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E Mekada

Publications and source records attributed to E Mekada.

65 records · Page 4Linked to original sources
PubMed1981

Methylamine stimulates the action of ricin toxin but inhibits that of diphtheria toxin.

The action of ricin toxin was stimulated by addition of methylamine or some other amines, as shown by measuring the inhibition of protein synthesis of cultured cells by the toxin. Under the same conditions, however, the action of diphtheria toxin was completely inhibited by the amines. In a cell-free protein-synthesizing system, methylamine had no effect on the action of the A chain of ricin toxin and fragment A of diphtheria toxin. Studies on the interactions of 125I-labeled toxins with cells revealed that methylamine did not alter toxin-receptor bindings, but affected the entry of the toxins into the cells. Studies were also made on the effects of methylamine on the actions of two hybrid toxins, formed from a subunit of Wistaria floribunda lectin and fragment A of diphtheria toxin and the A chain of ricin toxin, respectively. Results suggested that the processes of entry of ricin toxin and diphtheria toxin, or at least parts of these processes, are different.

Cell-Free System↗
PubMed1980

Hybrid toxin of the A chain of ricin toxin and a subunit of Wistaria floribunda lectin. Possible importance of the hydrophobic region for entry of toxin into the cell.

About 27% or more of the total amount of the A chain of ricin toxin associated with lipid vesicles, whereas less than 1.4% of the A fragment of diphtheria toxin associated with the vesicles under the various conditions tested. Two hybrid toxins were constituted from a subunit of Wistaria floribunda lectin and the A fragment of diphtheria toxin (Uchida, Y., Yamaizumi, M., Mekada, E., Okada, Y., Tsuda, M., Kurokawa, T., and Sugino, Y. (1978) J. Biol. Chem. 253, 6307-6310) and the A chain of ricin toxin, which has higher affinity to lipids than the A fragment of diphtheria toxin. The toxicity of the hybrid of the A chain of ricin and the subunit of W. floribunda lectin, estimated by measuring the inhibition of protein synthesis of cultured cells, was about 100 to 200 times that of the hybrid of the A fragment of diphtheria and the subunit of W. floribunda lectin. These findings may suggest that a hydrophobic region of the toxin is essential for entry of toxin into the cells.

Amino Acid Sequence↗
PubMed1978

Reconstitution of hybrid toxin from Fragment A of diphtheria toxin and a subunit of Wistaria floribunda lectin.

A hybrid protein was prepared in which Fragment A of diphtheria toxin was linked to a monovalent subunit of the lectin from Wistaria floribunda seeds by a disulfide bridge. The hybrid molecule was reconstituted effectively from a reduced mixture of Fragment A and the lectin subunit by oxidation with o-phenanthroline and CuSO4. The monovalent hybrid protein was partially purified and found to be toxic to L cells. The toxicity of the hybrid protein on the cells was blocked by the addition of either N-acetyl-D-galactosamine, which specifically binds to the lectin, or by anti-Fragment A antibody.

Acetylgalactosamine↗
PubMed1978

One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell.

Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that has fused with only one erythrocyte ghost were separated in a flourescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell. This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100-200 fold excess of fragment A-176 was needed to kill the cells.

Cell Survival↗
PubMed1978

An attempt to separate mononuclear cells fused with human red blood cell-ghosts from a cell mixture treated with HVJ (Sendai virus) using a fluorescence activated cell sorter (FACS II).

Nucleated cells (Ehrlich ascites tumor cells or L strain cells) and human red blood cells (RBC)-ghosts were mixed and fused by ultraviolet-inactivated HVJ (Sendai virus). The cell mixture was stained with FITC conjugated anti-RBC ghost antiserum and then applied to FACS II apparatus. The apparatus sorted mononuclear cells fused with RBC-ghosts from the cell mixture on the basis of both the light scattering and fluorescence profiles. When the same procedure was carried out on a mixture containing cells and intact human RBC, the cells sorted by this method were cells into which hemoglobin had been injected. The sorted cells were capable of forming colonies in culture. This sorting method may be useful for collecting cells in which macromolecules have been injected artificially by fusion of RBC-ghosts enclosing macromolecules.

Cell Fusion↗
PubMed1978

Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter.

FITC-conjugated bovine serum albumin (FITC-BSA) molecules were quantitatively introduced into human erythrocyte ghosts by gradual hemolysis. When the ghosts and L cells were fused with UV-inactivated HVJ (Sendai virus), FITC-BSA was introduced into the cytoplasm of the L cells and fluorescence could be observed inthe cells with a fluorescence microscope. A mixture of L cells and ghosts was introduced into a fluorescence activated cell sorter (FACS), which could separate the mononuclear cells on the basis of their light-scattering profile. Four distinct populations of mononuclear cells were found by fluorescence analysis. These populations were separated from the cell mixture and found to correspond to cells fused with one, two and three ghosts and unfused cells. After separation, the cells from each population could form colonies in culture. As a given macromolecule can be quantitatively introduced into erythrocyte ghosts with the FITC-BSA, after fusion of these ghosts with cells, this sorting method is useful for separating cells containing a definite number of macromolecules.

Cell Fusion↗