Validation of CHEMEST, an on-line system for the estimation of chemical properties.

CHEMEST is an on-line system for estimating physical/chemical properties important in the assessment of environmental fate of chemicals. Measured values of boiling point, melting point, vapor pressure, water solubility, and soil organic carbon partition coefficient (Koc) were compared to values estimated by CHEMEST, for more than 170 chemicals. The chemicals were composed of seven classes: alcohols, aldehydes and ketones, amines, carbamates, esters, ethers, and phenols. Mean errors for boiling and melting point were 22.6 and 36.2 degrees C, respectively, with relatively little variability among classes. Carbamates were an exception, accuracy being much lower for both properties. Vapor pressures were quite accurately estimated, measured and estimated values generally differing by less than a factor of two. Mean error factors for water solubility and Koc were somewhat higher, but nevertheless better than the order of magnitude accuracy commonly assumed. Solubility was very inaccurately estimated for the phenols, however. The results indicate that, for the chemicals and properties studied, estimated values were sufficiently accurate for screening-level fate assessment. TSTCHEM, a revised version of CHEMEST, out-performed the latter with respect to the accuracy of solubility and vapor pressure estimates.

Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.

A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media. Affi-Gel Blue chromatography, chromatofocusing, and anti-Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodinated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at Mr 31,500 (+/- 1,000), and a wide heavy band was observed between Mr 4,000 and 15,000. In the presence of 2-mercaptoethanol, bands were seen at Mr 31,500 (+/- 1,000), 27,000, and 4,000 to 15,000. Other similarities between purified E. coli H30 and Shiga 60R toxins included identical isoelectric points (7.03 +/- 0.02); comparable biological activities, i.e., cytotoxicity, lethality for mice, and enterotoxicity; and the same relative heat stabilities (up to 65 degrees C for 30 min). Nevertheless, the two toxins had apparently different molecular weights as determined by sucrose gradient analysis, by gel filtration, and by cross-linking experiments with dimethyl suberimidate. The Mr of native E. coli H30 toxin estimated from cross-linking studies was 48,000, whereas the estimated Mr of Shiga 60R toxin was 58,000. These results suggest that like the cholera-E. coli-heat-labile toxin family, a family of Shiga-like toxins exists.

Production of Shigella dysenteriae type 1-like cytotoxin by Escherichia coli.

Strains of Escherichia coli previously implicated or proven to be causes of diarrhea were examined for production of a toxin similar to that of Shigella dysenteriae type 1 (Shiga). Organisms grown in an iron-depleted broth were lysed by pressure disruption followed by ultracentrifugation. Saline-dialyzed extracts were tested for cytotoxic effects on HeLa cells that were neutralizable with antiserum to Shiga toxin. Among the 13 E. coli strains so analyzed, 11 made a Shiga-like cytotoxin in levels ranging from trace (two avirulent isolates) to amounts equivalent to S. dysenteriae type 1 (two noninvasive strains that did not make E. coli heat-labile or -stable enterotoxins but were isolated from infants with diarrhea). As with extracts of Shiga toxin, lysates of these E. coli strains that produced high levels of Shiga-like toxin were enterotoxic for rabbits, paralytic and lethal for mice, and inhibited protein synthesis in HeLa cells. Thus, these data suggest that Shiga-like toxin may be another heretofore undiscovered factor in the pathogenesis of diarrhea caused by some E. coli strains.

Immunochemical and cytotoxic activities of Shigella dysenteriae 1 (shiga) and shiga-like toxins.

Toxins in culture supernatants and bacterial lysates of S. dysenteriae 1 and S. flexneri were quantitated by a cytotoxicity assay and a newly developed radioimmunoassay. Cytotoxin titers paralleled toxin antigen levels. Thus, variations in cytotoxicity among shigellae probably reflect differences in toxin yield rather than specific activity (cytotoxicity per microgram of toxin antigen).

Characterization of Shigella dysenteriae 1 (Shiga) toxin purified by anti-Shiga toxin affinity chromatography.

Shigella dysenteriae 1 (Shiga) toxin was purified from whole-cell lysates by antitoxin affinity column chromatography, radioiodination, and Sephacryl S-200 gel filtration of 125I-labeled affinity column eluates. Two chromatographic peaks were observed. The percentage of radioactivity in peak I samples immunoprecipitated with antitoxin ranged from 95 to 100%. A pool of samples from this first peak contained over 90% of the HeLa-cell-cytotoxic units applied to the column and was enterotoxic for rabbit ileal loops and lethal for rabbits. This radiolabeled material migrated as a single cytotoxic band after nondenaturing polyacrylamide gel electrophoresis, but formed three bands, of 33,000, 29,000, and 4,000 to 7,000 daltons, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, material estimated as 7,000 daltons by Bio-Gel P-10 chromatography could be generated by treatment of S-200 peak I samples with 8 M urea. Pooled fractions from the second S-200 peak were separable into several low-molecular-weight peaks on a P-10 column. One of these P-10 peaks (7,000 daltons) was 27% immunoprecipitable with antitoxin. These data indicate that three of the known biological activities of Shiga toxin are associated with a 33,000-dalton substance which can be dissociated into 29,000- and 4,000- to 7,000-dalton components.