Gene therapy clinical trials in Belgium.
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Belgium↗
Biomedical subjects
W Moens
Publications and source records attributed to W Moens.
PCR typing of tetracycline resistance determinants (Tet A-E) in Salmonella enterica serotype Hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium.
The distribution of tetracycline resistance determinants Tet A-E was studied by PCR in 40 tetracycline-resistant Salmonella enterica serotype Hadar (S. hadar) isolates collected from human patients in 1996 and 1997, as well as in the microbial community originating from activated sludges of hospital and urban wastewater treatment facilities. A fast DNA extraction and purification method from activated sludges was used to provide amplifiable DNA. The method is based on the direct lysis of bacteria improved by bead-beating followed by DNA purification on polyvinylpolypyrrolidone spin columns to remove PCR inhibitors. The purified DNAs from salmonellae and activated sludges were characterized for the presence of tetracycline determinants with specific primer pairs designed on the basis of published sequences. The Tet A determinant was present in all clinical isolates and DNAs extracted from the bacterial community of the selected activated sludges. The Tet C determinant was identified in only one of the 40 clinical isolates and in six of the seven environmental samples. No signal was detected for Tet B, D and E determinants. This study revealed a high and stable prevalence of the Tet A determinant in both salmonellae clinical isolates and the microbial community of activated sludges from hospital and urban wastewater treatment facilities over a 2-year period.
Journal Article↗
Mutagenic activity of dichloroethylamino derivatives of nitronaphthofuran and some nitrobenzofurans in the Salmonella/microsome assay.
The mutagenic activity of five dichloroethylamino 2-nitrobenzofuran derivatives and one dichloroethylamino 2-nitronaphthofuran derivative was analysed in the Salmonella/microsome assay. We investigated the influence of the position of the dichloroethylamino and/or the methoxy groups on the mutagenic activity of these nitro arenofurans in S. typhimurium strain TA100 and its variant TA100NR, deficient in nitroreductase. Without metabolic activation 7-[bis(2-chloroethyl)amino]-2-nitronaphtho[2,1-b]furan (1), 4-[bis(2-chloroethyl)amino]-7-methoxy-2-nitrobenzofuran (2), 7-[bis(2-chloroethyl)amino]-4-methoxy-2-nitrobenzofuran (5) and 6-[bis(2-chloroethyl)amino]-2-nitrobenzofuran (6) are mutagenic in TA100, while 4-[bis(2-chloroethyl)amino]-5-methoxy-2-nitrobenzofuran (4) is weakly mutagenic and 5-[bis(2-chloroethyl)-amino]-2-nitrobenzofuran (3) toxic. In the NR deficient strain compounds 1, 3 and 6 are strong mutagens and 4 is weakly positive. The two isomers 2 and 5 are negative in that strain. The naphthofuran derivative 1 is highly mutagenic in the absence of S9 mix in both strains considered, but less than R7000 (7). A decrease in the electronic polarity of compound 1 versus compound 7 according to the hypothesis developed by Royer et al. is a possible explanation. After exogenous metabolic activation by S9 mix all the compounds tested are highly mutagenic in both Salmonella strains. The position of the dichloroethylamino group and/or the presence of a methoxyl on the alpha-nitroarenofuran derivatives seem to modify the activity of bacterial as well as exogenous nitroreductases or other activating enzymes.
Animals↗
The primary structure of human liver type phosphofructokinase and its comparison with other types of PFK.
The complete mRNA sequence of the human liver-type phosphofructokinase (hPFKL) was determined. The sequence included 55 nucleotides of 5' and 515 of 3' noncoding regions, as well as 2,337 nucleotides encoding the 779 amino acids of the hPFKL. Extensive similarity (approximately 90%) in the coding region was observed between the hPFKL and the mouse PFKL, whereas the degree of similarity between different types of PFK, i.e., hPFKL and human muscle-type PFK (hPFKM), was merely 68%. Nevertheless, striking similarity between these different types of PFK was noticed when the amino acid residues creating the various active sites of the enzyme were compared. Human PFK L- and M-specific probes were constructed and used to quantitate the mRNA levels in fetal and adult brains and fetal liver. It was found that while relative amount of PFKL mRNA in adult brain was one-fourth of that detected in fetal brain the level of PFKM mRNA in adult brain was slightly higher than in fetal tissue, suggesting that PFK expression might be controlled at the transcriptional level.
Amino Acid Sequence↗
In-vivo carcinogenicity of 2-nitro-oxaphenalenes.
2-Nitro-oxaphenalenes are synthetic chemicals which were synthesized in the authors' laboratory. They are the most efficient mutagenic compounds on mammalian cells in culture. They are chemically related to the nitro-naphthofuran family by the displacement of the heterocycle on the naphthalene ring. Since nitro-naphthofurans have a strong mutagenic activity in bacterial tests without metabolic activation and are active in-vivo carcinogens, the purpose of this study was to demonstrate the carcinogenic activity of two 2-nitro-oxaphenalenes. The two compounds were injected s.c. into Wistar rats initially 6-weeks-old. They were dissolved in dimethylsulfoxide (DMSO) at a concentration of 1 mg/ml. A s.c. injection of 0.5 ml containing 0.5 mg of carcinogen was given once a week in the neck of each animal tested. Five control animals were not injected and five animals received 0.5-ml injection of DMSO every week to serve as a control. The animals developed tumors only at the site of injection. The tumors were classified as high grade fibrosarcomas. This experiment demonstrates that: (i) 2-nitro-oxaphenalenes are very active in-vivo carcinogens in rats; (ii) there is a good correlation between the high mutagenic activity especially in mammalian tests and the strong carcinogenicity of the compounds; and (iii) the presence of a 6-methoxy group increases by two-fold the carcinogenic potential.
Animals↗
Genotoxic effects of glycidyltrimethylammonium chloride.
Evaluation of the genotoxicity of epoxides is best carried out on a case by case basis. Although glycidyltrimethylammonium chloride (GTAC) is widely used in several industrial applications, its genotoxicity is poorly documented. Therefore, we have evaluated GTAC in a battery of 4 in vitro short-term tests for genotoxicity. We report here that GTAC mediates the induction of base-pair substitutions in S. typhimurium, gene conversion in S. cerevisiae (D7), chromosomal aberrations in CHO cells and viral DNA amplification in Chinese hamster CO6O cells. In view of these results, it is advisable to consider GTAC a potential carcinogen.
Animals↗
Metabolic cooperation in SK-HEP-1 human hepatoma cells following treatment with benzodiazepine tranquilizers.
The ability of three benzodiazepine tranquilizers, diazepam, oxazepam and chlordiazepoxide chlorhydrate to block metabolic cooperation between human hepatoma cells was analysed using autoradiographical monitoring. Independent tests were performed with compound concentrations varying from 1 to 50 micrograms/ml. The results showed that at a toxic dose of 50 micrograms/ml diazepam and oxazepam markedly inhibited metabolic cooperation and that oxazepam remained effective at a lower, non-toxic, dose (10 micrograms/ml). Chlordiazepoxide chlorhydrate was found to be less toxic than diazepam and oxazepam and did not interfere with metabolic cooperation at any concentration up to 50 micrograms/ml.
Anti-Anxiety Agents↗
Induction of microchromosomes by chemical carcinogens correlates with SV40-DNA amplification in SV40-transformed Chinese hamster cells.
Radiations and chemical carcinogens induce the amplification of viral DNA inserts and of their cellular flanking sequences in SV40-transformed Chinese hamster embryo cells. In the cell line CO60, the phenomenon is easily measured by in situ hybridization using SV40-DNA as a probe. We found that the appearance of microchromosomes (MC) in CO60 metaphases correlated well with the induction of SV40-DNA amplification (SDA) mediated in the same cells by chemical carcinogens. SDA and MC formation had the same inducers and were essentially transient phenomena whose occurrence and disappearance were simultaneous. The banding properties of MC and the respective time courses of induction of chromosome aberrations and MC formation/disappearance indicated that MC were not chromosome or chromatid breaks but rather acentric double minute-like chromosomes. Double minute chromosomes (DM) have been shown to contain amplified genes. They specifically occur in established cell lines and in vivo tumor cells. Therefore, the reported correlations between SDA and MC formation and the homologies between MC and DM in CO60 cells further support the existence of specific relationships between mutagenesis, gene amplification, in vivo selective pressures and tumor cytogenetics.
Animals↗
Effects of tumour promoters on metabolic cooperation between human hepatoma cells.
Metabolic cooperation between cells from three human hepatoma cell lines was studied by the clonogenic method and by autoradiography. It was found that human HGPRT+/HGPRT- SK-HEP-1 cells only, showed a metabolic cooperation capacity that was inhibited by tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital, and was not inhibited by the non-promoter 4-O-methyl TPA, provided suitable experimental conditions (short exposure times) were used. This biological system might be the basis of a new in vitro short-term screening test for potential tumour-promoting chemicals.
Carcinogens↗
Carcinogen-mediated induction of SV40 DNA amplification is enhanced by acrylamide in Chinese hamster CO60 cells.
The exposure of SV40-transformed Chinese hamster cells (line CO60) to 50-150 micrograms/ml of monomeric acrylamide for 24 h resulted in a very weak induction of the amplification of the SV40 DNA inserts as measured by in situ hybridization with radioactive SV40 DNA as probe. The weak SV40 DNA amplification observed might result from a weak DNA-damaging activity as biologically illustrated by the capacity of high concentrations of acrylamide to irreversibly inhibit the DNA synthesis rate of CO60 cells. Moreover, acrylamide synergistically enhanced both the cytotoxicity and the induction of SV40 DNA synthesis by the carcinogens ethylmethane sulfonate, benzo[a]pyrene, mitomycin C, 4-nitroquinoline-1-oxide, 8-azaguanine and 5-fluorodeoxyuridine. Although a weak inducer of SV40 DNA amplification by itself, acrylamide potentiated the genotoxicity of a series of chemical carcinogens. This finding should be taken into consideration when assessing the risk of this widely used chemical.
Acrylamide↗
Evidence for unaltered structure and in vivo assembly of microtubules in transformed cells.
By using immunoperoxidase cytochemistry at the light and electron microscopic level, microtubles were visualized in a number of "normal" nontumorigenic and transformed tumorigenic cell lines. A well-defined cytoplasmic microtubule complex exists in both normal and transformed interphase cells. The distribution of this complex closely correlates with the cell shape and the degree of cell spreading. Our data support the idea that these properties determine the pattern of the cytoplasmic microtubule complex, rather than the reverse. Ultrastructural observations of immunoperoxidase-stained tumor cells showed characteristic microtubules in cells in which the microtubules were poorly resolved at the light microscopic level. The results suggest that microtubule assembly and structure are unaltered in transformed cells. However, this conclusion does not exclude the possibility that some of the microtubules' functions might be impaired in a yet-unknown way.
Cell Line↗
Cyclic AMP and cyclic GMP concentrations in serum- and density-restricted fibroblast cultures.
Mouse fibroblasts transformed by simian virus 40 (SV3T3 cells) are characterized by cyclic AMP and cyclic GMP levels, respectively, about half and twice those found in growing untransformed 3T3 cells. Density-dependent inhibition of growth is correlated with reduced cyclic GMP concentrations in 3T3 and four different density-restricted revertant lines derived from SV3T3. The levels of cyclic AMP are not increased at confluence. Upon serum restriction, serum-dependent cell lines show a greater increase in intracellular cAMP than serum-insensitive lines. Cyclic GMP levels are greatly reduced, even in serum-insensitive density revertants, but not in SV3T3. Serum readdition to all serum-dependent lines is followed by a rapid decrease in cyclic AMP and increase in cyclic GMP concentrations. The magnitude of these responses is decreased in SV3T3 and density revertants.
Animals↗
Cyclic nucleotides in transformation.
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Animals↗
Cyclic nucleotides and growth regulation in fibroblasts.
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Animals↗
Rapid detection and identification of pathogenic fungi by polymerase chain reaction amplification of large subunit ribosomal DNA.
We describe a polymerase chain reaction (PCR) based approach to the detection and identification of pathogenic fungi which has potential for the diagnosis of systemic mycoses. Primers to sequences of the large subunit ribosomal DNA genes, which are universally conserved within the fungal kingdom, were capable of amplifying DNA from 43 strains representing 20 species (12 genera) of medically important fungi. Sequence analysis of the products obtained from Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans allowed us to design species-specific primers which only amplified homologous DNA. The use of these two PCRs in tandem allows the detection (universal PCR) and identification (species-specific PCR) of a fungal pathogen within 8 h from simulated clinical specimens.
Base Sequence↗