Faculty Bibliography

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

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

The bacterial component responsible for the induction of transient cold agglutinin syndrome in rabbits after intravenous injection of heat-killed Listeria monocytogenes type 4B has been purified and biologically and chemically characterized. A purified immunoglobulin M cold agglutinin was prepared from high-titer sera resulting from the immunization of rabbits with heat-killed L. monocytogenes type 4B and was subsequently used to monitor the purification of the bacterial component responsible for its induction. The bacterial component was isolated from a hot phenol-water extract of lyophilized L. monocytogenes type 4B by multiple molecular sieve chromatography. Upon chemical analysis the purified material was found to be strikingly similar in chemical composition to gram-negative lipopolysaccharide endotoxins. The material contained 15% total fatty acid (of which 50% was beta-hydroxymyristic acid), 40 to 45% neutral sugar (glucose, galactose, and rhamnose), 11.5% amino sugar, 12% uronic acid, 2.5% 2-keto-3-deoxyoctonic acid, 2% heptose, 0.87% phosphorus, and 1.6% amino acid, thereby accounting for 85 to 90% of the weight of the component. Electron micrographs of the purified material were similar to those of lipopolysaccharide preparations from gram-negative organisms. The purified material exist in aqueous solutions as large aggregates, but can be dissociated into a single smaller subunit (3.1S) by dialysis against sodium dodecyl sulfate buffer. The listerial component was toxic and pyrogenic to rabbits, producing symptoms typical of gram-negative endotoxins. Activity in the limulus lysate gelation assay and in the carbocyanine dye assay provides a further link of this material with classical gram-negative endotoxins.

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

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