PubMed Health⌕ Search

Biomedical subjects

M Shimane

Publications and source records attributed to M Shimane.

32 records · Page 2Linked to original sources
PubMed1997

Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1.

cDNA for a novel proton/organic cation transporter, OCTN1, was cloned from human fetal liver and its transport activity was investigated. OCTN1 encodes a 551-amino acid protein with 11 transmembrane domains and one nucleotide binding site motif. It is strongly expressed in kidney, trachea, bone marrow and fetal liver and in several human cancer cell lines, but not in adult liver. When expressed in HEK293 cells, OCTN1 exhibited saturable and pH-dependent [3H]tetraethyl ammonium uptake with higher activity at neutral and alkaline pH than at acidic pH. Furthermore, treatment with metabolic inhibitors reduced the uptake, which is consistent with the presence of the nucleotide binding site sequence motif. Although its subcellular localization and detailed functional characteristics are not clear at present, OCTN1 appears to be a novel proton antiporter that functions for active secretion of cationic compounds across the renal epithelial brush-border membrane. It may play a role in the renal excretion of xenobiotics and their metabolites.

Adenosine Triphosphate↗
PubMed1997

Identification and characterization of BRDT: A testis-specific gene related to the bromodomain genes RING3 and Drosophila fsh.

The RING3 gene encodes a 90-kDa mitogen-activated nuclear protein. In proliferating cells, including in leukemia, RING3 has serine-threonine kinase and autophosphorylation activities. The cloning of D26362, a gene closely related to RING3, suggests a gene family. RING3 and D26362 are also related to the Drosophila developmental gene fsh. A database search for further members of the RING3 family identified an EST derived from a testis-specific library. cDNA clones representing the full coding sequence of the gene were isolated. The gene encodes a protein of 947 amino acids with extensive homology to RING3, D26362, and fsh. Similar to these proteins, it possesses two bromodomain motifs and a PEST sequence. Northern analysis of 16 normal tissues and eight cancer cell lines shows transcripts of 3.5 and 4.0 kb expressed specifically in testis. The gene has been named BRDT (for bromodomain, testis specific). PCR analysis of a panel of monochromosomal human/rodent hybrid cell lines and the GeneBridge 4 panel of radiation hybrids localizes the gene to chromosome 1p between markers WI-7719 and WI-3099 (D1S2154).

Amino Acid Sequence↗
PubMed1997

Identification of two novel human putative serine/threonine kinases, VRK1 and VRK2, with structural similarity to vaccinia virus B1R kinase.

A cDNA library enriched for human fetal-specific liver genes was constructed by suppressive subtractive hybridization. EST fls223 generated from this library was found to represent a novel putative serine/threonine (Ser/Thr) kinase. A full-length clone isolated for this gene encodes a protein of 396 amino acids. The amino acid sequence has 40% identity over 305 amino acids with the B1R Ser/Thr protein kinase of vaccinia virus. This gene has therefore been named VRK1 (vaccinia virus B1R kinase related kinase). VRK1 was also found to have sequence identity (62.0% over 481 nucleotides) to a database EST. A full-length clone for this EST was isolated and sequenced. Conceptual translation predicts a protein of 508 amino acids that, like VRK1, has similarity to B1R kinase (38.7% identity over 300 amino acids). This gene has been named VRK2. Comparison of VRK1 with VRK2 indicates that they encode structurally related putative Ser/Thr protein kinases. Northern analysis shows that expression of both genes is widespread and elevated in highly proliferative cells, such as testis, thymus, and fetal liver. B1R kinase is reported to be essential for DNA replication of vaccinia virus. The similarity of VRK1 and VRK2 to B1R indicates that these genes may have similar functions.

Amino Acid Sequence↗
PubMed1997

Molecular cloning of a novel vascular endothelial growth factor, VEGF-D.

We have identified and characterized a novel vascular endothelial growth factor (VEGF), VEGF-D, which is structurally related to vascular endothelial growth factor C. A full-length cDNA for human VEGF-D was cloned following the identification of an EST obtained through a TFASTA search of public EST databases. The murine VEGF-D was subsequently isolated from a mouse lung cDNA library. The human VEGF-D gene was mapped to human chromosome Xp22.31. Both human and mouse VEGF-D are strongly expressed in lung and encode the eight cysteine residues that are highly conserved among the members of this family. The high level of conservation between mouse and human VEGF-D may emphasize the biological importance of this gene. Recently the murine gene, FIGF, which is identical to mouse VEGF-D, was reported.

Amino Acid Sequence↗
PubMed1996

Effects of myeloid cell growth factors on alkaline phosphatase, myeloperoxidase, defensin and granulocyte colony-stimulating factor receptor mRNA expression in haemopoietic cells of normal individuals and myeloid disorders.

The mRNA expression of alkaline phosphatase (ALP), myeloperoxidase (MPO), defensin and G-CSF receptor (G-CSFR) in bone marrow cells of normal individuals and myeloid disorders, with or without in vitro stimulation by myeloid cell growth factors, i.e. G-CSF, GM-CSF and IL-3, were examined as markers for myeloid cell differentiation in both mononuclear cell (MNC) and polymorphonuclear cell (PMN) fractions. Without any stimulation, ALP mRNA was expressed only in PMNs, G-CSFR mRNA in PMNs were expressed stronger than in MNCs; both MPO and defensin mRNA were expressed to the same degree in both fractions. With stimulation, the ALP mRNA expression in both fractions was strongly enhanced by G-CSF, but the expression was inhibited by GM-CSF and/or IL-3. MPO mRNA expression was stimulated by G-CSF and/or GM-CSF in MNCs. G-CSFR mRNA expression was enhanced by G-CSF in both fractions. Defensin mRNA expression was inhibited by G-CSF. In cases of myelodysplastic syndrome and chronic myelogenous leukaemia which display a suppressed maturation of myeloid cells, our results demonstrated an almost normal response to these growth factors. Our results suggest that studies on these myeloid marker mRNA expressions would provide more knowledge about the differentiation state and cytokine reactivity of myeloid cells in normal individuals as well as various disorders.

Adult↗
PubMed1994

Retrovirus-mediated gene transfer of human pyruvate kinase (PK) cDNA into murine hematopoietic cells: implications for gene therapy of human PK deficiency.

With the advent of recent molecular studies, nonspherocytic hemolytic anemia caused by red blood cell pyruvate kinase (PK) deficiency is now considered to be caused by a structural mutation of the PK-LR gene. Because PK deficiency is a monogenic disorder, the introduction of the normal PK gene into a patient's bone marrow stem cells should cure the disorder. To study the feasibility of gene therapy for PK deficiency, we first constructed the PK retrovirus pMNSM-hPK using human liver-type PK (LPK) cDNA and obtained a producer cell line of E86/AmPK. By using the supernatant of this virus-producer cell, we transduced NIH/3T3 cells, mouse leukemic cells (NFS60, FDCP-2), and human leukemic cells (K562, HEL). The expression of human LPK enzyme activity was ascertained from the retrovirally transduced NIH/3T3 cells. Northern blot analysis demonstrated the expression of the human LPK mRNA in each transduced cell line. Furthermore, bone marrow stem cells (c-kit+, Lin-, Thy-1lo) sorted by fluorescence-activated cell sorting were also transduced by the producer cells in the presence of interleukin-3 and interleukin-6, and were transplanted into lethally irradiated C57BL/6 mice. Polymerase chain reaction analysis demonstrated the expression of human LPK mRNA in both the peripheral blood and hematopoietic organs on day 30 and on day 135 of bone marrow transplantation.

Animals↗
PubMed1994

Molecular cloning and characterization of G-CSF induced gene cDNA.

G-CSF (granulocyte colony-stimulating factor) is known to specifically stimulate the production and the functional activation of neutrophils. To investigate the intracellular signaling pathway of myeloid cells stimulated by G-CSF, we isolated new genes whose expression was induced by G-CSF. First of all, we constructed lambda gt10 cDNA library from G-CSF-stimulated mononuclear cells (MNC) of a chronic myelogenous leukemia (CML) patient (CML-MNC) and screened the cDNA library by a differential hybridization method. The 24 candidate clones which specifically hybridized with G-CSF-stimulated CML-MNC cDNA probes, but not with unstimulated CML-MNC cDNA probes, were obtained after 8 x 10(4) individual clones had been screened. One of these clones, GIG-1 (G-CSF-induced gene-1), was further characterized. The size of the GIG-1 mRNA was about 0.9kb. The GIG-1 mRNA was expressed mainly in the myeloid leukemic cell lines.

Amino Acid Sequence↗
PubMed1994

Recombinant human glycosylated granulocyte colony-stimulating factor (rhG-CSF)-combined regimen for allogeneic bone marrow transplantation in refractory acute myeloid leukemia.

Recombinant human glycosylated G-CSF (rhG-CSF) may stimulate proliferation of myeloid leukemia cells and thereby increase their susceptibility to anti-cancer agents. By in vitro colony assay, the rhG-CSF-responsive NFS-60 leukemic cell clones are more effectively killed by Ara C in the presence of rhG-CSF than in the absence of rhG-CSF, while the killing of the rhG-CSF-unresponsive HL-60 cell clones is unaffected by rhG-CSF. Leukemia cell colony forming units (L-CFU) derived from most AML patients demonstrate similar results to those of the NFS-60 cell clone when treated in vitro. Encouraged by these in vitro results, we used rhG-CSF as a component of a conditioning regimen for 15 relapsed AML patients who were receiving allogeneic BMT. The patients were conditioned with total body irradiation (TBI) and high-dose Ara C. rhG-CSF was infused continuously at a dose of 5 micrograms/kg/day from 24 h before the beginning of TBI to the end of Ara C therapy. Proliferation of the leukemia cells in vivo in response to rhG-CSF was confirmed in 7 of 14 patients tested and the combined use of rhG-CSF had no additional adverse effects. After BMT, four patients died of non-leukemic causes and three patients had leukemic relapse: the other eight patients have remained disease-free for 200-1600 (median 417) days. The actuarial probabilities of relapse and disease-free survival (DFS) at 4.4 years after BMT were 43.2% and 41.7%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

A marked decrease in defensin mRNA in the only case of congenital neutrophil-specific granule deficiency reported in Japan.

A deficiency of defensins (human neutrophil peptides, HNP) has been previously demonstrated in two individuals with congenital neutrophil-specific granule deficiency (SGD), but its genetic basis is not well understood. We have studied another case of SGD, the only case reported in Japan, to elucidate the molecular basis of defensin deficiency. Using Western blot analysis, we showed for the first time that HNP-4, a novel human defensin, was also deficient in the patient's neutrophils. Northern blot analysis demonstrated that the defensin mRNA was absent in the bone marrow cells of the patient. Limited Southern blot analysis, using a defensin cDNA probe, did not reveal any differences in fragment patterns of the genomic DNA between the patient and the control. We propose that the defensin deficiency in our patient is a consequence of a marked decrease in defensin mRNA in neutrophil precursors. Our findings were consistent with a previous speculation suggesting that the SGD defect occurs at the level of transcription.

Adult↗
PubMed1993

The production of granulocyte colony-stimulating factor and interleukin 6 by human bone marrow stromal cells in aplastic anemia.

Stromal cells are known to produce granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6). To assess the function of bone marrow stromal cells in the pathological state, we studied their in vitro ability to produce G-CSF and IL-6 in 9 patients with aplastic anemia and 5 normal volunteers, using an enzyme immunoassay method. Constitutive production of IL-6, but not G-CSF, was detected in almost all cases. Interestingly, the inducible production of G-CSF and IL-6 by stromal cells after stimulation with interleukin-1 and/or lipopolysaccharides was severely depressed in 3 patients with aplastic anemia. Our results may reflect poor hematopoietic activity in the bone marrow in some cases of aplastic anemia.

Adolescent↗
PubMed1993

[High-dose chemoradiotherapy combined with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) followed by autologous peripheral blood stem cell transplantation (PBSCT) in a case of refractory acute myelogenous leukemia (AML)].

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) stimulates the growth of myeloid leukemic cells and increases their susceptibility to cell-cycle specific agents. We treated a patient with acute myelogenous leukemia (AML) in a state of second resistant relapse, with high-dose chemoradiotherapy combined with rhGM-CSF (total body irradiation: TBI 3Gy x 4, on days -8 & -7; cytosine arabinoside: Ara-C 3g/m2, iv, q12h, on days -5-2; rhGM-CSF 250 micrograms/m2/day, cont.iv, on days -5-2) followed by autologous peripheral blood stem cell transplantation (PBSCT). In this case, rhGM-CSF enhanced the proliferation of leukemic cells in vitro. The test dose of rhGM-CSF (84 micrograms/m2 over 8 hours) also promoted leukemic cell proliferation in vivo, resulting in an increase in the percentage of leukemic cells in the peripheral blood and reappearance of chromosomal aberrations in the bone marrow. The toxicity of rhGM-CSF-combined conditioning regimen included fever and mild liver damage. The patient achieved a complete remission lasting for 2 months, then relapsed. The rhGM-CSF-combined conditioning regimen was tolerated by this patient, but further studies will be required to confirm not only its safety but also its effectiveness in the treatment of refractory AML.

Adult↗
PubMed1990

Expression of granulocyte and granulocyte-macrophage colony-stimulating factors by human non-hematopoietic tumor cells.

The expression of granulocyte colony-stimulating factor (G-CSF) mRNA was studied in human non-hematopoietic tumors, including 18 cases of lung cancers 10 cases of stomach cancers, three cases of glioblastomas, and one case each of breast phyllode sarcoma, thyroid cancer, and hepatocellular carcinoma. Northern blot analysis detected G-CSF mRNA in two of the lung cancer cases, in one of the glioblastoma cases, and in both the breast phyllode sarcoma and hepatocellular carcinoma cases. Since G-CSF receptors were not detected on the tumor cells by 125I-G-CSF binding assay, G-CSF autocrine loop are probably not involved in the growth of these G-CSF-producing tumors. Interestingly, granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA was concomitantly expressed in most of these G-CSF-producing tumors. No major gene deletions or rearrangements of G-CSF and GM-CSF genes were demonstrated by Southern blot analysis in the tumors expressing G-CSF and GM-CSF mRNAs except for one of the glioblastomas (G3) in which one chromosome 17 allele was deleted. Although the mechanism of the concomitant expression of G-CSF and GM-CSF mRNA is unknown, relatively high frequency of this phenomenon suggests the presence of common transcriptional factors acting on regulatory regions of G-CSF and GM-CSF genomes.

Cell Line↗
PubMed1989

Quantitative enzyme immunoassay for human granulocyte colony stimulating factor (G-CSF).

Human granulocyte colony stimulating factor (G-CSF) was detected at the pM level by a simple sequential sandwich enzyme immunoassay. The polyclonal antiserum obtained by immunizing rabbits with recombinant G-CSF did not recognize other colony stimulating factors. The EIA developed gave satisfactory reproducibility as judged by an intra-assay precision of 4.3-6.2% and an interassay precision of 6.2-9.0%. Results from this assay procedure correlated well with those of a bioassay. The method could be performed within 6 h.

Animals↗