dose-dependently inhibited tyrosine dephosphorylation of those substrates induced by OCL lysates. These findings suggest that tiludronate disrupts the preformed actin rings and suppresses bone-resorbing activity by inhibiting protein tyrosine phosphatases in osteoclasts.

Actins↗
PubMed1997

Essential roles of the Fas ligand in the development of hepatitis.

The Fas ligand (FasL) is expressed in activated T cells and induces apoptosis in Fas-bearing cells. A cytotoxic T lymphocyte (CTL) clone specific for hepatitis B surface antigen (HBsAg) causes an acute liver disease in HBsAg transgenic mice. Here we observed that the CTL clone killed hepatocytes expressing HBsAg in a Fas-dependent manner. Administration of the soluble form of Fas into HBsAg transgenic mice prevented the CTL-induced liver disease. In the second model, mice were primed with Propionibacterium acnes. A subsequent challenge with lipopolysaccharide (LPS) killed the mice by inducing liver injury. Neutralization of FasL rescued the mice from LPS-induced mortality, and Fas-null mice were resistant to LPS-induced mortality. These results suggest that FasL has an essential role in the development of hepatitis.

Animals↗
PubMed1997

Lymphohaematopoietic abnormalities and systemic lymphoproliferative disorder in interleukin-2 receptor gamma chain-deficient mice.

Interleukin-2 (IL-2) receptor gamma chain-deficient mice with a truncated mutation showed the absence or severe reduction of natural killer cells, decreased numbers of T- and B-cells, marked hypoplasia of the thymus and peripheral lymphoid tissues, defective formation of lymphoid follicles and germinal centre in the peripheral lymphoid tissues, and the absence of Peyer's patches in the intestinal mucosa. In addition, marked splenomegaly with extramedullary haematopoiesis, increased level of IgM and decreased levels of IgG and IgE in serum, severe reduction of conventional B cells (B-2) in the peripheral lymphoid tissues, the presence of IgM-producing CD5+ B cells (B-1) and their differentiation into plasma cells and Motto cells in the spleen, and increased production and differentiation of macrophages in various tissues were found in the mutant mice. However, the development of both marginal metallophilic macrophage populations in the spleen and of their related macrophages in the other tissues of the mutant mice was severely impaired. All these abnormalities seem to be induced by the loss-of-function of the IL-2 receptor gamma chain. From 8 weeks of age on, inflammatory changes occurred in the intestines, mesenteric lymph nodes, lungs, liver, and kidneys of the mutant mice. Besides the absence of Hassall's corpuscles, thymic cysts were frequently observed in the mutant mice. These pathological abnormalities suggest that the gamma chain is implicated not only in lymphoid and haematopoietic development but also in thymic epithelial cell ontogeny.

Animals↗
PubMed1997

Deregulated inflammatory response in mice lacking the STK/RON receptor tyrosine kinase.

Immune and inflammatory responses must be rightly regulated to maintain a homoeostatic balance between an effective immune response and tissue damage to the host. NO is a principal mediator of many of the cytokine-inducible macrophage activities during a normal cell-mediated immune response. STK, the murine homologue of the human RON receptor tyrosine kinase, is expressed on murine resident peritoneal macrophages. The ligand for STK, macrophage-stimulating protein (MSP), is a serum protein that is activated by members of the coagulation cascade in response to tissue damage. In addition to its potential to induce chemotaxis and phagocytosis of C3bi-coated erythrocytes, MSP has an inhibitory effect on the production of NO by activated peritoneal macrophages in vitro. Here we demonstrate that peritoneal macrophages from mice lacking STK produce elevated levels of NO in response to interferon (IFN)-gamma in a dose-dependent manner, without the need for a co-stimulus. However, production of pro-inflammatory cytokines by activated macrophages from stk -/- mice is unaltered. In vivo, stk -/- mice exhibit increased inflammation in an IFN-gamma-mediated delayed-type hypersensitivity reaction and increased susceptibility to lipopolysaccharide (LPS)-induced endotoxic shock. Furthermore, the levels of NO in the serum of mice injected with LPS are significantly higher than those in control littermates. Nevertheless, the serum levels of IFN-gamma and the intermediate cytokines generated by the inflammatory response, which have previously been shown to play a role in septicaemic shock, do not differ significantly from controls. These data suggest that the STK receptor suppresses NO production, therefore ameliorating the potentially tissue-damaging effects of a cell-mediated immune response, through negative regulation of the IFN-gamma signalling pathway.

Animals↗
PubMed1997

Flow cytometric analysis of nuclear DNA heterogeneity in gastric cancer.

Flow cytometric analysis of nuclear DNA ploidy was performed to evaluate the clinical significance of DNA-ploidy heterogeneity and DNA-index heterogeneity between the superficial layer and the deep layer of the tumor obtained from 88 advanced gastric cancer patients. DNA-ploidy heterogeneity was observed in 28 patients (31.8%) and characterized mainly by diploidy in the superficial layer and aneuploidy in the deep layer. More than 10% difference in the DNA index among aneuploidy (DNA-index heterogeneity) was observed in 10 (26.3%) of 38 patients with aneuploidy. There was no tendency for the DNA index to increase with deep infiltration. DNA-ploidy and DNA-index heterogeneities were not correlated with the various clinicopathological characteristics. Patients with aneuploidy had significantly poorer prognosis than did those with diploidy. The survival rate for patients with DNA heterogeneity was not significantly different from that for patients without DNA heterogeneity. These results suggest that the DNA-ploidy pattern may be an important prognostic factor, but that DNA heterogeneity may not have an impact on the survival in advanced gastric cancer.

Aneuploidy↗
PubMed1997

Telomerase activity and metastasis: expansion of cells having higher telomerase activity within culture lines and tumor tissues.

Tumor cells with metastatic potential may have a high telomerase activity that augments telomeric DNA repeats, allowing the cells to escape from the inhibition of cell proliferation due to shortened telomeres. We examined the expression level of telomerase activity using the telomeric repeat amplification protocol among a series of cell lines obtained by repeated transplantation of a mouse fibrosarcoma. The lines could be grouped into three; one has no metastatic potential, and the other two show metastatic abilities after intravenous or subcutaneous injection. Comparison of their telomerase activity indicated that more malignant lines had higher activity. A similar relation was seen in metastatic nodules formed through clonal expansion from the heterogeneous population of inoculated cells; clonality was monitored in terms of variable patterns of subtelomeric repeats. The results suggest that a high level of telomerase activity may not be requisite for metastasis, but may confer a propensity to dominate in a tumor tissue.

Animals↗
PubMed1997

Expression of estrogen receptor beta in rat bone.

A novel estrogen receptor, estrogen receptor beta (ERbeta), has recently been cloned from a rat prostate cDNA library. In bone, which is an important target tissue of estrogen, ER alpha has been reported to be present preferentially in osteoblasts, but the mechanism of action of estrogen in bone is still not known. In the present study, we examined expression of ERbeta mRNA in bone. Expression of ERbeta mRNA was evident in primary osteoblastic cells isolated from 1-day-old rat calvaria and rat osteosarcoma cells (ROS 17/2.8), and its level was higher than that of ER alpha mRNA. When osteoblastic cells were cultured for 28 days to induce differentiation into mature osteoblasts capable of forming bone nodules, ERbeta mRNA was constantly and highly expressed during the entire culture period. In contrast, the level of ER alpha mRNA was very low at the beginning of culture and it gradually increased during the differentiation of osteoblastic cells. Various tissues including bone were isolated from 8-week-old rats of both sexes, and total RNA was extracted to compare the tissue distribution of expression levels of ERbeta mRNA. In cancellous bone of the distal femoral metaphysis and lumbar vertebra, expression of ERbeta mRNA was obvious, and its level was equivalent to those in the uterus and testis, but lower than those in the ovary and prostate. The level of ERbeta mRNA in femoral cortical bone was lower than that in cancellous bone. There was no appreciable differences between female and male rats in the distribution and expression levels of ERbeta mRNA in bone. These results indicate that ERbeta mRNA is highly expressed in osteoblasts in rat bone, suggesting that there is a distinct mechanism of estrogen action mediated by ERbeta in bone.

Animals↗