A specific and potent immunotoxin composed of antibody ZME-018 and the plant toxin gelonin

Murine monoclonal antibody ZME-018 recognizes a 240 Kda glycoprotein present on the surface of most human melanoma cells and on over 80% of human biopsy specimens tested. Gelonin is a ribosome-inactivating plant toxin similar in nature and rivaling the activity of ricin A chain. ZME-018 was coupled to purified gelonin using the reagents SPDP and 2-iminothiolane. The ZME-gelonin conjugate was purified by S-300 Sephacryl and Blue Sepharose chromatography, removing unreacted gelonin and antibody, respectively. PAGE analysis showed that ZME was coupled to 1, 2, or 3 gelonin molecules. The ZME-gelonin conjugate was 10(6)-fold more active than gelonin itself in inhibiting the growth of log-phase human melanoma cells in culture. The immunoconjugate was not cytotoxic to antigen negative T-24 (human bladder carcinoma) cells. Treatment of melanoma cells with recombinant IFN-alpha or TNF substantially augmented the cytotoxicity of the immunoconjugate while treatment with IFN-gamma had a minor effect. Using the human tumor colony assay of melanoma cells obtained from fresh biopsy specimens, greater than 90% growth suppression was observed in 2 of 4 samples tested at a concentration of 250 ng/ml. In addition, 25% growth suppression was observed with a third sample tested, and no growth suppression was observed in 1 sample. Thus, clonogenic melanoma cells are sensitive in vitro to the cytotoxic activity of this immunotoxin at concentrations which we presume are pharmacologically relevant.     

The expression of granulocyte/macrophage colony-stimulating factor in activated mouse lymphocytes declines with age

The expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) was studied in spleen lymphocytes isolated from male C57BL/6J mice of 6, 20, and 29 months of age. GM-CSF expression (biological activity and mRNA level) was maximum after culturing the lymphocytes for 45 hr with concanavalin A and phorbol myristate acetate. The induction of both GM-CSF activity and mRNA levels was observed to decline over 60% between 6 and 29 months of age. The age-related decline in the level of GM-CSF paralleled the age-related decline in the mRNA levels of interleukin-2 and interleukin-3.     

Lethal effect of polymyxin B sulfate in experimental Proteus rettgeri infection in mice

The mouse model of intraperitoneal sepsis with Proteus rettgeri was used to evaluate the anti-endotoxic effect of polymyxin B sulfate. An unexpected reversal of the usual protective effect of polymyxin in experimental enterobacterial sepsis was observed in which the lethality of the infection was enhanced.     

The reversal of inhibition of protein synthesis by double-stranded RNA in lysed rabbit reticulocytes with fructose 6-phosphate.

Fructose 6-phosphate (1.4 mM – 3.0 mM) effectively prevents the inhibition of protein synthesis in unfractionated rabbit reticulocyte lysates by the presence of double-stranded RNA (poly rI:poly rC, 1 μg/ml). Glucose 6-phosphate, but not fructose 1,6-diphosphate, is equally as effective as fructose 6-phosphate. The data suggest that fructose 6-phosphate prevents the formation of a protein synthesis inhibitor induced by double-stranded RNA.     

Reaction of phenylglyoxal with arginine. The effect of buffers and pH.

Phenylglyoxal reacts much more rapidly with N²-acetylarginine than with either N²-acetyllysine or N-acetylcysteine. The rate of the reaction of phenylglyoxal with either N-acetylarginine or arginine increases with increasing pH from 7.5 to 11.5. The model reaction with arginine is much faster in bicarbonate, diethylamine, or triethylamine buffer than in N-ethylmorpholine, borate, phosphate, or Tris buffer. This activation by various buffers should be taken into consideration when glyoxal derivatives are used to modify arginyl residues.     

Purification and properties of the hemagglutinin from Wistaria floribunda seeds.

Extracts of Wistaria floribunda seeds contain separable erythroagglutinating and lymphocyte mitogenic activities. We wish to report the purification and characterization of the erythroagglutinating lectin of these seeds. A phosphate-buffered saline (PBS) extract of the ground seeds was made to 50% ethanol and the precipitate, which contained both the agglutinin and mitogen, was dissolved in PBS. The erythroagglutinating activity was adsorbed onto insoluble polyleycyl derivatized A + H active hog gastric mucin. After desorption with 0.2 M D-galactose and removal of the sugar by dialysis, the eluate displayed three protein bands on polyacrylamide gel electrophoresis. The major component represented 85% of the mixture. Immunoelectrophoresis of the mixture demonstrated immunochemical identity among the proteins. Gel filtration through Sephadex G-200 resulted in purification of the major component. Based upon the composition and subunit molecular weight, it was concluded that the three components represent a dimer, tetramer, and octamer of a single glycopolypeptide chain of 28 000. The erythroagglutinin has a pI at pH 5.4 and one cystine per dimeric unit.     

Colchicine and cytochalasin B (CB) effects on random movement, spreading and adhesion of mouse macrophages

The role of microtubules and microfilaments in the control of random movement of mouse peritoneal macrophages was examined by studying the colchicine and cytochalasin B (CB) effects. Colchicine in the concentration range of 10^?8–10^?4 M enhances the random movement of these cells. Enhanced movement of macrophages is observed only at colchicine concentrations which cause inhibition of their spreading and adhesion. CB does not enhance random movement at any concentration; it inhibits movement at 10^?7 M and higher concentrations. Furthermore, though 10^?8 M CB alone has no effect on the migration of macrophages, when present along with colchicine, the two drugs act synergistically and enhance random movement to a greater extent than colchicine alone. These findings suggest a cooperative interaction between microtubules and microfilaments in the control of movement of macrophages. A model is presented to explain the nature of this interaction.     

Fluorescence studies of 1,N6-ethenoadenosine triphosphate bound to G-actin: the nucleotide base is inaccessible to water

When 1,N6-ethenoadenosine triphosphate (epsilon-ATP) is free in solution, its fluorescence is collisionally quenched by iodide ion, by methionine, by tryptophan, and by cysteine. None of these quenches the fluorescence of epsilon-ATP bound to G-actin. Thus, the ethenoadenine base is bound in a region of the protein which is inaccessible to collisions with these reagents. Since we have previously shown that the fluorescence of epsilon-ATP is quenched by water, the long lifetime of epsilon-ATP bound to G-actin (36 nsec, vs 27 nsec for epsilon-ATP in water) indicates that the bound nucleotide base is inaccessible to collisional quenching by water molecules.