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T Nakamura

Publications and source records attributed to T Nakamura.

At least 19 recordsLinked to original sources
PubMed1992

Hepatocyte growth factor is linked by O-glycosylated oligosaccharide on the alpha chain.

The glycosylation site and the structure of O-glycosylated oligosaccharide of recombinant human HGF were investigated. N-acetylgalactosamine (GalNAc) in the alpha chain suggested the presence of O-glycosylated oligosaccharide. Sugar analysis and amino acid sequence analysis of peptide fragments produced by limited degradation revealed that O-glycosylated oligosaccharide linked to Thr445 of the alpha chain. The molecular weight of the oligosaccharide was determined with ion spray mass spectrometry. From these studies, the structure of the O-glycosylated oligosaccharide on the alpha chain of HGF was concluded as [formula: see text].

Amino Acid Sequence↗
PubMed1992

Proteolytic activation of a single-chain precursor of hepatocyte growth factor by extracellular serine-protease.

Hepatocyte growth factor (HGF) is biosynthesized as a single-chain precursor (pro-HGF) and is proteolytically processed to a two-chain mature form. When MRC-5 fibroblasts were pulse-radiolabeled under serum-free conditions, pro-HGF was the predominant molecular form of HGF in the culture medium. CHO cells transfected with an expression plasmid containing a full-size human HGF cDNA produced pro-HGF when these cells were cultured in serum-free medium. These findings suggest that HGF is secreted as a pro-form, which is then converted to a two-chain form by extracellular protease. Single-chain HGF exhibited mitogenic activity on cultured hepatocytes, with a potency similar to that of mature HGF, but this activity was remarkably inhibited by leupeptin. We postulate that inactive pro-HGF is converted to an active two-chain form by a leupeptin-sensitive serine-protease expressed by hepatocytes. Neither plasminogen activators nor plasmin showed any processing activity of pro-HGF in vitro.

Animals↗
PubMed1992

Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression.

Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 ng/ml and maximal at 5 ng/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha, whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth.

Animals↗
PubMed1992

Negative regulation of hepatocyte growth factor gene expression in human lung fibroblasts and leukemic cells by transforming growth factor-beta 1 and glucocorticoids.

Hepatocyte growth factor (HGF), a mesenchymal-derived factor which regulates growth, motility, and morphogenesis of epithelial and endothelial cells, functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. We have now obtained evidence that transforming growth factor-beta 1 (TGF-beta 1) and glucocorticoids are negative regulators for HGF gene expression. When TGF-beta 1 or dexamethasone was added to cultures of MRC-5 human embryonic lung fibroblasts and HL-60 human promyelocytic leukemic cells, the amount of HGF secreted into the culture medium was inhibited to 30-40% of that of control cultures by 10 ng/ml TGF-beta 1 and to 40-50% by 10(-6) M dexamethasone. The inhibitory effect of TGF-beta 1 and dexamethasone on HGF synthesis in MRC-5 cells was additive, thereby suggesting that TGF-beta 1 and dexamethasone exert effects through distinct mechanisms. Hydrocortisone also inhibited HGF synthesis with the same potency as dexamethasone; however, testosterone, estriol, and beta-estradiol had no effect. The rate of HGF synthesis in MRC-5 cells, as measured by pulse labeling with [35S]methionine and subsequent immunoprecipitation, was suppressed to 30-40% of the control with 10 ng/ml TGF-beta 1, and to 30-45% by 10(-6) M dexamethasone. HGF mRNA levels in MRC-5 cells and HL-60 cells were dose-dependently suppressed by TGF-beta 1 and dexamethasone; 10 ng/ml TGF-beta 1 suppressed HGF mRNA levels to 32% and 35% of control culture, respectively, in MRC-5 cells and HL-60 cells, and 10(-6) M dexamethasone suppressed to 43% and 38%, respectively. Thus, TGF-beta 1 and glucocorticoids seem to inhibit HGF synthesis by suppressing the expression of the HGF gene. We propose that a negative regulation of HGF gene expression by TGF-beta 1 or glucocorticoids may be involved in physiological or pathological processes during tissue regeneration.

Animals↗
PubMed1992

Hepatocyte growth factor down-regulates the alpha-fetoprotein gene expression in PLC/PRF/5 human hepatoma cells.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the alpha-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of beta-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.

Animals↗
PubMed1992

Developmental changes in hepatocyte growth factor mRNA and its receptor in rat liver, kidney and lung.

Hepatocyte growth factor (HGF) is a mesenchymal-derived factor which induces mitosis, cell movement and morphogenesis of tissue-like structure. We analyzed changes in HGF mRNA and its receptor, the c-met proto-oncogene product, in the liver, kidney and lung during late fetal and postnatal development in rats. In the liver, the HGF-mRNA level was very low during late gestation and in neonates, it increased remarkably and reached a maximum two weeks postnatally, to be followed by a decrease to 33% of the maximum. HGF mRNA in the kidney and lung was either undetectable or very low during late gestation and the neonatal period and increased markedly to reach a maximum, respectively, 3-4 weeks postnatally. HGF-mRNA level in the adult rat lung was fivefold higher than that in the liver and kidney. The number of HGF receptors on plasma membranes of these tissues was low in neonates but there was a rapid increase after birth and a maximum was reached within three weeks. The number of HGF receptors/ng plasma membrane protein at the maximal level was highest in the liver and lowest in the lung. c-met/HGF-receptor mRNA in the liver was also low during late-gestation or in early neonatal periods and increased postnatally. Since HGF-mRNA and HGF-receptor levels changed differently in liver, kidney and lung, the expression of HGF and its receptor may be independently regulated in each organ. However, in these organs, HGF mRNA and the HGF receptor increased within a few weeks of birth, HGF may play roles in organ growth, organ maturation and the maintenance of tissue homeostasis during the postnatal period, presumably through its potential to act as mitogen, motogen and morphogen.

Aging↗
PubMed1992

The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene.

The ALL-1 gene located at human chromosome 11 band q23 is rearranged in acute leukemias with interstitial deletions or reciprocal translocations between this region and chromosomes 1, 4, 6, 9, 10, or 19. The gene spans approximately 100 kb of DNA and contains at least 21 exons. It encodes a protein of more than 3910 amino acids containing three regions with homology to sequences within the Drosophila trithorax gene, including cysteine-rich regions that can be folded into six zinc finger-like domains. The breakpoint cluster region within ALL-1 spans 8 kb and encompasses several small exons, most of which begin in the same phase of the open reading frame. The t(4;11) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-1 and from a gene on chromosome 4. This suggests that each 11q23 abnormality gives rise to a specific oncogenic fusion protein.

Amino Acid Sequence↗
PubMed1992

The (4;11)(q21;q23) chromosome translocations in acute leukemias involve the VDJ recombinase.

Chromosomal region 11q23 is frequently rearranged in acute lymphocytic leukemias (ALLs) and in acute myeloid leukemias (AMLs), mostly in reciprocal exchanges with various translocation partners. The most common of these translocations is t(4;11)(q21;q23). It is present in approximately 10% of ALL patients, most frequently in very young children. We have recently cloned a region of chromosome 11, the ALL-1 locus, found to be rearranged in malignant cells from patients with the t(4;11), t(9;11), t(11;19), t(1;11), t(6;11), t(10;11), and del(11q23) chromosomal abnormalities. Here we report the cloning and characterization of chromosomal breakpoints from leukemic cells with t(4;11) aberrations. The breakpoints cluster in regions of 7-8 kilobases on both chromosomes 4 and 11. The presence of heptamer- and nonamer-like sequences at the sites of breakage suggests that the VDJ recombinase utilized for immunoglobulin gene rearrangement is also directly involved in these translocations. We also show that leukemic cells with t(4;11) express altered RNAs transcribed from the derivative chromosomes 11 and 4.

Acute Disease↗
PubMed1992

Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts.

Hepatocyte growth factor (HGF) functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. When 1 ng/ml of interleukin-1 alpha (IL-1 alpha) or interleukin-1 beta (IL-1 beta) was added to cultures of human skin fibroblasts, the production of HGF was 5-6 fold higher than levels in the controls. HGF mRNA level in the cells was increased to 4-fold higher levels at 6 h after exposure to IL-1 alpha. Tumor necrosis factor-alpha and interferon-gamma but no other cytokine tested had slightly stimulatory effects on HGF production. The tumor promoter, tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the stimulatory effect of IL-1 alpha and IL-1 beta on the production of HGF. The stimulatory effect of both IL-1 alpha and IL-1 beta and the synergistical stimulation with TPA were completely abrogated by 10 ng/ml TGF-beta 1 or 1 microM dexamethasone. These results suggest that IL-1 alpha and IL-1 beta are positive regulators for expression of the HGF gene and are likely have a role in regeneration of tissues following the occurrence of inflammatory diseases.

Animals↗
PubMed1992

Hepatocyte growth factor increases urokinase-type plasminogen activator (u-PA) and u-PA receptor expression in Madin-Darby canine kidney epithelial cells.

We have recently demonstrated that fibroblast-conditioned medium induces Madin-Darby canine kidney (MDCK) epithelial cells to form branching tubules when grown in three-dimensional collagen or fibrin gels (Montesano, R., Schaller, G., and Orci, L. (1991) Cell 66, 697-711), and that this morphogenetic effect is mediated by hepatocyte growth factor (HGF), also known as scatter factor (Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908). In fibrin gels, this effect is inhibited by addition of exogenous serine protease inhibitors, which suggests a role for plasminogen activators (PAs) in the matrix remodeling required for tubulogenesis. In the studies reported in this paper, we have investigated the effect of fibroblast-conditioned medium (CM) and HGF on the production of PAs by MDCK cells. We have found that urokinase-type PA (u-PA) activity and mRNA are increased 4.9-fold by CM from human MRC-5 fibroblasts, which has tubulogenic activity, but not by CM from human Detroit-550 fibroblasts, which lacks tubulogenic activity. The u-PA inductive property of MRC-5 CM was completely inhibited by preincubation with antibodies to recombinant human HGF (rhHGF). Exogenously added rhHGF also increased u-PA activity and mRNA 5.9-fold in MDCK cells, with an optimal effect at approximately 10 ng/ml. MRC-5 CM also increased u-PA receptor mRNA 34.9-fold in MDCK cells, an effect which was inhibited by 71% by preincubating the CM with antibodies to rhHGF, and which was mimicked by exogenously added rhHGF (31.3-fold increase). These results demonstrate that HGF, which induces tubulogenesis by MDCK cells in vitro, also increases u-PA and u-PA receptor expression in these cells. Taken together with our previous observations, this suggests that the resulting increase in extracellular proteolysis, appropriately localized to the cell surface, is required for epithelial morphogenesis.

Animals↗
PubMed1992

Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.

Animals↗
PubMed1992

Role of the adenylate cyclase, phosphoinositidase C and receptor tyrosyl kinase systems in the control of hepatocyte proliferation by hepatocyte growth factor.

Hepatocyte growth factor (HGF) is the most potent known mitogen for hepatocytes in primary culture. However, the mechanisms through which HGF induces hepatocyte proliferation have not been defined. Here we have investigated the role of the adenylate cyclase, phosphoinositidase C and tyrosine kinase signalling systems in the control of hepatocyte proliferation by HGF using freshly isolated or cultured adult rat hepatocytes. We show that human recombinant HGF caused a dose-dependent increase in hepatocyte DNA synthesis with a maximal effect at 10 ng/mL and an EC50 of 5.9 ng/mL. HGF had no effect on hepatocyte adenylate cyclase activity or intracellular cAMP levels. Elevation of hepatocyte cAMP levels resulted in inhibition of HGF-stimulated DNA synthesis. HGF stimulated inositol phospholipid hydrolysis with a maximal effect at 25 ng/mL and potentiated the effect of vasopressin (10(-8) and 10(-9)M). HGF (100 ng/mL) caused an increase in the phosphorylation on tyrosine of an unknown hepatocyte protein with a molecular mass of 36 kDa. Thus, we have shown that HGF, like epidermal growth factor (EGF), can activate the phosphoinositidase C and tyrosine kinase systems in rat hepatocytes. As with EGF, these intracellular signalling systems may underlie HGF-induced hepatocyte proliferation.

Adenylyl Cyclases↗
PubMed1992

Heterogeneity of immunoglobulin-associated molecules on human B cells identified by monoclonal antibodies.

Two covalently linked transmembrane molecules, encoded in mice by the mb-1 and B29 genes, have been defined as integral components of the antibody receptor units expressed on B cells. We have produced monoclonal antibodies against an exposed extracellular epitope on the putative human equivalent of the mouse B29 product. These antibodies, CB3-1 and -2, were used to show that cytoplasmic expression of this molecule begins in human pro-B cells (terminal deoxynucleotidyltransferase-positive, mu chain-negative), whereas surface expression coincides strictly with surface immunoglobulin expression of all isotypes. Immunochemical analysis of the human immunoglobulin-associated molecules revealed greater molecular heterogeneity than has been noted for the murine analogues. This molecular heterogeneity of immunoglobulin-associated molecules varied as a function of differentiation stage and the immunoglobulin isotypes expressed by B-lineage cells. Our data support the hypothesis that biochemical heterogeneity of the surface immunoglobulin-associated molecules may contribute to the variability in biological effects of antigen receptor crosslinkage on B cells of different maturational stages. Because the CB3 antibodies are capable of down-modulating the antigen receptors on all B cells, they may prove therapeutically useful as universal B-cell suppressants.

Antibodies, Monoclonal↗
PubMed1992

Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity.

The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa. The amino acid sequence of the first 18 NH2-terminal residues revealed that the receptor is a member of the activin receptor family. The purified receptor phosphorylated itself and exogenous substrate proteins on serine, threonine, and tyrosine residues, indicating that the activin receptor is a transmembrane serine/threonine/tyrosine protein kinase. These results suggest that signal transduction of activin employs a novel pathway via a new class of cellular receptor in EC P19 cells.

Activin Receptors↗
PubMed1992

Ca2+/calmodulin-activated protein phosphatase (PP2B) of Saccharomyces cerevisiae. PP2B activity is not essential for growth.

Protein phosphatase (PP2B) whose activity is stimulated 12-20-fold by Ca2+/calmodulin (CaM) was partially purified by CaM-Sepharose and heparin-agarose chromatographies from cell extract of the yeast Saccharomyces cerevisiae. PP2B activity was not detectable in a mutant in which two genes (CMP1 and CMP2) encoding homologs of mammalian PP2B catalytic subunit were disrupted. We have previously shown that the double gene disruption has no significant effect on the growth of yeast [1991, Mol. Gen. Genet. 227, 52-59]. The results indicated that CMP1 and CMP2 are the only genes that encode the PP2B catalytic polypeptide in S. cerevisiae, and PP2B activity is not essential for the growth of the yeast under normal conditions.

Calcium↗
PubMed1992

Marked induction of hepatocyte growth factor mRNA in intact kidney and spleen in response to injury of distant organs.

Hepatocyte growth factor (HGF) is a potent mitogen for various epithelial cells, including mature hepatocytes and renal tubular cells. Here, HGF mRNA was found to be markedly increased in non-injured kidney and spleen, when the liver or kidney in rats was injured by 70% partial hepatectomy or unilateral nephrectomy. HGF mRNA increased to 3-4 fold higher level than the normal in the kidney and spleen as well as in the remnant liver after partial hepatectomy. Similarly, HGF mRNA markedly increased in the spleen as well as in the remnant kidney after unilateral nephrectomy. These results suggest that the onset of injury to the liver or kidney may be recognized by distal non-injured organs by the signalling of a humoral factor and that HGF derived from these organs may be involved in the regeneration of liver or kidney, through an endocrine mechanism.

Animals↗