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Biomedical subjects

A Tojo

Publications and source records attributed to A Tojo.

158 records · Page 9Linked to original sources
PubMed1987

Interleukin 3-specific tyrosine phosphorylation of a membrane glycoprotein of Mr 150,000 in multi-factor-dependent myeloid cell lines.

Tyrosine phosphorylation of cellular proteins induced by various hematopoietic growth factors such as interleukin 3 (IL3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL4) was studied in several multi-factor-dependent myeloid cell lines. Among the growth factors, IL3 specifically induced rapid tyrosine phosphorylation of a membrane glycoprotein of mol. wt 150 kd (gpp150) in the IL3-dependent cell lines, IC2 and DA-1. The IL3-induced tyrosine phosphorylation of gpp150 was detected within 30 s, reached a maximum at 3 min and decreased thereafter. The concentration of IL3 required for half-maximum stimulation of gpp150 tyrosine phosphorylation with 2.5 x 10(6)/ml cells was approximately 200 pM, which is the same as the dissociation constant for 125I-labeled IL3 binding. gpp150 was constitutively phosphorylated on tyrosine residue(s) in growth factor independent variants, IC2Tr and DA-1Tr, derived from IC2 and DA-1 respectively. Neither variant synthesized IL3. The present findings suggest that tyrosine phosphorylation of gpp150 is a critical event involved in both IL3-dependent and -independent growth.

Animals↗
PubMed1987

Identification of erythropoietin receptors on fetal liver erythroid cells.

Erythropoietin (EPO) has a central role in the growth and development of erythroid cells. Using a biologically active radioiodinated derivative, EPO receptors were identified on fetal mouse liver cells mostly consisting of erythroid cells. 125I-EPO was cross-linked to two receptors forms with apparent molecular masses of 110 and 95 kilodaltons, respectively and both having similar affinity toward EPO.

Animals↗
PubMed1987

Internalization of radioiodinated erythropoietin and the ligand-induced modulation of its receptor in murine erythroleukemia cells.

We have studied the internalization of 125I-erythropoietin (Epo) and regulation of Epo receptors by the ligand in a murine erythroleukemia cell clone, TSA8. To determine internalization, a high-salt acid wash was performed. Internalization of 125I-Epo was found in TSA8 cells as well as in fetal mouse liver cells (FMLC), although the percentage of internalized radioactivity reached 40% after incubation at 37 degrees C for 150 min and was lower than that in FMLC. Exposure of TSA8 cells to unlabeled Epo resulted in a rapid, time-dependent reduction in 125I-Epo binding activity. The net loss of the activity was related to the ambient Epo concentration and 5 X 10(-8)M Epo induced approximately 80% loss of total binding capacity. Scatchard analysis of the binding data revealed that the high-affinity receptor number was decreased but the affinity was increased in the Epo-treated cells. In low-affinity receptors, however, the receptor affinity was decreased and the receptor number was not changed much by preincubation with Epo. These results suggest that the decrease in 125I-Epo binding activity after preincubation with unlabeled Epo is mainly accounted for by a decrease in the number of high-affinity receptors, and the high-affinity receptors play an important role in the biological response to Epo.

Animals↗
PubMed1987

Vanadate can replace interleukin 3 for transient growth of factor-dependent cells.

A mouse interleukin 3 (IL-3)-dependent cell line, IC2, could survive and proliferate over a 48-h period in the absence of IL-3 when incubated with micromolar concentrations of sodium orthovanadate. The greatest response was obtained at 12.5 microM, as judged by stimulation of cell growth. Vanadate also stimulated synthesis of nucleotides and protein in IC2 cells. After 6 h of culture in the absence of IL-3, the intracellular ATP levels of IC2 cells fell dramatically; this fall was prevented by addition of vanadate to the culture. Studies with recombinant IL-3 revealed that vanadate potentiated the effect of submaximal doses of IL-3 on the growth of IC2 cells, but did not appear to act synergistically with IL-3 to give a maximal proliferative response of the cells. After 48 h of IL-3 replacement with vanadate, IC2 cells could respond to IL-3 with no loss of proliferative integrity. These results suggest that IL-3 dependence of IC2 cells is transiently substituted for by vanadate, and it may be a useful tool to understand the mechanism of action of IL-3.

Adenosine Triphosphate↗
PubMed1987

Binding properties and proliferative potency of insulin-like growth factor I in fetal mouse liver cells.

Specific high-affinity receptor(s) for insulin-like growth factor I have been identified in fetal mouse liver cells (FMLC) rich in late erythroid progenitors (CFU-E). Competition for [125I]IGF-I binding by IGFs and insulin demonstrated the presence of Type-I IGF receptors. Scatchard analysis of the binding data revealed a single class of receptors (Kd, 1.2 nM; R0, 600 sites per cell). Erythroid colony formation and DNA synthesis by these cells were enhanced by IGF-I alone or in combination with erythropoietin (Epo). Subfractionations of FMLC using Percoll density gradients showed that a significant part of [125I]IGF-I binding was observed in the CFU-E-enriched fraction and that the erythroid colony formation was mostly enhanced by IGF-I in the same fraction. IGF-I stimulated the phosphorylation of the beta-subunit of the Type-I receptors. These results indicate that IGF-I modulates the Epo-stimulated proliferation and differentiation of erythroid progenitors via its specific receptors.

Animals↗
PubMed1987

Binding of erythropoietin to CFU-E derived from fetal mouse liver cells.

The binding of recombinant erythropoietin (EPO) to fetal mouse liver cells (FMLC) was investigated using a radioiodinated derivative which retained full biological activity. FMLC were fractionated using a preformed Percoll density gradient. Using the fractionated FMLC, the ability to form CFU-E colonies in a semisolid culture was examined, and the binding of [125I]EPO was measured. The highest specific binding of [125I]EPO was observed in a fraction with a density between 1.062 and 1.076 g/ml. The same fraction showed the highest ability to form CFU-E-derived colonies. After suspension culture of FMLC with EPO for 2 days, differentiated erythroid cells with higher density markedly increased. The specific binding of [125I]EPO to these cells almost disappeared with differentiation. Scatchard analysis with cells of the CFU-E-enriched fraction showed a nonlinear curve, suggesting the existence of two classes of binding sites. One binding site was high-affinity (Kd1 = 0.41 nM), and the other low-affinity (Kd2 = 3.13 nM). These results suggest that the expression of EPO receptors on the erythroid cells is highest in CFU-E.

Animals↗
PubMed1987

Pure erythropoietic colony and burst formations in serum-free culture and their enhancement by insulin-like growth factor I.

Recombinant human insulin-like growth factor I (IGF-I) increased human and murine erythropoietic colony formation in serum-free culture. In order to investigate the effects of purified factors such as IGF-I on hemopoietic progenitor cells, we have established a serum-free culture system which supports the clonal growth of CFU-E- and BFU-E-derived colonies. Exogenously supplied ingredients were bovine serum albumin (BSA), transferrin, lipid suspensions, 2-mercaptoethanol, and recombinant human erythropoietin (epo). Among these, BSA and cholesterol were found to be essential ingredients. The optimum concentration of BSA sufficient to grow BFU-E was 3%. Erythroid colony and burst formation of human and murine marrow cells was enhanced twofold (p less than 0.05) by a physiological concentration of recombinant human IGF-I. Potentiation was observed in a dose-dependent manner between 10(-9) and 10(-7) M. A few murine CFU-E colonies were formed in the absence of epo. These results suggest that IGF-I has a supportive effect on the proliferation and differentiation of erythroid precursor cells stimulated by epo and that its action is synergistic with that of epo.

Animals↗
PubMed1986

Mode of Action of Bipyramidal delta-Endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1.

The mode of action of the toxic fragment (P-59) derived from bipyramidal-shaped delta-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 on the silkworm Bombyx mori was investigated. An enzyme-linked immunosorbent assay showed that there was no translocation of P-59 from the gut lumen to the hemocoel. When membrane vesicles prepared from silkworm midgut were incubated with P-59, normally smooth surface of vesicles became rough, and patch formation was observed on the surface. Vesicles treated with P-59 tended to agglutinate. The vesicle-denaturing activity of a 130,000-dalton subunit protein of bipyramidal toxin was enhanced by treatment with a gut juice protease of the silkworm. P-59 did not cause any uncoupling effect on mitochondria of the silkworm midgut. These results suggest that the attacking site of this toxin is not the mitochondrion but the cell membrane of the susceptible cell.

Journal Article↗
PubMed1983

Dissolution and Degradation of Bacillus thuringiensis delta-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori.

The dissolution and degradation of dagger-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.

Journal Article↗