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Biomedical subjects

A Tojo

Publications and source records attributed to A Tojo.

At least 145 records · Page 8Linked to original sources
PubMed1990

Effects of atrial natriuretic peptide on renal arterioles: morphometric analysis using microvascular casts.

In normal rat kidneys, the effect of atrial natriuretic peptide (ANP) on the diameter of the arterioles was evaluated by scanning electron microscopy of vascular casts. Acryl resin was infused into rat kidneys during the administration of ANP, either alone or with norepinephrine (NE). ANP infusion constricted the proximal efferent arteriole in the superficial cortex. Although NE constricted the proximal and distal segments of the afferent arteriole in the superficial cortex, the addition of ANP reversed the constriction and further constricted the efferent arteriole. In the deep cortex, only the proximal segment of the afferent arteriole was dilated by ANP when infused with NE. In a separate set of experiments, ANP increased both the glomerular filtration rate (GFR) and urinary sodium excretion (UNaV), and NE decreased the renal blood flow (RBF). However, administration of ANP after NE recovered RBF and increased GFR as well as UNaV. Results indicate that ANP increases GFR and natriuresis by constricting the efferent arteriole. NE appears to decrease RBF by constricting the afferent arteriole. ANP antagonizes the renal effects of NE primarily by dilating afferent arterioles.

Animals↗
PubMed1990

Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colony-stimulating factor.

The receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the surfaces of normal and leukemic myeloid cells were characterized using 125I-labeled bacterially synthesized GM-CSF. The binding was rapid, specific, time dependent, and saturable. Scatchard analysis of the 125I-GM-CSF binding to peripheral blood neutrophils indicated the presence of a single class of binding site (Kd = 99 +/- 21 pM; 2,304 +/- 953 sites/cell). However, for peripheral blood monocytes and two GM-CSF-responsive myeloid cell lines (U-937 and TF-1), the Scatchard plots were biphasic curvilinear, which were best fit by curves derived from two binding site model: one with high affinity (Kd1 = 10-40 pM) and the other with low affinity (Kd2 = 0.9-2.0 nM). For U-937 cells, the number of high-affinity receptors was 1,058 +/- 402 sites/cell and that of low-affinity receptors was estimated to be 10,834 +/- 2,396 sites/cell. Cross-linking studies yielded three major bands with molecular masses of 150 kDa, 115 kDa, and 95 kDa, which were displaced by an excess amount of unlabeled GM-CSF, suggesting 135-kDa, 100-kDa, and 80-kDa species for the individual components of the human GM-CSF receptor. These bands comigrated for different cell types including peripheral blood neutrophils, U-937 cells and TF-1 cells. In experiments using U-937 cells, only the latter two bands appeared to be labeled in a dose-dependent manner in a low-affinity state. These results suggest that the human GM-CSF receptor possibly forms a multichain complex.

Binding, Competitive↗
PubMed1990

Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family.

A new human gene encoding a receptor-type tyrosine kinase was isolated by a weak cross-hybridization with v-ros oncogene. A cDNA of about 7.7 kb carried a 4.2 kb open reading frame, and the predicted amino acid sequence of 1338 residues contained extracellular, transmembrane and tyrosine kinase domains. Although its extracellular domain is approximately 220 amino acids longer than those of the products of the fms family, i.e. c-fms, c-kit and platelet-derived growth factor receptor genes, the overall structure including cysteine motifs in its extracellular domain and a long peptide insertion in its tyrosine kinase domain indicates that this new gene is closely related to the fms family. Consequently, the gene was designated as flt (fms-like tyrosine kinase) gene. The expression of the flt gene was strongly suppressed in most of the tumor cell lines examined so far, whereas this mRNA was expressed in a variety of normal tissues of adult rat.

Amino Acid Sequence↗
PubMed1990

Factitious proteinuria in a young girl.

A 16-year-old girl showed waxing and waning proteinuria and fat globules in urine for three years. There were no other abnormal findings except for the urinalysis. The renal biopsy findings did not indicate glomerular disease. Electrophoresis of the urinary protein showed two abnormal fractions at the alpha and beta globulins. Immunoelectrophoresis demonstrated that these abnormal proteins were not derived from human serum proteins, but were egg proteins. It appears that the proteinuria was factitious and that egg proteins were injected into the bladder, as they were also present in the bladder urine.

Adolescent↗
PubMed1989

Characterization of cellular receptors for erythroid differentiation factor on murine erythroleukemia cells.

Erythroid differentiation factor (EDF), which is structurally related to transforming growth factor-beta family and induces differentiation of murine erythroleukemia cell clone F5-5, has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-EDF to F5-5 cells is time- and temperature-dependent, specific, saturable, and reversible. Transforming growth factor-beta 1 has no significant effects on growth of F5-5 cells and binding of 125I-EDF to F5-5 cells. Scatchard analysis of the binding data indicated that F5-5 cells have a single class of binding sites (3,200/cell) with an apparent Kd of 3.1 X 10(-10) M. Affinity cross-linking experiments demonstrated three radiolabeled components of 140,000, 76,000, and 67,000 daltons under both reducing and nonreducing conditions. Labeling of these three components has been inhibited by incubation of the cells with excess unlabeled EDF. These results imply molecular weights of 115,000, 51,000, and 42,000 for the EDF receptors on this cell line.

Activins↗
PubMed1989

Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line.

Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors.

Cell Line↗
PubMed1989

Multi-copy introduction and high-level expression of interleukin-3 genes by retroviral vector superinfection.

We constructed a retroviral expression vector carrying multiple cloning sites. This vector was found to express efficiently the cloned gene. Using this vector and a helper virus-free system, a murine interleukin-3 (mlL-3) high-producing cell line was established by multiple cycles of infection with recombinant retroviruses carrying mlL-3 cDNA. The infected cells produced a considerable amount of mlL-3 and the concentration of mlL-3 in culture media increased as a function of the frequency of infection. High levels of mlL-3 cDNA, mRNA and protein in this cell line were confirmed by Southern, Northern and biological assays, respectively. These results suggest that artificial gene amplification is possible in a helper-free retroviral system. This should be applicable to efficient expression of bioactive molecules in a wide variety of mammalian cells including suspension cells.

Animals↗
PubMed1989

Variations in arterioles in spontaneously hypertensive rats. Morphometric analysis of afferent and efferent arterioles.

In the present study, the diameters of afferent and efferent arterioles of kidneys from spontaneously hypertensive rats (SHR) were evaluated and compared with those from Wistar Kyoto rats. (WKY) using a vascular cast model. At 4 weeks of age, the blood pressure was slightly higher in SHR than in WKY (124 +/- 1 vs 116 +/- 7 mmHg, ns). The diameters of afferent arterioles in SHR were smaller than those in WKY (10.3 +/- 0.6 vs 12.3 +/- 0.7 microns, P less than 0.001), whereas the diameters of efferent arterioles were comparable in the two strains. At 20 weeks of age, the blood pressure was markedly elevated in SHR than in WKY (192 +/- 5 vs 140 +/- 4 mmHg, P less than 0.001). The diameters of afferent arterioles in SHR at this age were much smaller than those in WKY (14.3 +/- 0.5 vs 17.1 +/- 0.6 microns, P less than 0.01). The diameters of efferent arterioles in SHR were, however, larger than those in WKY (15.4 +/- 1.2 vs 12.9 +/- 0.4 microns, P less than 0.05). The net effect of these changes in arteriolar size helps to maintain normal intraglomerular pressure and to protect glomeruli from damage due to hypertension.

Aging↗
PubMed1989

Binding properties and proliferative effects of human recombinant granulocyte-macrophage colony-stimulating factor in primary leukemia and lymphoma.

Binding of radiolabeled human granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied with blast cells from eight patients with acute myeloblastic leukemia (AML), and neoplastic lymphoid cells from one patient with acute lymphoblastic leukemia (ALL), two patients with chronic lymphocytic leukemia (CLL) and one patient with undiagnosed B cell neoplasia. In all AML cases studied, Scatchard graphs of the direct binding data were curvilinear, and were best fitted by curves derived from a two-binding-site model; one site with high affinity (Kd1 = 12-71 pM; 174-602 sites/cell) and the other with low affinity (Kd2 = 0.5-2.7 nM; 1137-6020 sites/cell). A cross-linking study on blast cells from one AML patient demonstrated specific bands which were similar to those reported for peripheral blood neutrophils. Furthermore, blast colony assays for the same preparations showed remarkable proliferative response to GM-CSF in the concentration range from 0.3 nM to 7.0 nM (ED50 greater than 0.7 nM). This concentration range is approximately one order of magnitude higher than that which is effective for colony formation from normal bone marrow progenitors (ED50 in equilibrium 0.1 nM). No significant correlation could be observed between the responsiveness of blast progenitors to GM-CSF, and the numbers or affinities of GM-CSF binding sites demonstrated on blast cells. In studies with neoplastic lymphoid cells from four patients, 125I-GM-CSF also specifically bound in two cases, while response to GM-CSF was not observed in these cases. These results indicate that the expression of GM-CSF receptor is not restricted to the GM-CSF-responsive AML blast cells, but can be observed in other AML blast cells and even in neoplastic lymphoid cells.

Binding Sites↗
PubMed1989

Binding of iodinated erythropoietin to rat bone marrow cells under normal and anemic conditions.

Specific binding sites for erythropoietin (Epo) were shown in normal and anemic rat bone marrow cells using [125I]labeled human recombinant Epo. When rats were treated once or several times with phenylhydrazine or malotilate, or by phlebotomy, the serum Epo level determined by RIA began to increase rapidly. Thereafter, both the number of erythroid colony-forming unit (CFU-E)-derived colonies and the Epo binding capacity of bone marrow cells increased almost simultaneously in response to induced anemic states, suggesting that the amount of Epo binding in bone marrow cells may reflect in vivo erythropoiesis. Scatchard analysis of the binding data from normal rats revealed the presence of a single class of binding sites (Kd = 0.18 +/- 0.04 nM, 38 +/- 5 sites/cell). In anemic states, the apparent average receptor number per cell increased (52-62 sites/cell) without changing in binding affinity toward Epo. Furthermore, [125I]Epo was cross-linked to the cell surface molecule of approximately 165 kd in nonreducing conditions and 75 kd in reducing conditions. Autoradiographic analysis indicated that Epo receptors were distributed on immature erythroid cells. Proerythroblasts were the most heavily labeled, whereas orthochromatic erythroblasts and cells of myeloid and lymphoid lineages were not labeled. Calculations based on Scatchard and autoradiographic analysis showed that proerythroblasts have 390 receptor sites per cell, twice as many as basophilic or polychromatophilic erythroblasts have. These results are consistent with the stage-specific action of Epo in physiological differentiation of erythroid cells.

Anemia↗
PubMed1989

Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1.

We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

Cell Division↗
PubMed1989

Tunicamycin modulates binding of 125I-erythropoietin to Friend erythroleukemia cells.

The effects of tunicamycin and neuraminidase treatment on the specific binding of 125I-erythropoietin to a murine erythroleukemia cell clone, B8, were investigated. Neuraminidase treatment of B8 cells did not affect the specific binding of erythropoietin, but tunicamycin treatment caused a 2.5 to 4-fold increase in the amount of 125I-erythropoietin binding. Scatchard analysis of the binding data showed that the increase in the amount of binding resulted from increased affinity of the receptor. These results suggest that N-linked sugars of the erythropoietin receptor protein are involved in the interaction of erythropoietin with the cell-surface receptors on B8 cells.

Animals↗
PubMed1989

Isolation and characterization of new mammalian kinase genes by cross hybridization with a tyrosine kinase probe.

We isolated two novel mammalian kinase genes by weak cross-hybridization with v-ros oncogene. (1) A cDNA of about 7.7 kb obtained from a human placenta cDNA library carried a 4.2 kb open reading frame, and the predicted amino acid sequence of 1338 residues contained extracellular, transmembrane and tyrosine kinase domains. The overall structure including cysteine motifs in its extracellular domain and a long peptide insertion in its tyrosine kinase domain indicates that this new gene is closely related to the fms family. Consequently, the gene was designated as flt(fms-like tyrosine kinase) gene. The expression of the flt gene was examined in normal and transformed cells, and it was mapped to human chromosome 13q12-13. (2) Another new gene was found to be expressed almost exclusively in testis. The cDNA sequence analysis revealed that the predicted product carried protein kinase consensus motifs in its amino-terminal region. Comparison of the deduced amino acid sequence of this gene in the kinase domain with those of other protein kinase genes will be discussed.

Amino Acid Sequence↗
PubMed1988

Induction of the receptor for erythropoietin in murine erythroleukemia cells after dimethyl sulfoxide treatment.

Biologically active 125I-labeled human recombinant erythropoietin (EPO) was used to demonstrate specific receptors for this erythroid-specific hemopoietic growth factor on the cell surface of murine erythroleukemia cell clone B8. The binding of radioiodinated EPO to these cells was time and temperature dependent, specific, saturable, and reversible. During erythroid differentiation by dimethyl sulfoxide, B8 cells displayed a rapid and marked increase in the amount of specific 125I-EPO binding before the appearance of hemoglobin-containing cells. Scatchard analysis of the saturation binding data revealed that B8 cells had a single class and low number (350 to 650) of EPO receptors per cell with an apparent Kd of 1.2 to 1.4 nM. In addition, the number of EPO receptors on B8 cells was increased twice by induction with DMSO for 1 day, but the binding affinity of EPO toward its receptors did not change significantly. Affinity cross-linking experiments with disuccinimidyl suberate demonstrated two radiolabeled components with apparent molecular weights of 145,000 and 130,000 under both reducing and nonreducing conditions. Labeling of the two components was inhibited by incubation of cells with unlabeled EPO. These results suggest that some murine erythroleukemia cells potentially express EPO receptors as a differentiation marker of erythroid lineage, which contain two polypeptides with molecular weights of 109,000 and 94,000.

Affinity Labels↗
PubMed1988

Expression of the functional erythropoietin receptors on interleukin 3-dependent murine cell lines.

Two distinct hemopoietic growth factors, interleukin 3 (IL-3) and erythropoietin (EPO), support the growth and development of erythroid cells in a sequential manner in vitro. Stimulation of multipotential stem cells by IL-3 appears to develop committed erythroid progenitor cells that respond to EPO. When several murine IL-3-dependent cell lines were assayed for their ability to respond to EPO, the growth and survival of the three cell lines showing the profiles of either myeloid or mast cell lineage (IC-2, DA-1, FDC-P2) were stimulated by EPO in a dose-dependent fashion. To determine whether the biologic effects were mediated through the specific receptors for EPO, we performed binding experiments on these cells with radioiodinated EPO. All of these cells displayed significant levels of specific binding for EPO. Among a family of hemopoietic growth factors, only unlabeled EPO was able to compete for the binding of radioiodinated EPO to the cells. Analysis of the binding data revealed the existence of a single case of binding sites in extremely low abundance. IC-2 cells were used to study the effects of IL-3 on the regulation of expression of EPO receptors. It was demonstrated that a decrease in IL-3 concentration in the culture medium increased the responsiveness to EPO and the amount in specific binding of EPO as well. These results suggest that some IL-3-dependent cell lines have functional EPO receptors and their expression may be modulated by IL-3.

Animals↗
PubMed1988

Effects of type beta transforming growth factors on haematopoietic progenitor cells.

The effects of type beta transforming growth factors (TGF-beta s) on normal human and murine haematopoietic progenitor cells were examined using bone marrow colony assays. In erythroid colony assays, TGF-beta 1 inhibited human CFU-E derived colony formation, BFU-E derived burst formation, and murine BFU-E derived burst formation in a dose dependent manner between 0.1 and 5.0 ng/ml. However, murine CFU-E derived colony formation was unaffected even at a concentration of 5.0 ng/ml TGF-beta 1. In myeloid colony assays, different sensitivity of progenitor cells to the inhibitory effects of TGF-beta s was observed between both species. TGF-beta 1 inhibited murine granulocyte-macrophage colony (GM-colony) formation and granulocyte colony (G-colony) formation in a dose dependent manner between 0.1 and 5.0 ng/ml, but had no remarkable effects on human GM-colony and G-colony formation. TGF-beta 2 also had similar inhibitory effects on haematopoietic progenitor cells, while its inhibitory effect was less potent than that of TGF-beta 1. Thus our data suggest that TGF-beta may be involved in negative regulation of haematopoiesis and that its inhibitory action may be restricted in lineage and/or species specific manner.

Animals↗