A partially purified preparation of the heatstable enterotoxin of EsMherichia coli caused a rapid and persistent increase in electric potential difference and short-circuit current when added in vitro to the luminal surface of isolated rabbit ileal mucosa. As little as 1 ng/ml produced an easily detectable response. Under short-circuit condition, the enterotoxin abolished net Cl-absorption; this change was half that produced by theophylline, which stimulated net secretion. The enterotoxin did not change cyclic AMP concentration but caused large and persistent increases in cyclic GMP concentration. The electrical and nucleotide responses exhibited similar and unusually broad concentration-ependences and maximal effects could not be demonstrated. Theophylline elevated cyclic GMP concentration 3fold both in the presence and absence of the enterotoxin, suggesting no effect of the toxin on cyclic GMP hosphodiesterase. Guanylate cyclase [GTP pyrophosphatelyase~cycizing); EC 4.6.1.21 activity in a crude membrane fraction from intestinal epithelial cells was stimulated 7-fold by the enterotoxin. These results suggest that guanylate cyclase stimulation is the basis for the toxin's diarrheagenic effect.Two enterotoxins have been identified among the extracellular products of Escherichia colh isolated from humans and other mammals with diarrheal disease-one heat-labile (1, 2) and the other heat-stable (1, 3, 4). The former is immunologically crossreactive with cholera toxin (5) and, like cholera toxin, stimulates adenylate cyclase (6). The latter acts more rapidly (7) and has a lower molecular weight [5000 or less (8) been equilibrated with 99% methanol/1% acetic acid. A broad peak of toxin activity appeared just behind the void volume.The eluate was reconcentrated and again filtered on Sephadex LH-20 that had been equilibrated with water. Toxin was then eluted with water and the eluate was stored in a refrigerator with preservatives. Even when stored for 6 wk at room temperature, no loss of activity could be detected. The final material was 200-to 1000-fold purified but still gave several peaks on silica gel chromatography. A detailed description of this procedure will be published elsewhere (W. J. Laird and D. M. Gill, unpublished data).Enterotoxin activity was assayed in suckling mice (9). Fifty-microliter aliquots of toxin in water were introduced by transabdominal injection into the stomachs of 2 to 4-day-old suckling mice (CD-1 Swiss white from Charles River Laboratories, Boston, MA). After 60 min the mice were killed with CHC13 and ratios of total intestinal weight to total body weight were determined. Control ratios were about 0.06 and maximal ratios were about 0.13. A mouse unit was defined as the amount of toxin producing a half-maximal increase in ratio. Serial 1:1 dilutions of toxin were assayed; three mice were used for each dilution.Three separately prepared batches of toxin were used for various phases of the present study. An aliquot from one of these batches was lyophilized and weighed: one mouse ...

Virulent strains of Yersinia pestis, Y. pseudotuberculosis and Yersinia enterocolitica invariably autoagglutinated in tissue culture media when grown at 36°C. Avirulent strains did not possess this property. Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica are animal pathogens which can cause a diversity of human disease. Y. pestis, the causative agent of bubonic plague in humans, is highly infectious and lethal to most rodents and many small mammals. Y. pseudotuberculosis and Y. enterocolitica, which pro

Recombinant human interleukin 2 purified from Escherichia coli has limited solubility at neutral pH and a short circulatory half-life. This recombinant interleukin 2 was chemically modified by an active ester of polyethylene glycol. The modified interleukin 2 was purified by hydrophobic interaction chromatography and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and isoelectric focusing. This conjugate was compared to unmodified recombinant interleukin 2 in vitro and in vivo. Covalent attachment of the hydrophilic polymer polyethylene glycol enhanced the solubility of interleukin 2, decreased its plasma clearance, and increased its antitumor potency in the Meth A murine sarcoma model.Interleukin 2 (IL-2), a glycosylated lymphokine with approximate molecular weight 15,000 (1), has therapeutic potential in treating cancers and infectious diseases (2-4). Human IL-2 has been obtained from genetically engineered Escherichia coli as an unglycosylated protein, rIL-2 (5-7), with biological activities equivalent to those of native glycosylated IL-2 (6, 8). However, the IL-2 expressed in E. coli is contained in insoluble refractile bodies within the bacteria and therefore denaturants are required during its purification. Both native IL-2 and rIL-2 are rapidly cleared from the circulation of mice (9-12) and rats (13), resulting in limited bioavailability of the protein. Nevertheless, Rosenberg and co-workers have shown that systemic administration of rIL-2 in high doses causes regression of established metastases in mice (14) and, in conjunction with lymphokine-activated killer cells, in humans (15). This suggests that increasing the bioavailability of rIL-2 may increase its potency, thus facilitating more effective use of this protein as a drug.We have modified rIL-2 to increase its solubility and plasma half-life. This has been accomplished by chemically attaching the polymer monomethoxy polyethylene glycol (PEG) to the protein. This straight-chain amphiphilic polymer has been used to covalently modify several proteins (16, 17). Abuchowski, Davis, and co-workers have modified several enzymes with PEG, including asparaginase and uricase, resulting in partially active conjugates with longer half-lives in vivo (18,19).We report here the preparation, purification, and characterization of rIL-2 covalently modified by monomethoxy polyethylene glycol. This PEG-rIL-2 conjugate is soluble in aqueous medium at neutral pH, is as active as IL-2 in three in vitro bioassays, exhibits an increased circulatory half-life in vivo, and is efficacious in vivo in a mouse tumor model. MATERIALS AND METHODSPreparation of an Active Ester of PEG. Monomethoxy PEG of average molecular weight 5000 (Aldrich) was used without further purification. PEG was converted to the active ester in two steps. In the first step, PEG-glutarate was prepared from PEG and glutaric anhydride as described by Zalipsky et al. (20). The second step was the preparation of the Nhydroxysuccinimide ester of 21). This ester reacts predomin...

A prophage map for corynebacteriophage beta consisting of seven markers has been constructed and compared with the vegetative map. The mapping system utilizes heteroimmune double lysogens and capitalizes on the fact that these double lysogens are very unstable and throw off monolysogenic segregants. The prophage map, produced by characterizing the recombinant phage in these monolysogenic segregants, appears to be a cyclic permutation of the vegetative map with the gene for toxin at one end of the prophage map and the gene for phage immunity at the other. This permutation is in accord with the Campbell model for insertion of lambda phage if a site between the toxin and immunity genes in the vegetative map is designated as the phage attachment site. The position of the gene for toxin in the prophage map suggests that converting phages may have originated as specialized transducing phages for this gene.

The orientation of the gene for diphtheria toxin, tox, in the prophage of converting corynebacteriophage beta has been determined. The orientation of tox in prophage and that reported simultaneously by Holmes (1976) for vegetative phage are compatible with the hypothesis that beta phage is inserted into the chromosome of its bacterial host by means of a mechanism similar to that described for lambda phage, and that the phage attachment site lies between the tox and imm genes. The position of three tox mutations that are phenotypically CRMhas also been determined. Relative to the tox-45 mutation, they are located more proximally to the end of the tox structural gene that corresponds to the amino terminal of diphtheria toxin.

The purpose of the present study was to develop immunotoxins directed against human ovarian carcinoma cells. Four monoclonal antibodies (260F9, 454(11, 2801)11, and 245E7) were chosen because they were found to bind to various ovarian carcinoma cell lines. These antibodies were covalently linked to either Pseudomonas exotoxin (PE) or ricin A chain (RTA), and the conjugates were tested against five ovarian cancer cell lines (OVCAR-2, -3, 4, -5; A1847). The ability of the immunotoxins to inhibit both protein synthesis and colony formation was tevaluated. Qualitatively similar results were obtained for both types of assays. Usually, PE conjugates were more toxic than their corresponding RTA conjugates. 454011-PE was very toxic for all ovarian carcinoma lines, whereas 454C11-RTA had low activity. Both 260F9-PE and 260F9-RTA were active in all OV-CAR cell lines but not in A1847 cells. 280D11-PE was toxic for OVCAR4; otherwise, 280D)1 -PE and RTA conjugates of both 280D11 and 245E7 had little activity. Specificity of immuiniotoxin action was shown by competition by excess antibody, nontoxicity in nontarget cells, and inactivity of an irrelevant immunotoxin.To investigate the basis of antibody-dependent differences in activity of the various immunotoxins, antibody uptake was studied in OVCAR-2 cells, and the results indicate that antibody internalization is one important factor in the activity of immunotoxins.

Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have not been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct from the structural gene for toxin, and that the CRMstrains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed.

a The top 11 markers are presented in the same order as in table 4 in Maraganore et al. 1 b Counts of genotype 11, 12, and 22. c Minor-allele frequency. d Two-sided P value for all strata and for female and male substrata in rs7878232.