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Biomedical subjects

N Komatsu

Publications and source records attributed to N Komatsu.

At least 91 records · Page 5Linked to original sources
PubMed1997

Identification of functional domains of the human thrombopoietin receptor required for growth and differentiation of megakaryocytic cells.

To identify the functional domains of the human thrombopoietin (TPO) receptor essential for proliferation and megakaryocytic differentiation, we introduced human wild type c-mpl cDNA and deletion mutants of c-mpl cDNA into the human erythropoietin (EPO)-dependent cell line UT-7/EPO that does not express endogenous c-Mpl. TPO induced the proliferation and megakaryocytic differentiation of UT-7/EPO expressing wild type c-Mpl, as evidenced by increased levels of the CD41 antigen specific for cells of the megakaryocytic lineage and by changes in morphology. Mutational analysis of the cytoplasmic domain of c-Mpl identified four functional regions: (a) two C-terminal regions (amino acids 575-586 and 615-630) containing a domain essential for cell proliferation and megakaryocytic differentiation but not for DNA synthesis; (b) a region (amino acids 587-614) containing a negative domain for TPO-induced cell proliferation and megakaryocytic differentiation; and (c) a region (amino acids 565-574) including a box2 motif that is required for DNA synthesis. These deletion mutants will provide useful materials for analyzing the signals specific for TPO-induced proliferation and megakaryocytic differentiation.

Cell Differentiation↗
PubMed1997

Megakaryocytopoietic activity of a truncated variant of mouse thrombopoietin.

Thrombopoietin (TPO) is a hemopoietic cytokine that specifically stimulates the growth and development of megakaryocytes. In addition to full-length TPO (corresponding to TPO-1), cDNAs of three truncated variants (TPO-2 to 4) have been isolated. However, whether or not these variants have biological activities has not been clarified presumably because of poor secretion into extracellular milieu. To examine the biological significance of the shortest variants, TPO-4, we prepared two fusion cDNAs of TPO-1 and TPO-4, each of which was combined with the finger-growth factor-kringle 1 (FGK1) domains of tissue plasminogen activator (tPA) as a tag and transfected them into baby hamster kidney (BHK) cells. An immunodetection assay for FGK1 antigen revealed that the amount of secreted TPO-4-FGK1 fusion protein was about 2% of that of TPO-1-FGK1. These fusion proteins after affinity purification had comparable activities on a molar basis in both megakaryocyte colony stimulating activity towards mouse bone marrow cells and growth promotion activity towards the TPO-responsive cell line UT-7/GM. These results indicate that the TPO-4 is biologically active in vitro, although its primary structure of the C-terminal half after the 111th amino acid residues is completely different from authentic TPO-1 due to frame shift.

Animals↗
PubMed1997

Cell-cycle-dependent regulation of erythropoietin receptor gene.

To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

Cell Cycle↗
PubMed1997

Protein kinase C and c-myc gene activation pathways in thrombopoietin signal transduction.

Thrombopoietin (TPO) is the major regulator of the proliferation and differentiation of megakaryocyte precursors through interaction with its receptor encoded by the c-mpl protooncogene. We established the human TPO-dependent leukemia cell line, UT-7/TPO (Blood 87, 4552, 1996). In these cells, TPO activated protein kinase C (PKC) in a time dependent manner. Subsequently, the c-myc gene was transiently induced to a maximal level 60-90 minutes after TPO exposure. In addition, we found that stimulating UT-7/TPO cells with TPO rapidly induces the significant accumulation of inositol 1, 4, 5-trisphosphate (Ins-P3), leading to the mobilization of calcium from intracellular stores. Taken together, the activation of PKC and subsequent c-myc gene induction are involved in the TPO-induced cellular response(s), presumably through the activation of PLC.

Calcium↗
PubMed1997

The production of tissue inhibitors of metalloproteinases (TIMPs) in megakaryopoiesis: possible role of platelet- and megakaryocyte-derived TIMPs in bone marrow fibrosis.

We quantified tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in serum and plasma in normal control subjects and patients with a low or high platelet count, using one-step sandwich enzyme immunoassays. The serum levels of TIMP-1 and TIMP-2 were 101.1 +/- 13.3 ng/ml, and 82.7 +/- 26.3 ng/ml, respectively, in normal subjects. In patients with an elevated platelet count, such as in essential thrombocytosis, polycythaemia vera, and myelofibrosis, serum levels of TIMP-1 and TIMP-2 were 351.6 +/- 200.9 ng/ml and 148.9 +/- 84.0 ng/ml, respectively. Serum levels of TIMP-1 and TIMP-2 in patients with a low platelet count, such as in aplastic anaemia and idiopathic thrombocytopenic purpura, were 57.2 +/- 25.8 ng/ml and 19.7 +/- 7.68 ng/ml, respectively. The serum level of TIMP-1 was significantly correlated with the platelet count in all subjects. The correlation between the serum level of TIMP-2 and the platelet count was not as strong. The level of TIMP-1 in platelet-depleted plasma was not correlated with the platelet count. Immunohistochemical staining using monoclonal antibodies against TIMP-1 and TIMP-2 showed that megakaryocytes and platelets were positive for both TIMP-1 and TIMP-2, confirming that they are rich sources of TIMPs. TIMP-1 and TIMP-2 stimulated the proliferation of bone marrow fibroblasts, although their effect was less potent than that of TGF-beta and PDGF. Erythroleukaemia and megakaryoblastic cell lines showed the highest secretion of TIMP-1 among the leukaemia cell lines examined. There was no lineage specificity for TIMP-2 secretion. These results suggest that TIMPs released from megakaryocytes or from local platelet coagulation may be important in the development of bone marrow fibrosis.

Blood Platelet Disorders↗
PubMed1997

Recovery from neuroendocrinological abnormalities and frontal hypoperfusion after remission in a case with rapid cycling bipolar disorder.

A 51-year-old Japanese woman who had been suffering from a rapid cycling affective disorder (RCAD) for 24 years responded to combined clonazepam and carbamazepine therapy. Before remission, she showed neuroendocrinological and neuroimaging abnormalities such as subclinical hypothyroidism with exaggerated response to thyrotropin releasing hormone (TRH) injection, non-suppression on the dexamethasone suppression test (DST) and hypofrontality in cerebral blood flow. Her symptoms improved remarkably soon after adjunctive clonazepam treatment. After remission, her biological markers gradually returned to normal. First, subclinical hypothyroidism improved 2 months after remission. Next, hypofrontality disappeared 18 months later. Furthermore, non-suppression on the DST normalized 24 months later. The normalization of biological markers with apparent recovery from RCAD suggests a decreased risk of relapse into mood disorder. These findings reiterate the importance of following-up on the biological markers in RCAD for years after remission.

Anticonvulsants↗
PubMed1997

Effects of MRI abnormalities on WAIS-R performance in solvent abusers.

OBJECTIVE: To clarify how chronic solvent abuse affects cognitive function using magnetic resonance imaging as an index of brain damage. MATERIAL AND METHODS: A total of 25 chronic solvent abusers underwent magnetic resonance imaging with quantitative neurometry and Wechsler Adult Intelligence Scales revised. RESULTS: The abusers with white matter change (n=10) showed significantly (P<0.05) lower performance IQ, especially in Digit Symbol subtest (P<0.01). Also, the severity of pontine atrophy was significantly correlated with PIQ (r=0.60, P<0.01). CONCLUSIONS: These data indicate that solvent abuse causes the decline in the test performance reflecting cognitive and fine motor dysfunction and that white matter changes and pontine atrophy may have some roles in this decline.

Adult↗
PubMed1997

Inhibitory effect of dideoxyforskolin on cell death induced by ricin, modeccin, diphtheria toxin, and Pseudomonas toxin in MDCK cells.

We have found that 1,9-Dideoxyforskolin (DDF) strongly inhibited the cell death induced by ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in MDCK cells, suggesting that these protein toxins have a DDF-sensitive common pathway leading to cell death. However, no significant effect of forskolin on these toxins was observed, implying that cAMP-independent DDF specific mechanism is responsible for the inhibitory effect. The protective effect of DDF against ricin-induced cell death was significantly reversed by the increase in extracellular Ca2+ concentrations. The addition of brefeldin A (BFA) also reversed the protective effect of DDF, while BFA alone slightly increased the cytotoxicity of ricin. The protein synthesis inhibitory activity of modeccin was strongly inhibited by DDF, while only partial inhibition of the activities of ricin and diphtheria toxin was observed. However, the activity of Pseudomonas toxin was enhanced by DDF rather than inhibited. Thus, the process leading to cell death and protein synthesis inhibition by these toxins may be separately affected by DDF, and the protective effect of DDF against toxin-induced cell death is distinct from its effect on protein synthesis inhibition by toxins. Forskolin and DDF slightly increased rather than inhibited the binding and the internalization of ricin to MDCK cells. Despite the strong inhibitory effect of DDF on toxin-induced cell death, DDF did not block toxin-induced DNA fragmentation. These results suggest that DNA fragmentation and cell death may be triggered through separate pathways during apoptosis caused by these toxins, and that a DDF-sensitive specific step may be present in the pathway leading to cell death.

Animals↗
PubMed1997

Cell lysis induced by ricin D and ricin E in various cell lines.

Ricin D, one of the two isolectins from small caster beans showed stronger cytotoxicity than another one, ricin E, based on the inhibition of colony formation and the inhibition of protein synthesis. Both ricin D and ricin E induced cell lysis to different extents in each cell line tested, albeit ricin E was slightly less effective than ricin D. DNA fragmentation, a characteristic feature of apoptosis, was also induced by ricin D and ricin E in Vero cells. Scatchard plot analysis showed that ricin D binds to cells with higher affinity than ricin E, while the number of binding sites per cell was not much different, suggesting that the differences in the cytotoxicity between ricin D and ricin E is mainly due to their differential binding affinity to cells. In Vero cells, the cytolytic activities of ricin D and ricin E were inhibited by brefeldin A (BFA), which is known to effect the Golgi apparatus, but not significant effect of BFA was observed in a BFA-resistant cell line, MDCK cells. These results suggest that the Golgi apparatus may be involved in ricin-induced cell lysis.

Animals↗
PubMed1997

Tec protein-tyrosine kinase is involved in the thrombopoietin/c-Mpl signaling pathway.

Thrombopoietin (TPO) is the ligand of the proto-oncogene product, c-Mpl, and supports the growth and maturation of megakaryocytic cells. Although much attention has been paid to the in vivo role of the TPO/c-Mpl system in the context of maintenance of circulating platelets, still little is understood about how activation of c-Mpl leads to mitogenesis and differentiation inside cells. Tec protein-tyrosine kinase (PTK) is the prototype of a recently emerging subfamily of nonreceptor type PTKs. Tec has been demonstrated to be activated by a wide range of cytokine stimulations. In this paper we show that TPO stimulation also rapidly enhances tyrosine-phosphorylation and activity of the Tec kinase in a human TPO-dependent cell line. In addition, the Vav protein, a blood cell-specific signaling molecule, is shown to be tyrosine-phosphorylated in response to TPO and to be constitutively associated with the Tec protein. From this evidence, we conclude that Tec is involved in the intracellular signaling system of TPO/c-Mpl.

Cell Cycle Proteins↗
PubMed1997

[Double autologous peripheral blood stem cell transplantation (double APBSCT) for lymphoblastic lymphoma].

A 24-year-old male was admitted to our hospital, complaining of right back pain, in May 1995. Chest X-ray films showed an abnormal mass in the mediastinum. Computed tomography revealed massive effusion in the pleural and pericardial space. A biopsy specimen of the pleural lesion demonstrated lymphoblastic lymphoma of T cell type. After the completion of intensive chemotherapy by our original protocol, he entered into partial remission. Peripheral blood stem cells (PBSC) were harvested using a high-dose cytarabine (Ara-C) followed by granulocyte-colony stimulating factor (G-CSF) mobilization regimen. The total number of collected PBSC was enough to perform two courses of PBSCT. In January 1996, following the conditioning regimen of nimustine hydrochloride, etoposide (VP-16), Ara-C, thiotepa, he received PBSCT. Complete remission was achieved after the 1st PBSCT. In March 1996, he received the 2nd PBSCT following the conditioning regimen of carboplatin, VP-16, ifosfamide. No regimen-related toxity or delayed engrafment was observed. Subsequently, he received irradiation to his neck and mediastinum, the primary site of the disease. As of February 1997, he has no evidence of the disease.

Adult↗
PubMed1997

[Clinical efficacy in pediatrics sinusitis infections of cefditoren pivoxil granule therapy and its in vitro antibacterial activity against clinically isolated strains].

We investigated the clinical efficacy in pediatrics sinusitis infections of cefditoren pivoxil granule therapy and its in vitro antibacterial activity against clinically isolated strains. The results are summarized as follows. The specimens from 343 patients were cultured and 595 strains of bacteria were isolated and identified. Oral doses of 3 and 5 mg/kg of CDTR-PI were clinically effective at high percentages, 85.1% and 89.5%, respectively, of treated patients. CDTR-PI at 3 mg/kg orally was clinically effective in 80.8% of patients with PCG intermediate S. pneumoniae (PISP) infections, 80.0% of those with PCG susceptible S. pneumoniae (PSSP) infections, 81.8% of those with H. influenzae infections and 78.3% of those with M. (B.) catarrhalis infections among the infections by major causative agents. The frequent isolates included S. pneumoniae accounting for 33.1%, H. influenzae accounting for 32.1%, M. (B.) catarrhalis accounting for 17.6% and S. pyogenes accounting 3.7% of all the isolates. PISP accounted for 16.1% of all the isolates and for 49.8% of the isolates of S. pneumoniae, and were isolated from 28.6% of the 343 patients. The isolation of PISP was frequent from children of 4 and under especially, and especially frequent from those below age 2. Of the isolates of S. pneumoniae, the biotype frequencies among PSSP were in the order of type I > type II > type III, while those among PISP were in the order of type I < type II with none of type III. Bacteriologically, an eradication rate of 89.4% was achieved with 3 mg/kg and 93.5% with 5 mg/kg of CDTR-PI.

Administration, Oral↗
PubMed1996

Cloning and characterization of Xenopus laevis xSox7 cDNA.

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding a novel Sox protein from Xenopus laevis ovary. This cDNA contains an open reading frame (ORF) coding for 362 amino acids, which encompasses an HMG box and exhibits a strong (90%) identity to that of mouse Sox7; the cDNA was named xSox7 in this study. Northern analysis revealed that the xSox7 mRNA was 2.0 kb in length. Various adult frog tissues were tested by reverse transcription/polymerase chain reaction for xSox7 mRNA, and the results showed that xSox7 is expressed in a wide range of tissues. Furthermore, electrophoretic mobility shif assay indicated that recombinant xSox7 is capable of binding to AACAAT sequence.

Animals↗
PubMed1996

Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO.

Cell Differentiation↗
PubMed1996

Role of c-jun in the inhibition of erythropoietin receptor-mediated apoptosis.

Human bone marrow cells express both a truncated and full-length form of the erythropoietin receptor (EpoR-T and EpoR-F, respectively). Transfection experiments using the murine interleukin (IL)-3-dependent cell line, Ba/F3, revealed that the cells coexpressing EpoR-F and EpoR-T (Ba/F3-FT) were more likely to undergo programmed cell death (apoptosis) than cells expressing EpoR-F (Ba/F3-FF), even in the presence of erythropoietin (Epo). When Ba/F3-FF cells were stimulated with Epo or IL-3, rapid induction of c-myc, c-fos, c-jun and junB genes was observed. A similar effect was also seen in IL-3 stimulated Ba/F3-Ft cells. However, in Ba/F3-FT cells expression of the c-jun gene was not induced by Epo stimulation. Administration of Epo could prevent apoptosis induced by IL-3 deprivation in Ba/F3-FT cells expressing ectopic c-Jun protein. These results indicate that induction of c-Jun through the Epo signaling pathway has an important role in the inhibition of apoptosis.

Animals↗
PubMed1996

Cloning and expression of Xenopus laevis xSox12 cDNA.

A family of SRY-related genes has been termed SOX. We have isolated and sequenced a cDNA encoding xSox12 from Xenopus laevis ovary. The cDNA contained an open reading frame (ORF) coding for 470 amino acids encompassing an HMG box characteristic of the SOX family, a leucine zipper motif and glutamine-rich segments. The size of the xSox12 mRNA was determined to be 3.0 knt by Northern analysis. The ovary was the most prominent in the expression of the Sox mRNA among the various tissues of adult frog as far as examined.

Amino Acid Sequence↗