Stimulation of human gamma interferon production by diterpene esters.

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.

Lectins in the United States diet: a survey of lectins in commonly consumed foods and a review of the literature.

Plant lectins or phytohemagglutinins possess potent in vivo biological activities. Some, primarily of the family Leguminosae, have been shown to have deleterious nutritional effects. Little information exists, however, regarding the prevalence of lectins or the specific foods that contain lectins in the United States diet. In the present study the edible parts of 29 of 88 foods tested, including common salad ingredients, fresh fruits, roasted nuts, and processed cereals were found to possess significant lectin-like activity as assessed by hemagglutination and bacterial agglutination assays. Based on this survey and a review of the literature we conclude that dietary exposure to plant lectins is widespread. The spectrum of nutritional consequences of such exposure remains to be determined.

Chloride self exchange in Ehrlich ascites cells. Inhibition by furosemide and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid.

The effects of furosemide and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) on steady-state Cl- flux were studied in Ehrlich mouse ascites cells. At 10 mM, furosemide inhibited isotopically-determined Cl- flux by 86% without changing cell Cl- content, indicating that influx and efflux were depressed by the same amount. These results suggest that at least 86% of the steady-state Cl- flux may occur as a one for one exchange. Half of the inhibitory effect was not reversed by vigorous washing with albumin-Ringer. A smaller portion of steady-state Cl- flux was inhibited by SITS. The maximum effect of SITS was reached near 0.6 mM; at this concentration Cl- flux was reduced by 37% without an alteration in cell Cl- content. Possible competition of environment Cl- and SITS was investigated by replacing environment Cl- with acetate or NO3. These anions reduced the efficacy of SITS because they depressed cell Cl- turnover themselves, apparently acting on the same exchange process.

Altered erythrocyte osmotic fragility in mice with Ehrlich ascites tumor.

The osmotic fragility of erythrocytes from mice with Ehrlich ascites tumor was compared with that of erythrocytes from normal control animals. Erythrocytes collected from mice 15 days after the ip injection of tumor cells exhibited a uniform pattern of abnormal resistance to hemolysis in hypotonic saline, with 50% hemolysis occurring at an average saline concentration of 0.44% compared to an average of 0.49% for 11 controls a significant difference (P less than 0.0001). Erythrocytes from mice with this tumor apparently undergo an alteration in some component of the cell membrane that regulates either permeability to cations and water or distensibility of the cell.

Identification and quantitation of solubilized I blood group substance by wheat germ agglutinin using quantitative immunoelectrophoresis.

Wheat germ agglutinin (WGA) has been shown to react specifically with solubilized I blood group substance, purified from papain treated human erythrocyte membranes. WGA and I react to form an affinity precipitate in immunodiffusion gels, a reaction which can be blocked by the incorporation of N-acetyl glucosamine into the gel. The I material was a strong inhibitor of both anti-I cold hemagglutination and WGA hemagglutination reactions. Utilizing the techniques of crossed immunoelectrophoresis we have clearly established that WGA and anti-I IgM cold antibody are reacting with the same membrane macromolecule (I antigen). WGA was then used in a rocket affinoelectrophoretic assay system to quantitate I substance. The limits of detection in this system was 25 ng.

Nature of lectin-induced alteration of potassium transfer in Ehrlich ascites tumor cells.

The way in which the lectins concanavalin A (Con A) and Ricinus communis agglutinin (Ricin) alter the K+ content of Ehrlich ascites tumor cells was investigated. Unidrectional and net fluxes were determined in unwashed cells during a time course following lectin addition. Total influx, ouabain sensitive influx, Mg++- and Na+-K+-ATPase activity were all unaffected. Cell ATP content was normal for at least 19 minutes after exposure to Con A. Early after contact with Ricin or Con A efflux was stimulated 2-3-fold, resulting in net K+ loss, but after 20 minutes efflux had returned to normal. Ricin and Con A acted similarly although Ricin was present at only 1/50 the concentration of Con A. When the findings are evaluated together with previous work it is suggested that a particular membrane glycoprotein may be concerned in the efflux alteration observed.

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.

Multiple specificities of mammalian blood group substances comparatively studied with human isoagglutinins and fractionated anti-H lectins.

Purified blood group-active substances derived from different pig, horse, baboon, Rhesus monkey and human tissues were quantitatively studied for their haemagglutination inhibiting potency with: (1) human IgM anti-A and anti-B; (2) human anti-Lea and anti-Leb; (3) Ulex europaeus extracts separated into lectin fractions with respective L-fucose-inhibitable ('anti-HF') and chitobiose-cellobiose-inhibitable ('anti-HC') combining sites. Irrespective of species origin, A and B blood group activity per milligram of purified material tended to be strikingly higher in substances low in, or devoid of, Lewis blood group activity. Most of the blood group substances displayed variable but about equally balanced amounts of Ulex anti-HF and anti-HC inhibiting activity. In contrast, pig submaxillary gland mucins displayed strikingly high levels of Ulex anti-HC inihibiting activity, even in the complete absence of Ulex anti-HF inhibiting activity. These serological findings are consistent with current biochemical concepts regarding the heterosaccharide microheterogeneity of blood group-active glycoproteins.

Immunological properties of Micrococcus lysodeikticus membranes.

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.