Suspension Assay Research Articles

Mutagenicity of methyl-, ethyl-, propyl- and butylnitrosourea towards Escherichia coli WP2 strains with varying DNA repair capabilities

Methyl- (MNUA), ethyl- (ENUA), propyl- (PNUA) and butylnitrosourea (BNUA) have been tested for toxicity and mutation in a liquid suspension assay towards Escherichia coli WP2 and some of its repair deficient derivatives. A comparison of survival rates after nitrosourea exposure between WP2 and WP2 uvrA showed no difference between the two strains but a consistent difference in potency between the various nitrosoureas studied. Toxicity increased in the order MNUA less than PNUA less than ENUA less than BNUA. ENUA and PNUA induced a greater number of trp+ revertants in both strains than did MNUA and BNUA, particularly at low survival rates. None of these differences in biological potency could be accounted for by differences in rates of hydrolysis. ENUA, PNUA and BNUA were non-mutagenic towards WP2 lexA, WP2 recA and WP2 uvrA lexA, whereas MNUA did induce mutations. Ethyl methanesulphonate (EMS) was able to mutate WP2 lexA. These results are discussed in the light of current theories regarding the mechanism of action of these compounds.

Concanavalin A-Induced Agglutinability of Normal, Preneoplastic, and Neoplastic Mouse Mammary Cells<xref ref-type="fn" rid="FN2">2</xref>

The concanavalin A (Con A)-induced agglutinability of normal, preneoplastic, and neoplastic mouse mammary epithelial cells was examined. Cells freshly dissociated from normal mammary glands, hyperplastic alveolar nodules, or primary mammary adenocarcinomas by collagenase digestion in the presence of bovine serum albumin were strongly agglutinated by low concentrations of Con A. After short-term culture in vitro, however, cells from all three types of tissue were only weakly agglutinated by Con A, as measured by both suspension and hemadsorption assays. By comparison, cells of three established mammary tumor culture lines agglutinated strongly in the presence of the lectin. Treatment of the normal, preneoplastic, and neoplastic mammary cells in primary cultures with either trypsin or collagenase had little or no effect on their agglutinability, whereas hyaluronidase significantly increased their reactivity. Studies with fluorescein-tagged Con A indicated that all three cell types were capable of binding the lectin. The results were consistent with previous evidence suggesting that neoplastic transformation of mouse mammary epithelial cells is not manifested in vitro by several of the alterations in growth patterns, intercellular interactions, and surface properties that usually accompany transformation of fibroblastic cells.

Cell surface glycosyltransferases—do they exist?

The presence of glycosyltransferases on surfaces of mammalian cells has been reported by many investigators and a biological role for these enzymes in cell adhesion and cell recognition has been postulated. Critical analysis, however, showed 2 major complications regarding the assay for cell surface glycosyltransferases: 1) hydrolysis of the nucleotide sugar by cell surface enzymes and subsequent intracellular use of the free sugar and 2) loss of cell integrity if trypsinized or EDTA-treated cells were used in suspension assays. We have assayed intact, viable cells in monolayer for cell surface glycosyltransferases using conditions under which intracellular utilization of free sugars generated by hydrolysis of the nucleotide sugar was prevented. Our data demonstrate that the presence of galactosyltransferases on the surface of a variety of cells, including established (normal and virally transformed) as well as nonestablished cells, is unlikely. No evidence for the existence of cell surface fucosyl- and sialytransferases could be obtained, but our data do not exclude the possibility that low levels of these enzymes are present.

Activation of fibrinolysis by certain phospholipids

The fibrinolytic activity of plasmin was found to be significantly increased by phosphatidyl serine and inositol phosphatide. Only these two of a wide variety of lipid-related and micelle-forming agents were effective in enhancing fibrinolysis, though calcium, even at low concentrations, prevented the enhancement effect. Plasmin fibrinolytic activity was assessed by decreased turbidity in a fibrin powder suspension. The effect was not due to stabilization of plasmin by phospholipid under the conditions used. At temperatures near 48°C plasmin itself was destroyed by inositol phosphatide more rapidly than normal. This was taken to indicate some direct interaction between the enzyme protein and the phospholipid, rather than between the fibrin substrate and the phospholipid. The fibrin powder suspension assay was easily manipulated to give a range of substrate concentrations. Rates of fibrinolysis determined at several fibrin levels were expressed as a reciprocal plot assuming a normal three-step enzyme mechanism. Kinetic analysis suggested that the phosphatidyl inositol did not affect the first step of the mechanism, i.e. that involving binding of plasmin to fibrin, but increased the rate of one of the subsequent steps.