Effects of deficiency in p53 or bcl-2 on the sensitivity of clonogenic cells in the small intestine to low dose-rate irradiation.

PURPOSE: To study the role of p53 and bcl-2 in the response of the small intestine to irradiation delivered at low dose-rate. MATERIALS AND METHODS: Mice homozygous for p53 or bcl-2 deletion (-/-), their respective heterozygotes (+/-), and their wild-type littermates (+/+) including a previously used hybrid strain (B6D2F1), were irradiated to the whole-body using 60Co gamma-rays at 1 Gy h(-1). Crypt survival levels in the small intestine were measured at day 3 after the end of irradiation. RESULTS: Crypt survival levels were higher in p53 -/- mice than in the other p53 genotypes after 25-30 Gy, but not after lower or higher doses. Similar experiments with the three genotypes for bcl-2 status showed lower crypt survival after all doses used in the -/- mice, compared with the +/- and +/+ mice, which were similar in response. The marked degree of curvature in the survival curve observed for the p53 genotypes was also observed in B6D2F1 hybrid mice, was particularly striking in the p53 -/- mice, but was not seen to the same extent in the bcl-2 genotypes. The heterozygotes for p53 or for bcl-2 were nearer in response to their respective +/+ genotypes rather than the -/- genotypes. CONCLUSION: The increased crypt survival levels at some radiation dose levels in the p53 nulls contrasts with the lack of change reported previously using irradiation at high dose-rate. The decreased survival in the bcl-2 nulls is consistent with the known 'survival' function of bcl-2, although bcl-2 expression has not been detected immunohistochemically in this intestinal site. The marked degree of curvature in the dose-response curve at high dose levels for some genotypes was unexpected at this low dose-rate.

The effect of lincomycin on exoprotein production by Vibrio cholerae.

Lincomycin has a differential effect on exoprotein production by Vibrio cholerae. The production of some proteins, such as cholera toxin and deoxyribonuclease, is stimulated by low concentrations of the drug while production of other proteins, such as protease and alkaline phosphatase, is unaffected. Possible mechanisms of the lincomycin effect are discussed.

Effect of extracellular protease production on bacteriophage sensitivity of Vibrio cholerae.

The effect of varying levels of extracellular protease production on the bacteriophage type of Vibrio cholerae 1621 serotype 01, biotype E1 Tor, has been investigated. It has been shown that the production of high levels of exoprotease can alter the apparent type of the strain by rendering it insensitive to infection by a number of bacteriophages. Prevention of a productive infection appears to be due to altered surface characteristics rather than to specific damage to the bacteriophages. Such altered surface characteristics may be due to auto-digestion. The importance of this observation to epidemiological studies of V. cholerae is noted and discussed.

The effect of influenza virus on the adherence of Haemophilus influenzae to human cells in tissue culture.

The adherence of eleven strains of Haemophilus influenzae to MRC5 cells was studied and compared with adherence of the same eleven strains to MRC5 cells infected with influenza A/NWS/33 virus. Per cent Adhesion (the proportion of cells to which more than two bacteria were adhering) was estimated. Organisms grown on solid media adhered better than those grown in liquid media though the difference was not statistically significant (t test for independent means). A wide range of % Adhesion values for organisms grown on solid media to control cells was exhibited (1-88%). Ten of eleven strains grown on solid media or in broth showed increased adherence to influenza virus infected cells; this difference was significant (P less than 0.05, t test for independent means). The effect of virus infection in increasing % Adhesion was inversely proportional to the adhesiveness of the strain in question to uninfected cells. Strains that adhered most efficiently to control cells showed little increase in % Adhesion following virus infection, while strains that adhered poorly to control cells showed large increases in % Adhesion following virus infection.

The expression of biologically active cholera toxin in Escherichia coli.

Chromosomal DNA from Vibrio cholerae E1 Tor strain 1621 was digested with Hind III and the fragments obtained fractionated by centrifugation through a sucrose gradient. A 15Kb fragment which contained the toxin gene of V. cholerae was identified by its homology with the heat labile toxin (LT) gene of toxigenic E. coli. This fragment was cloned in E. coli using pAT153 and subsequently characterized by restriction endonuclease digestion. Sequences homologous to the LT gene were identified by hybridization and then sub-cloned using either pAT153 or pACYC184. Expression of the cloned CT gene in E. coli was detected using both cell culture and ELISA assays. One recombinant plasmid coded for the synthesis of an immunologically active but biologically inactive derivative of CT.

Biochemical characterization of extracellular proteases from Vibrio cholerae.

Isoelectric focusing of culture supernatants from Vibrio cholerae El Tor 1621 and high protease-producing mutant strain 1621 hip revealed the presence of three different types of extracellular protease. Type I protease was the major activity in the wild-type strain and was inhibited by phenylmethylsulfonyl fluoride and by the lima bean trypsin inhibitor. Type II protease was present in the wild type and was the major activity in the high protease-producing mutant. It was resistant to inhibitors of metalloproteases and serine proteases. Two peaks of type II protease differed by 1.2 pI units in isoelectric point and by 1,500 in molecular weight. Type II protease had broad specificity, acted as a mucinase, and caused degradation of some other V. cholerae extracellular proteins, including DNase and cholera toxin. Type III protease was EDTA inhibitable and was detected only in the high protease producer. Possible roles of extracellular proteases as virulence factors in cholera pathogenesis are discussed.

The expression of biologically active cholera toxin in Escherichia coli.

Chromosomal DNA from Vibrio cholerae El Tor strain 1621 was digested with Hind III and the products fractionated by centrifugation through a sucrose gradient. A 15kb fragment containing the toxin gene of V. cholerae was identified by its homology with the heat labile toxin (LT) gene of toxigenic E. coli. This fragment was cloned in E. coli using pAT153 and subsequently characterised by digestion with different restriction endonucleases. Sequences homologous to the LT gene were identified by hybridisation and then sub-cloned using either pAT153 or pACYC184. Expression of the cloned CT gene in E. coli was detected using both cell culture and ELISA assays. One recombinant plasmid coded for the synthesis of an immunologically active but biologically inactive derivative of CT.

Survival of intestinal crypts after treatment by adriamycin alone or with radiation.

A survival curve has been established for jejunal crypts of BDF1 mice treated by single i.p. doses of the antibiotic agent adriamycin. The threshold dose and Do were twice that for marrow CFU-S of these mice. The overall extrapolation number of the crypt survival curve was very low (1.3 +/- 0.13) compared to the value for gamma radiation. This observation is discussed with respect to the interpretation of crypt survival curves. We were unable to demonstrate any enhancement by adriamycin (reduction in Dq) of the response of microcolony-forming cells to radiation given immediately before the drug.