Biological response modifiers and chemotherapeutic agents that alter interleukin 2 activities.

IL2 immunotherapy alone or with LAK cells represents a novel approach to the treatment of metastatic cancers. Similarly, other BRMs and classical chemotherapeutic drugs through their molecular effects on the components of the immune system reveal new and exciting prospects for the better use of these agents. Both approaches converge in that they restore balance among numerous components of the immune surveillance system. This review raises the possibility of improved protocols through the judicious use of these agents and stresses the need for further investigation of combined use of chemotherapy and immunotherapy.

Solubilization of multilamellar liposomes in the presence of non-ionized drug.

The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37 degrees C by 7.3 nM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 h at pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilization was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.

Induction of adhesiveness in human endothelial cells by Plasmodium falciparum-infected erythrocytes.

Cytoadhesion of infected erythrocytes to endothelium plays an important role in the pathogenesis of Plasmodium falciparum malaria. In vitro assays of cytoadhesion have helped to identify putative host ligands, namely thrombospondin, platelet glycoprotein IV (CD36), and intercellular adhesion molecule-1 (CD54) as possible mediators of cytoadhesion. However, the presence of these ligands on some host cells to which infected erythrocytes do not adhere raises the possibility that other molecules or factors may be involved. In the present study, we investigated the effects of prolonged incubation of endothelial cells (EC) with infected erythrocytes on adhesiveness of EC. We also studied the effects of tumor necrosis factor (TNF), interleukin-1 (IL-1), and phorbol myristate acetate (PMA). We found that when EC were incubated in contact with ring-infected erythrocytes for 24 hr during which the rings developed into trophozoites, adhesiveness was enhanced up to 250%. Incubation of EC with IL-1 or TNF for 12 hr increased adhesiveness by 50% at minimum doses of 5 U/ml and 50 U/ml, respectively, while PMA decreased adhesiveness in a consistent and dose-dependent manner. These results show that host EC adhesive ligands for infected erythrocytes can be induced, most notably by direct contact between the EC and infected erythrocytes containing developing parasites. The cultured human EC used in this study lacked surface CD36 detectable by immunofluorescence assay, suggesting that CD36 is not required for endothelial adhesiveness.

Regulation of polyamine-responsive protein kinase by certain highly specific polyamines and charged carbohydrates.

Polyamine-responsive protein kinase, a cyclic nucleotide-independent protein kinase from the cytosol of Morris hepatoma 3924A, was stimulated 8-9 fold by several different polymers of polylysine, polyornithine and random copolymers of lysine-alanine; spermidine, spermine, and mixtures of spermine and spermidine stimulated 2, 3, and 5 fold, respectively. The protein kinase was not stimulated by poly-carboxybenzyl-lysine, random copolymer of lysine-tyrosine, polyhistidine, polymethionine, polyglutamic acid, polyaspartic acid, dipeptide (Lys-Lys), lysine, ornithine, and putresine. The polyamine stimulation of the protein kinase was prevented by certain specific charged carbohydrates: heparin, chondroitin sulfates A, B, and C, dextran sulfate and hyaluronic acid. It was not prevented by noncharged carbohydrates: dextran, glycogen, starch, sucrose, etc; or by sulfate salts: ammonium sulfate, potassium sulfate, sodium thiosulfate, etc. The inhibition was reversed by increased polylysine. Heparin was non-competitive inhibitor of Mg2+-ATP. It would appear that this enzyme is regulated by certain highly specific molecules with certain sizes and charges; plus charge is stimulatory, negative charge prevents the stimulation.

Calmodulin stimulates polyamine-responsive protein kinase in the absence of Ca2+.

A cyclic nucleotide-independent, polyamine-responsive protein kinase from the cytosol of Morris hepatoma 3924A, which phosphorylated heat-stable endogenous substrates and casein in the presence of polyamines (Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Res. 38, 3540-3545) was observed to be stimulated by an endogenous protein activator. This protein activator was identified to be calmodulin. the polyamine-responsive protein kinase was also stimulated by purified calmodulin, but only in the presence of polyamines such as polylysine. This action of calmodulin did not require Ca2+ for activation of the enzyme; and activation occurred in the presence of EGTA. DNA and RNA inhibited the polyamine-responsive protein kinase, either in the presence or absence of Ca2+. Purified calmodulin, in the presence of cyclic AMP or cyclic GMP, did not activate the protein kinase. Therefore, polyamines such as polylysine are an absolute requirement for this expression of calmodulin action. The increased enzyme activity by calmodulin was accompanied with an increased Vmax and with no changes in the Km (ATP). High levels of cation, up to 100 mM Mg2+, did not effect the action of calmodulin. These results indicate that tumor cytosolic polyamine-responsive protein kinase is regulated by calmodulin, the latter being increased in the tumor tissue.

Interaction of bisulfite with unsaturated fatty acids.

Sodium bisulfite reacted with unsaturated fatty acids, significantly increasing their polarity as determined by behavior on silica gel thin-layer chromatography. The ultraviolet absorption spectra of unsaturated fatty acids (due to the presence of double bonds) were abolished as a result of the reaction. Fatty acids containing more than one double bond (arachidonate, linolinate, and linoleate) reacted more rapidly with bisulfite than did oleate. When arachidonate double bonds were titrated with bisulfite there was a much larger spectral decrease with the first equivalent of bisulfite added than with each subsequent addition. Vitamin E, vitamin E nicotinate, and butylated hydroxytoluene significantly inhibited the reaction of bisulfite with unsaturated fatty acids. It is suggested that the reaction of bisulfite with unsaturated fatty acids may be a mechanism of SO2 toxicity.

Identification and characterization of endogenous inhibitors of polyamine-responsive protein kinase activity.

Polyamine-responsive protein kinase activity was inhibited by two heat stable inhibitors which were purified from Morris hepatoma 3924A. They were of low molecular weight (inhibitor I-1,600 to 2,000, inhibitor II-600 to 800 daltons). The inhibitors, when purified, passed through a DEAE-cellulose column; however, the crude complex of inhibitors and protein kinase activity bound to the DEAE-cellulose column. This suggests that the inhibitors are bound to the polyamine-responsive protein kinase in vivo. Both inhibitors completely inhibited the polyamine stimulated activity, but did not affect the basal enzymatic activity. They were not affected by treatment at 90 degrees for 2 min. These results demonstrate the presence of two new protein kinase inhibitors in mammalian cells.