Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Insertional activation of a promoterless thymidine kinase gene.

A plasmid carrying a promoterless herpes simplex virus thymidine kinase gene was transfected via calcium phosphate precipitation into LM (tk-) mouse fibroblast cells. The transfected gene was efficiently expressed, as the transfected cells grew perfectly well in selective hypoxanthine-aminopterin-thymidine medium, suggesting that the thymidine kinase-coding region became linked to a promoterlike element on integration into the recipient genome. To investigate the structure of the surrogate promoter, we first isolated the integrated gene from a genomic library. The nucleotide sequence of the DNA adjacent to the thymidine kinase-coding sequence was then determined. We found, first, that the integration of the transfected DNA apparently occurred by a blunt end ligation mechanism involving no obvious sequence similarities between integrated and recipient DNA and, second, that the 5'-flanking region included a TATA box, two CCAAT boxes, and a GC box element. However, the TATA box motif and the most proximal CCAAT box appeared to be sufficient for full promoter activity, as determined by the transfection efficiencies of appropriate plasmid constructs. Except for these canonical promoter elements, the surrogate promoter had no obvious similarities to known thymidine kinase gene promoters.     

Diazepam metabolism and albumin secretion of porcine hepatocytes in collagen-sandwich after cryopreservation

Cryopreserved porcine hepatocytes in collagen cultures secreted albumin (up to 98 ± 3 g ml^–1; non-cryopreserved controls: 100 ± 9 g ml^–1) and metabolised diazepam (67.5% ± 7.5% of initially applied diazepam are metabolised; 77.5% ± 10% in non-cryopreserved controls) for up to 14 days after thawing. The cultures resembled the recently developed flat membrane bioreactors. Addition of 5% (v/v) serum did not effect diazepam metabolism. Hepatocytes in collagen-sandwich cultures offer a storable liver support system for potential clinical use.     

Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction

The use of hepatocyte cultures is well established for the study of drug-drug interactions. However, the major hindrance for the use of human hepatocyte cultures is that human hepatocytes are only occasionally available. This problem could be overcome by cryopreservation. Although cryopreserved hepatocytes have been recommended for short term applications in suspension, studies on induction of enzyme activity, requiring a more prolonged maintenance of cryopreserved hepatocytes in culture, represent a new field of research. In the present study, we established a technique that allows preparation of rat hepatocyte co-cultures, using cryopreserved hepatocytes. After incubation with phenobarbital (0.75 mM; 72 h) induction factors for the isoenzyme-dependent regio and stereoselective testosterone hydroxylations were 1.6, 2.2, 1.0, 2.1, 5.6, 2.4, 3.6, 4.5 and 0.9 for 2alpha-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone and 4-androsten-3,17 dione. Regarding induction factors of less than 2-fold, as questionable these induction factors were similar to those of cultures with freshly isolated hepatocytes and the induction pattern of the individual hydroxylation products was similar to the in vivo situation. In addition 3-methylcholanthrene (5 microM; 72 h) induced exclusively the formation of 7alpha-hydroxytestosterone (6.6-fold) in cultures with cryopreserved hepatocytes. This specificity also correlates to that obtained in rats. Although these induction factors were clearly satisfactory in cryopreserved cultures, the absolute activities of the main testosterone hydroxylation products were reduced when compared to fresh cultures. For instance, 6beta-hydroxytestosterone, the main metabolite in solvent controls was reduced to 79%, 7alpha-hydroxytestosterone, the main metabolite after induction with 3-MC, was reduced to 66% and 16beta-hydroxytestosterone, the main metabolite after induction with PB, was reduced to 52%. Similarly, EROD activity after induction with 3-methylcholanthrene in cryopreserved cultures was reduced to 62%, compared with that in fresh cultures. Although further optimization and validation is required, the data show that cytochrome P450 activities can clearly be induced in co-cultures of cryopreserved hepatocytes, in a fashion which for the investigated inducers, is similar to that in cultures from freshly isolated hepatocytes and similar to the in vivo situation.     

Comparative cytotoxicity of fumonisin B1 in two cell lines derived from normal human bronchial epithelial cells using four distinct bioassay techniques

This study focuses on the cytotoxic effects of fumonisin B₁ (FB₁) on both immortalised and immortalised and subsequently transfected normal human bronchial epithelial (NHBE) cells of human origin using four bioassays. While the MTT, Neutral Red and hexosaminidase colorimetric assays showed little difference between the toxic effects on the two related cell lines, the clonogenic assay, measuring cell survival and proliferation, indicated that FB₁ had a more toxic effect on the nontransfected cells. This kind ofin vitro approach using cells which retain many characteristics of normal cell growth and differentiation can go some way to developing evaluation models for food safety in the case of mycotoxin contamination without resorting totally to whole animal testing. Nevertheless, one or two cytotoxicity tests may be inadequate for a complete appraisal of toxic potential: rather, as wide a range of methodologies as feasible should be employed initially before meaningful conclusions may be drawn.     

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.