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Biomedical subjects

T Tsujimura

Publications and source records attributed to T Tsujimura.

At least 73 records · Page 4Linked to original sources
PubMed1996

Role of aspartic acid 814 in the function and expression of c-kit receptor tyrosine kinase.

The c-kit receptor tyrosine kinase (KIT) is constitutively activated in three different types of neoplastic mast cell lines by naturally occurring mutations that result in substitutions of Val or Tyr for Asp814 in the phosphotransferase domain. In an effort to characterize the role of the Asp814 residue, we have investigated the properties of mutant KITs in which the Asp814 residue was deleted or mutated to a series of other amino acids. With the exception of rare instances, mutant KITs with substitutions of Asp814 were found to be constitutively phosphorylated on tyrosine and activated in the absence of the ligand, stem cell factor (SCF), whereas a deletion mutant lacking Asp814 (KITDel-Asp-814) did not exhibit tyrosine phosphorylation and activation even after treatment with SCF. In addition to constitutive activation, furthermore, both highly activated substitution mutants (KITVal-814 and KITTyr-814) and modestly activated substitution mutants (KITGly-814 and KITHis-814) were continuously degraded in the absence of SCF, whereas wild-type KIT (KITWild) required SCF stimulation to undergo degradation. These results suggested that the Asp814 residue may play a crucial role in regulating enzymatic activity and expression of KIT and that various types of mutations at the Asp814 residue may generate oncogenic protein with constitutive activation and degradation.

Amino Acid Sequence↗
PubMed1996

Expression of multiple c-kit receptor messenger ribonucleic acid transcripts during postnatal development of the rat testis.

The c-kit protooncogene is a transmembrane tyrosine kinase receptor expressed during gametogenesis. Using the polymerase chain reaction (PCR), we have identified the c-kit receptor mRNA transcripts in the rat testis and studied their expression during postnatal development of the testis. Five different transcripts were identified using sets of primers encoding within the extracellular domain. Two transcripts were obtained from primer sets encoding regions within the cytoplasmic domain and the primer set encoding the entire length of the c-kit receptor. We have compared the levels of expression of these transcripts on different days during postnatal development. The level of expression of a particular transcript varied depending upon the developmental stage of the testis. In summary, our results suggest that multiple forms of mRNAs exist for the c-kit receptor in the rat testis, and they are regulated differentially during postnatal development.

Aging↗
PubMed1996

Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain.

A peculiar point mutation results in constitutive activation of c-kit receptor tyrosine kinase (KIT) in three different tumor mast cell lines; ie, the HMC-1, P-815, and RBL-2H3. Because constitutive activation of KIT was also observed in the FMA3 mouse mastocytoma cell line, we investigated the molecular mechanism. Sequencing of the whole coding region of the c-kit showed that the point mutation found in HMC-1, P-815, and RBL-2H3 cells was absent in FMA3 cells and that the c-kit cDNA of FMA3 cells carried an in-frame deletion of 21 base pairs (bp) encoding Thr-Gln-Leu-Pro-Tyr-Asp-His at codons 573 to 579 at the juxtamembrane domain. The FMA3-type c-kit cDNA with 21 bp deletion was introduced into the IC-2 cell line, which was derived from murine cultured mast cells. IC-2 cells were dependent on interleukin (IL)-3 and did not express KIT on the surface. In IC-2 cells introduced with the FMA3-type c-kit cDNA, KIT was constitutively phosphorylated on tyrosines and activated. Moreover, the FMA3-type KIT was dimerized without the stimulation by stem cell factor (SCF), a ligand for KIT. The spontaneously dimerized FMA3-type KIT without SCF binding was not internalized even after the activation. IC-2 cells expressing the FMA3-type KIT grew in suspension culture without IL-3 and SCF and became leukemic in nude athymic mice. The deletion of seven amino acids at the juxtamembrane domain appeared to be a new activating mutation of KIT that might be involved in neoplastic growth of mast cells.

Amino Acid Sequence↗
PubMed1996

O6-methylguanine induces intrachromosomal homologous recombination in human cells.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which alkylates many positions in DNA including the 06 position of guanine, efficiently induces intrachromosomal homologous recombination in mouse L-cells. To investigate the role of 06-methylguanine in the induction of homologous recombination in human cells, three cell strains containing duplicated copies of the Herpes simplex virus I thymidine kinase (Htk) gene and three cell strains containing duplicated copies of the gene coding for hygromycin phosphotransferase (hyg) were treated with MNNG. Neither the Htk genes nor the hyg genes code for a functional enzyme because each contains an insertion mutation at a unique site, i.e. 8-bp XhoI linker insertions in the Htk genes and 10-bp HindIII linker insertions in the hyg genes. These cell strains differ in their level of 06-alkylguanine-DNA alkyltransferase (AGT), which specifically removes the methyl group from the 06 position of guanine. Generation of a functional Htk or hyg gene has been shown to require intrachromosomal homologous recombination between the two mutant Htk genes or the two mutant hyg genes. In all six cell strains, MNNG induced a dose-dependent increase in the frequency of homologous recombination. In each set, there was an inverse correlation between the frequency of MNNG-induced recombination and the level of AGT activity. To further study the role of 06-methylguanine in the induction of homologous recombination, we used 06-benzylguanine to inactivate AGT in two additional human cell strains containing the hyg recombination substrate. After depletion of AGT activity by 06-benzylguanine, both cell strains showed a significantly elevated level of MNNG-induced homologous recombination. These results indicate that 06-methylguanine is the principal lesion responsible for the induction of homologous recombination in these human cells by this methylating agent.

Cells, Cultured↗
PubMed1996

Inhibition of growth and metastasis of ovarian carcinoma by administering a drug capable of interfering with vascular endothelial growth factor activity.

The present study investigates the relationship between in vivo growth/metastasis of tumor cells and their capacity to produce the vascular endothelial growth factor (VEGF), as well as the regulation of tumor growth/metastasis using an angiogenesis-inhibitory drug. Two cloned tumor cell lines designated OV-LM and OV-HM were isolated from a murine ovarian carcinoma OV2944. OV-LM and OV-HM cells grew in cultures at comparable rates. However, when transplanted s.c. into syngeneic mice, OV-HM exhibited a faster growth rate and a much higher incidence of metastasis to lymph nodes and lung. Histologically, intense neovascularization was detected in sections of OV-HM but not of OV-LM tumor. OV-HM and OV-LM tumor cells obtained from in vitro cultures expressed high and low levels of VEGF mRNA, respectively. A difference in VEGF mRNA expression was much more clearly observed between RNAs prepared from fresh OV-HM and OV-LM tumor masses: RNA from OV-HM contained larger amounts of VEGF mRNA, whereas RNA from OV-LM exhibited only marginal levels of VEGF mRNA. An angiogenesis-inhibitory drug, FR118487 inhibited the VEGF-mediated in vitro growth of endothelial cells but did not affect the expression in vitro of VEGF mRNA by OV-HM tumor cells. Intraperitoneal injections of FR118487 into mice bearing OV-HM tumors resulted in: (i) a subsequent growth inhibition of primary tumors; (ii) a marked decrease in neovascularization inside tumor masses expressing comparable levels of VEGF mRNA to those detected in control OV-HM masses; and (iii) almost complete inhibition of metastasis to lymph nodes and lung. These results indicate that growth/metastasis of tumor cells correlates with their VEGF-producing capacity and that an angiogenesis inhibitor, FR118487, inhibits tumor growth and metastasis through mechanism(s) including the suppression of VEGF function in vivo.

Animals↗
PubMed1996

Role of c-kit receptor tyrosine kinase in the development, survival and neoplastic transformation of mast cells.

The c-kit gene is allelic with the dominant spotting (W) locus on mouse chromosome 5 and encodes a receptor tyrosine kinase. The ligand for c-kit receptor is stem cell factor (SCF), which is the principal growth factor for mast cells. The loss-of-function mutations of c-kit receptor affect the development of mast cells, thereby resulting in a depletion of mast cells. The abundant expression of c-kit receptor is indispensable for the survival of mast cells. In addition, the gain-of-function mutations of c-kit receptor were found in several tumor mast cell lines. When these gain-of-function mutations were introduced to cells of murine interleukin (IL)-3-dependent cell lines, the expression of c-kit receptor with constitutive tyrosine kinase activity not only abrogated the IL-3 requirement of the cells, but also caused them to become tumorigenic in nude athymic mice. The gain-of-function mutations of c-kit receptor appear to result in the malignant transformation of mast cells. Taken together, the signals from the c-kit receptor are essential for the development, survival, and malignant transformation of mast cells.

Animals↗
PubMed1996

Phosphaturic mesenchymal tumor, mixed connective tissue variant (oncogenic osteomalacia).

A case of tumor-induced phosphaturic osteomalacia in a 54 year old man is reported. The patient was admitted because of progressive muscle spasms with pain and weakness in the bilateral thighs. Laboratory data showed hypophosphatemia, decreased tubular resorption of phosphate (TRP), a low 1,25-dihydroxyvitamin D level, and a high serum alkaline phosphatase level. Radiologic examinations revealed multiple lesions of osteomalacia in the ribs, and a small mass in the lower posterior mediastinum. After removal of the tumor, clinical symptoms disappeared and hypophosphatemia, decreased TRP, and the 1,25-dihydroxyvitamin D level were corrected. Microscopical examination revealed that the tumor was composed of mature adipose tissues, osseous tissues, and primitive stromal zones including osteoclast-like giant cells, non-mineralized woven bone, and various sized blood vessels. Patho-physiologic observations suggested that the tumor secreted some humoral substances inhibiting 25-hydroxyvitamin D-1 alpha-hydroxylase activity, renal phosphate resorption, and parathyroid hormone production.

Alkaline Phosphatase↗
PubMed1996

Transforming and differentiation-inducing potential of constitutively activated c-kit mutant genes in the IC-2 murine interleukin-3-dependent mast cell line.

Two mutations of c-kit receptor tyrosine kinase (KIT), valine-559 to glycine (G559) and aspartic acid-814 to valine (V814), resulted in its constitutive activation. To examine the transforming and differentiation-inducing potential of the mutant KIT, we used the murine interleukin-3-dependent IC-2 mast cell line as a transfectant. The IC-2 cells contained few basophilic granules and did not express KIT on the surface. The KITG559 or KITV814 gene was introduced into IC-2 cells using a retroviral vector. KITG559 and KITV814 expressed in IC-2 cells were constitutively phosphorylated on tyrosine and demonstrated kinase activity in the absence of stem cell factor, which is a ligand for KIT. IC-2 cells expressing either KITG559 or KITV814 (IC-2G559 or IC-2V814 cells) showed factor-independent growth in suspension culture and produced tumors in nude athymic mice. In addition, IC-2G559 and IC-2V814 cells showed a more mature phenotype compared with the phenotype of the original IC-2 cells, especially after transplantation into nude mice. The number of basophilic granules and the content of histamine increased remarkably. KITG559 and KITV814 also influenced the transcriptional phenotype of mouse mast cell proteases (MMCP) in IC-2 cells. The expression of MMCP-2, MMCP-4, and MMCP-6 was much greater in IC-2G559 and IC-2V814 cells than in the original IC-2 cells. The results indicated that constitutively activated KIT had not only oncogenic activity but also differentiation-inducing activity in mast cells.

Animals↗
PubMed1996

Kimura disease: CT and MR findings.

The lesions of Kimura disease showed slightly high and very high intensity on T2-weighted MR, and low and intermediate intensity, respectively, on T1-weighted images. The degree of enhancement also differed between the two cases. These discrepancies may be attributable to differing degrees of fibrosis and vascular proliferation.

Adolescent↗
PubMed1995

Pyruvate kinase deficiency of mice associated with nonspherocytic hemolytic anemia and cure of the anemia by marrow transplantation without host irradiation.

Mutant mice with splenomegaly and nonspherocytic hemolytic anemia were found in an inbred colony of the CBA/N (hereafter CBA) strain maintained in the Japan SLC Haruno farm (Shuchi-gun, Shizuoka, Japan). The activity of pyruvate kinase (PK) in red blood cells (RBCs) of the anemic mutants decreased to 16.2% of normal (+/+) CBA mice. Because the mutant CBA mice showed a remarkable reticulocytosis (41.6%) and because the PK activity of reticulocytes is much higher than that of mature RBCs, the PK activity in mature RBCs of the mutant CBA mice was calculated to be 2.8% that of mature RBCs of CBA-(+/+) mice. Because RBC type PK is encoded by the Pk-1 locus of the mouse (chromosome 3), we designated the mutant locus as Pk-1slc. The anemia and PK deficiency of CBA-Pk-1slc/Pk-1slc mice were cured by bone marrow transplantation (BMT) from CBA-(+/+) mice. Prior irradiation was not necessary for the curative BMT. On the other hand, the BMT from CBA-Pk-1slc/Pk-1slc mice to nonirradiated CBA-(+/+) mice did not result in the decrease of RBCs and the reduction of PK activity. The present results indicate that CBA-Pk-1slc/Pk-1slc mice are a potentially useful animal model for studying pathophysiology of PK deficiency and for developing new therapeutic methods to correct PK deficiency.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1995

Primary structure of murine red blood cell-type pyruvate kinase (PK) and molecular characterization of PK deficiency identified in the CBA strain.

To clarify the molecular abnormality of pyruvate kinase (PK) deficiency identified in the mutant mice of CBA-Pk-1slc/Pk-1slc, we cloned murine red blood cell-type PK (R-PK) cDNA of those animals. The cDNA sequence spans 1827 bp, including an open reading frame that can encode 574 amino acids. Homology in the coding sequences between murine and human R-PK was 86.1% at nucleotide and 91.5% at amino acid levels. A homozygous missense mutation at nucleotide 1013 GGT-->GAT was identified in the cDNA sequence of the mutant, causing a single amino acid substitution at no. 338Gly-->Asp of the murine R-PK. Six amino acid residues, 335Val-336Ala-337Arg-338Gly-339Asp-340L eu, were encoded in exon 8 of both human and rat L (liver-type)/R-PK genes and were evolutionarily conserved in PK from bacteria through humans. 337Arg was reported to be important for substrate binding, suggesting that the amino acid change would impair substrate affinity of the PK subunit. A homozygous missense mutation at the catalytic domain has been identified in a human PK variant, PK Hong Kong (941ATT-->ACT, 314 Ile-->Thr). Although both 1013A and 941C gave rise to an amino acid change adjacent to the active site and may interfere with substrate binding to the subunit, the degree of anemia was much more severe in the human case. The erythroid-progenitor cell number increased in the spleen of Pk-1slc/Pk-1slc mice to a level approximately 66 times higher than that in normal CBA mice, suggesting that compensatory extramedullary erythropoiesis in the spleen of the mutant mice, but not in the human variant, might account for the observed difference in the phenotype.

Amino Acid Sequence↗
PubMed1995

Growth response of acute myeloblastic leukemia cells to recombinant human thrombopoietin.

Thrombopoietin (TPO) is a newly identified hematopoietic growth factor that stimulates both megakaryopoiesis and thrombopoiesis through its interaction with a specific cell surface receptor encoded by the c-mpl proto-oncogene. In an effort to investigate the effect of TPO on human myeloid leukemia cells, the expression of c-mpl and the proliferative response to recombinant human (rh) TPO were investigated in a series of patients with acute myeloblastic leukemia (AML). Of 50 cases of AML, the c-mpl mRNA was detectable by means of Northern blot analysis in 26 cases, and the in vitro treatment with rhTPO led to proliferation of AML cells in 22 cases. The c-mpl expression and proliferative response to rhTPO was observed in all subtypes of AML and did not correlate with French-American-British classification, whereas all cases of M7-type AML cells expressed c-mpl and proliferated in response to rhTPO. Furthermore, rhTPO-induced proliferation of AML cells was augmented with the addition of interleukin-3 (IL-3), IL-6, stem cell factor, or granulocyte-macrophage colony-stimulating factor. These results suggested that c-mpl may be functional in terms of supporting proliferation of various types of AML cells and that TPO may contribute, at least in part, to abnormal growth of the cells, especially in combination with other hematopoietic growth factors.

Acute Disease↗
PubMed1995

Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines.

The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val-559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.

Amino Acid Sequence↗
PubMed1995

bcl-2 deficiency in mice leads to pleiotropic abnormalities: accelerated lymphoid cell death in thymus and spleen, polycystic kidney, hair hypopigmentation, and distorted small intestine.

Transgenic mice homozygously lacking in the bcl-2 gene were generated using homologous recombination in embryonal stem cells. The complete absence of Bcl-2 alpha and -beta proteins did not interfere with normal embryonic development. Abnormalities became evident after birth, although the severity varied among homozygous null mice, bcl-2-/- mice displayed pleiotropic abnormalities similar to those in the previously described bcl-2-/- mice, including growth retardation, smaller ears, short lives, polycystic kidney, atrophic thymus and spleen with accelerated apoptotic cell death of lymphocytes, and hair hypopigmentation in the second hair follicle cycle. Our bcl-2-/- mice also revealed novel defects in the small intestine, characterized by retarded development, accelerated exfoliation of epithelial cells, and very few mitotic progenitor cells.

Abnormalities, Multiple↗
PubMed1995

Silicone plate-induced granuloma presenting pituitary apoplexy-like symptoms: case report.

BACKGROUND: Granuloma induced by silicone plate is rare. We present a case of silicone plate-induced granuloma in the sellar and suprasellar region confirmed by histologic examination at surgery. CASE DESCRIPTION: A 31-year-old woman suddenly complained of severe headache, polyuria, and visual disturbance 8 years and 9 months after an initial transsphenoidal surgery for pituitary adenoma. At surgery, granuloma and hematoma around the dimethylpolysiloxan (silicone) plate, which had been placed on the sella floor at the initial surgery, were found. Postoperative course was uneventful. CONCLUSIONS: Our presented case showed one of the disadvantages for silicone placement for repairing the sella floor, and the diagnostic possibility of silicone-induced granuloma in the sellar region.

Adult↗
PubMed1995

Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3.

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.

Amino Acid Sequence↗
PubMed1995

Recurrent primary central nervous system lymphoma mimicking neurodegenerative disease--an autopsy case report.

A 42-year-old female presented with recurrent primary central nervous system lymphoma mimicking the roentgenographic appearance of diffuse brain degeneration. Betamethazone was administered, but her condition worsened. Biopsy of a swollen neck lymph node demonstrated lymphoma cells. Computed tomography and magnetic resonance (MR) imaging showed no contrast-enhanced lesions. T2-weighted MR imaging revealed diffuse, high intensity areas in the white matter of the bilateral cerebrum, basal ganglia, and brainstem. Despite chemotherapy for systemic lymphoma, she died of respiratory arrest. Histological examination of autopsy specimens showed diffuse infiltration of lymphoma cells in the perivascular space of the cerebral cortex, but little neovascularization. The absence of contrast enhancement may have been due to preservation of the blood-brain barrier. Histological confirmation of roentgenographic findings of brain degeneration is important in patients treated for primary central nervous system lymphoma.

Adult↗