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Biomedical subjects

Y Ichikawa

Publications and source records attributed to Y Ichikawa.

At least 775 records · Page 43Linked to original sources
PubMed1982

Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin.

A myeloid leukemia cell line, M1, differentiates to macrophage and gains locomotive and phagocytic activity when incubated with conditioned medium (CM) from a fibroblast culture and bacterial endotoxin. To characterize the actin molecules before and after differentiation, the actin was purified through three sequential steps: DEAE-sephadex A- 50, polymerization/depolymerization, and sephadex G-150 chromatography. There were no essential differences between the inhibitory activity of actins from control M1 cells and CM-treated M1 cells on both DNase I and heavy meromyosin (HMMM) K(+)-EDTA-ATPase; the same dose response as with skeletal muscle actin took place. After the treatment with CM, however, the specific activity for the activation of HMMM Mg(2+)- ATPase by actin became two-fold that of untreated M1 actin, which was one third of the value for skeletal muscle actin. The V(max) for the control and the CM-treated M1 cell, as well as the skeletal muscle actins, proved to be the same. By contrast, the K(app) values for the control and CM-treated M1-cell actins were 3- and 1.5-fold the value for skeletal-muscle actin. This means that CM treatment of the M1 actin produced a twofold affinity for the Mg(2+)-ATPase of skeletal-muscle myosin. The critical concentrations for polymerization were compared under different salt concentrations and temperatures. Although no marked difference was found for the presence of 2 mM MgCl(2), 0.1 M KCl in place of MgCl(2) at 5 degrees C gave the following values: 0.1 mg/ml for skeletal-muscle actin, 0.7 mg/ml for control M1 actin, 0,5 mg/ml for CM- treated M1 actin, and 1.0 mg/ml for the D(-) subline that is insensitive to CM. Although the critical concentration of D(-) actin is extraordinarily high, this actin showed normal polymerization above the critical concentration. This together with the data presented in our previous paper, that the D(-) actin in the crude extract did not polymerize, suggests that an inhibitor for actin polymerization is present in the subline. The kinetics experiment at 0.1 M KCl and 25 degrees C revealed a slower polymerization of untreated M1- and D(-)-cell actins as compared with CM-treated M1 actin. This delayed polymerization was due to a delay during the nucleation stage, not during the elongation stage. By isoelectric focusing, the ratios of beta- to gamma-actin showed a marked difference depending on the states of cells: about 4.9 for control M1, 2.8 for CM-treated M1, and 7.6 for D(-)-subline actins. Tryptic peptide maps also revealed the presence of different peptides. Thus, the functional differences of actin before and after the differentiation was accompanied by some chemical changes in actin molecules.

Actins↗
PubMed1982

Changes in myosin during differentiation of myeloid leukemia cells.

Changes in cellular myosin were followed during the differentiation into macrophages of a myeloid leukemia cell line (Ml) which can be induced by conditioned medium (CM) from a rat embryo culture. To extract the myosin, we used three different procedures, all of which gave a lower yield of myosin for the differentiated than for the undifferentiated Ml cells. This low extractability we attributed to increased binding of the myosin to the plasma membrane. Taking the different extractabilities into consideration, we calculated the myosin contents in the total cellular protein from the densitometry of SDS-polyacrylamide electrophoresis, 0.6% for the untreated Ml cells and 1.0% for the differentiated ones. The three ATPase activities of the Ml cell myosin were in the order, K+-EDTA-=Ca2+- much greater than Mg2+-ATPase in the presence of 0.6 M KCl, whether or not there was treatment with CM. Myosin was purified through fractionation with 25-55% saturated ammonium sulfate, then gel filtration with Sepharose 4B followed by affinity chromatography on F actin-Sepharose 4B. The Ml cell myosin consists of 1 heavy chain (H) and 3 light chains (L1, L2, L3), with molecular ratios of L1 + L2/H not equal to and L3/H not equal to 1. The ratio of L1/L2 was about 1.2 for the untreated Ml cells, but it decreased to about 0.7 after differentiation.

Adenosine Triphosphatases↗
PubMed1982

Purification and properties of the tightly bound reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase of bovine adrenocortical mitochondria.

Two forms of NADPH-adrenodoxin reductase, tightly and loosely bound forms, were found in bovine adrenocortical mitochondria. The loosely bound NADPH-adrenodoxin reductase was solubilized by ultrasonication and its content was about 65% of the total activity in bovine adrenocortical mitochondria. The tightly bound NADPH-adrenodoxin reductase could not be solubilized by mechanical sonication and required detergent treatment. The tightly bound NADPH-adrenodoxin reductase was purified to homogeneity, judging by sodium dodecyl sulfate (SDS) gel electrophoresis, from bovine adrenocortical mitochondria using sodium cholate solubilization and chromatography on DEAE-Sepharose and 2'5'-ADP-Sepharose columns. Its physicochemical properties were similar to those of the loosely bound form, except for its carbohydrate composition.

Adrenal Cortex↗
PubMed1982

A cofactor protein required for actin activation of myosin Mg2+ATPase activity in leukemic myeloblasts.

The Mg2+ATPase activity of the myosin of a myeloid leukemia cell line (Ml) was not activated by purified Ml actin or by muscle actin alone. Activation required the presence of a cellular fraction as a cofactor in addition to the actin, when Mg2+ATPase was stimulated as much as 20-fold. The cofactor was partially purified and characterized. 1) Its molecular weight was estimated as 45,000 to 55,000 daltons by gel filtration and as 45,000 daltons by SDS polyacrylamide gel electrophoresis. 2) The cofactor was a light chain kinase that phosphorylated both the L1 and L2 light chains of the Ml cell myosin, but not the L3 or heavy chain.

Actins↗
PubMed1982

Isolation of chicken eosinophils and their migration response in chick embryos.

Almost pure eosinophils have been isolated from chicken bursa, spleen, thymus or blood using Ficoll metrizoate and Ficoll gradient (eosinophil p.c. 71.4-87.6). Centrifugation by Ficoll discontinuous gradient yielded clear cut interface layer and had no deleterious influence on eosinophil viability. Purified eosinophil populations were examined for eosinophil migration response in vitro by exposure of the eosinophil population to lymphocytes sensitized in ovo with dinitrophenylated bovine serum albumin (DNP-BSA), DNP or BSA. Embryonic lymphocytes sensitized with antigens used led a eosinophil migration in vitro. From these findings, it is suggested that immunological maturation of the lymphocytes in the production of chemotactic factor may occur during the developing stage of chick embryos by antigenic stimulation.

Animals↗
PubMed1982

Differentiation of myeloid leukemia cells and changes in their contractile proteins.

In this report we tried to draw a sketch of our recent work on the changes in actomyosin system during the differentiation of a myeloid leukemia cell line (M1). Before the differentiation, the M1 cells do not have a locomotive and phagocytic activity but do after the differentiation. The rather peculiar properties of actin shown by the leukemic M1 cells, low polymerizability and low affinity to myosin ATPase as well as the presence of polymerization inhibitor may explain their inability of motility and phagocytosis. Enhanced binding of actin and myosin on the plasma membrane looks necessary to carry out those differentiated functions which must need vigorous movement of the membrane. The cell movement also anticipates cell deformability which accompanies gelation in a part of cytoplasm and solation in another part. We have already isolated several gelation-factors as well as inhibitors. Not only chemical modifications of actin and myosin molecules but also quantitative variations in those regulatory proteins must be taken into consideration. Thus, studies on the cellular contractile proteins are casting a new light on the process of differentiation of the M1 cell line.

Actin Depolymerizing Factors↗
PubMed1982

Experimental induction of sialadenitis in rats by heterologous anti-salivary gland antibodies.

The purpose of the present study was to investigate the possibility of experimental induction of sialadenitis in rats by heterologous anti-salivary gland antibodies. Two fractions (Fraction I and II) were separated from rat submaxillary gland by Sephadex-G 200 gel-filtration, and then rabbits were immunized with each fraction. Although cross-reactivity of anti-FrI antiserum obtained from the rabbits against rat liver and rat serum was shown, specific reactivity of both anti-FrI and anti-FrII antisera against rat submaxillary gland was demonstrated by immunodiffusion or the indirect immunofluorescence method. Activity of antibody-dependent cell-mediated cytotoxicity against fresh rat submaxillary gland was also demonstrated using the anti-FrII antiserum in vitro. Sialadenitis with periductal cell infiltrations was induced in the submaxillary glands of many rats injected intraperitoneally with each heterologous antiserum. Similar lesions, however, were also induced in the parotid glands, livers, or kidneys of these rats. By a direct immunofluorescence method, granular deposits of rabbit gammaglobulin were detected in cytoplasma of the infiltrating cells and the small vessels around the lesions of the submaxillary glands. It was suggested, therefore, that the lesions are induced by circulating immune complexes probably formed by non-organspecific antibodies contaminating each antiserum. Salivary gland lesions induced by a similar mechanism were also suggested in patients with Sjögren's syndrome, although the pathological significance in vivo of the anti-salivary gland antibody as an organspecific antibody could not be confirmed by the present study.

Animals↗
PubMed1981

Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes.

The steroid 21-hydroxylase system was purified from bovine adrenocortical microsomes. The physicochemical properties of NADPH-cytochrome P-450s21 were studied. The properties of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) of bovine adrenocortical microsomes have been reported previously (Hiwatashi, A. and Ichikawa, Y. (1979) Biochim. Biophys. Acta 580, 44-63). The steroid 21-hydroxylase system was reconstituted by cytochrome P-450s21 and NADPH-cytochrome P-450 reductase in the presence of detergent. The substrate specificity of the cytochrome P-450s21 was examined with the reconstituted system. The enzyme system was active in the 21-hydroxylations of 17 alpha-hydroxyprogesterone and progesterone, and the N-demethylation of (+)-benzphetamine. The cytochrome P-450s21 purified to as high as 16-17 nmol per mg protein was an electrophoretically pure glycoprotein. The molecular weight of the cytochrome P-450s21 was estimated to be 47 500 by SDS-polyacrylamide gel electrophoresis. Although cytochrome P-450scc or cytochrome P-45011 beta of bovine adrenocortical mitochondria did not react with antibody to cytochrome P-450BPA of bovine liver microsomes, the cytochrome P-450s21 formed an immunoprecipitin line against the antibody to cytochrome P-450BPA in the Ouchterlony double diffusion test.

Adrenal Cortex↗