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See detailStudies of the Domains II and III of Bacillus subtilis PBP4a in relation with the protein localization
Vanden Broeck, Arnaud ULg; Van Der Heiden, Edwige ULg; Sauvage, Eric ULg et al

Poster (2014, April 23)

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been ... [more ▼]

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been mutated in glutamine residues (Q). Both proteins (WT and Mut4KQ PBP4a) have been produced without signal peptide in E. coli and their sub-cellular localizations determined by measuring the DD-carboxypeptidase activities in the different compartments (cytoplasmic vs membrane attached proteins). In order to detect a possible influence of the PBP4a domain III in the localization of the protein, its encoding sequence has been cloned into pET-28b-BlaP, a vector allowing the production of WT BlaP β-lactamase or BlaP/DIII chimeric protein (with domain III inserted in a permissive loop of BlaP). The nitrocefin hydrolysis activities of BlaP or BlaP/DIII have been measured in the different cellular compartments. [less ▲]

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See detailEffect of land use on pollution status and risk of fish endocrine disruption in small farmland ponds
Mandiki; Gillardin; Martens et al

in Hydrobiologia (2014), 723

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See detailStudy of the Bacillus subtilis ATCC21332 pbpE-racX operon in relation with the formation or disassembly of biofilms
Vanden Broeck, Arnaud ULg; Van Der Heiden, Edwige ULg; Joris, Bernard ULg et al

Poster (2013, December 20)

Bacillus subtilis is a PGPR (Plant Growth Promoting Rhizobacterium) Gram positive bacterium and a model for studying the in vitro formation or disruption of biofilms. At the liquid/air interface of ... [more ▼]

Bacillus subtilis is a PGPR (Plant Growth Promoting Rhizobacterium) Gram positive bacterium and a model for studying the in vitro formation or disruption of biofilms. At the liquid/air interface of standing cultures, B. subtilis forms thick pellicles of limited lifetimes. Some D-amino acids have been reported among the factors playing a role in the disassembly of B. subtilis biofilms and ylmE or racX mutants (in which the racemases YlmE or RacX are absent) show a delay in pellicle disruption [I. Kolodkin et al. Science (2010) 328:627-629]. The racX encoding gene is part of a bicistronic operon in which the first gene (pbpE) codes for a Penicillin-Binding Protein, the PBP4* whose function is not characterized. Our studies aim to delete the complete pbpE-racX operon and compare the phenotypes of mutants and parental strains ATCC21332 or ATCC6051 in standing cultures. The substrate specificity of the purified RacX racemase is currently under investigation as well as the functional characterization of PBP4*, a protein possessing a lipocalin-like domain. [less ▲]

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See detailInsertion of domain III of Bacillus subtilis and Bacillus amyloliquefaciens PBP4a in the Bacillus licheniformis BlaP β-lactamase to study the binding to peptidoglycan and whole bacteria
Van Der Heiden, Edwige ULg; Hoebreck, Charline ULg; Freichels, Régine ULg et al

Poster (2013, October 03)

Domain III of Bacillus subtilis and B. amyloliquefaciens DD-endopeptidase PBP4a was introduced in the BlaP beta-lactamse of Bacillus licheniformis. Domain III of Bacillus licheniformis binds to ... [more ▼]

Domain III of Bacillus subtilis and B. amyloliquefaciens DD-endopeptidase PBP4a was introduced in the BlaP beta-lactamse of Bacillus licheniformis. Domain III of Bacillus licheniformis binds to peptidoglycan of Bacillus subtilis 168 and to itself whole cells. [less ▲]

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See detailA Pathway closely related to the D-tagatose pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis
Van Der Heiden, Edwige ULg; Delmarcelle, Michaël ULg; Lebrun, Sarah ULg et al

Poster (2013, June)

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium ... [more ▼]

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. [less ▲]

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See detailA Pathway closely related to the D-tagatose pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis
Van Der Heiden, Edwige ULg; Delmarcelle, Michaël ULg; Lebrun, Sarah ULg et al

in Applied and Environmental microbiology (2013), 79(11), 3511-3515

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium ... [more ▼]

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. [less ▲]

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See detailFood interactions : effects on health, consumer perception and impact on agro-food industries “FOODINTER”.
Scippo, Marie-Louise ULg; Blust, Ronny; Boniver, Delphine et al

Report (2011)

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See detailPotential of an in vitro toolbox combined with exposure data as a first step for the risk assessment of dietary chemical contaminants
Ribonnet, Laurence; Van Der Heiden, Edwige ULg; Nobels, Ingrid et al

in Food Additives & Contaminants (2011), 28(9), 1136-1158

In vitro risk assessment of dietary contaminants has become a priority in human food safety. This paper proposes an in vitro approach associating different complementary tools in an original toolbox and ... [more ▼]

In vitro risk assessment of dietary contaminants has become a priority in human food safety. This paper proposes an in vitro approach associating different complementary tools in an original toolbox and aims to improve the assessment of the toxicological impact of dietary contaminants at realistic human exposure levels, with a special focus on the intestinal compartment. The system is based on the use of four complementary cellular tools, namely stress gene induction in transgenic strains of Escherichia coli, modulation of the activity of key biotransformation enzymes (cytochrome P-450 (CYP) 1A1 and 3A4) in a human intestinal cell line, and activation of aryl hydrocarbon receptor (AhR) and oestrogenic receptor (ER)-dependent genes in agonistic and antagonistic assays with luciferase reporter cells. It was applied to four chosen model molecules: ochratoxin A (OTA) and deoxynivalenol (DON), two common food-borne mycotoxins, and imazalil (IMA) and benomyl (BEN), two fungicides widely occurring in foodstuffs. All these assays were performed at or around a realistic intestinal concentration, determined through a deterministic approach based on the calculation of a theoretical maximum daily intake (TMDI). Using the four model molecules, it is clearly highlighted that induction of CYP1A1 activity and inhibition of CYP3A4 activity occurred in Caco-2 cells at a realistic intestinal concentration of IMA. Furthermore, some bacterial stress genes were induced in a range of realistic concentrations, following exposure to DON and IMA. In addition, BEN clearly provoked an ER agonistic activity in a human oestrogen sensitive reporter cell line. All these results are in accordance with the literature, suggesting that the in vitro toolbox constitutes an interesting approach in order to obtain a first 'fingerprint' of dietary contaminants at realistic human exposure for further risk assessment. [less ▲]

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See detailRemoval of Androgens and Estrogens from Water by Reactive Materials
Kai, Cai; Phillips, D. H.; Elliott, Christian et al

in Journal of Water Resource and Protection (2010), 2(11), 990-993

Nowadays, endocrine disruptor compounds in the water system have become a concern due to the risk of contamination to wild life and humans even at the nanogram level. Excess estrogens and androgens are a ... [more ▼]

Nowadays, endocrine disruptor compounds in the water system have become a concern due to the risk of contamination to wild life and humans even at the nanogram level. Excess estrogens and androgens are a major contributor group of endocrine compounds. Statistical surveys have shown that dairy farms contribute to over 90% of the total estrogens in the UK and US. An analytical system is being developed to assess the efficiency of reactive materials to remove target hormonal contaminants from dairy farm effluent. This can be achieved using reporter gene assays (RGAs) to detect low level steroid hormones. A preliminary study comparing the efficiency of granular activated carbon, zero-valent iron, and organoclay was carried out using bench-scale evaluations in negative control HPLC water. Their potential ability to remove testosterone and 17-β- estradiol spiked at reported environmental levels was measured by androgenic and estrogenic luminescent reporter assay respectively. [less ▲]

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See detailUnderstanding the gap between the estrogenicity of an effluent and its real impact into the wild
Nadzialek, Stéphanie; Vanparys; Van Der Heiden, Edwige ULg et al

in Science of the Total Environment (2010), 408(4), 812-821

To study the reliability between in vitro and in vivo data collected downstream 2 sewage treatment plants (STP) as well as from bleached kraft mill industry (BKME), 5 rivers (3 impacted and 2 references ... [more ▼]

To study the reliability between in vitro and in vivo data collected downstream 2 sewage treatment plants (STP) as well as from bleached kraft mill industry (BKME), 5 rivers (3 impacted and 2 references) were investigated in the Walloon region (southern of Belgium). For the in vitro part of the work, water samples were collected to measure the estrogenicity of the ‘out’ effluent compared to reference sample point by MCF-7 assay. Results indicated significant estrogenicity of effluents from STP and BKME and a weak estrogenicity in reference sites. However, estradiol equivalents (EEQ) estimated into rivers were probably too low to impact wild population. Chemical analysis of 13 compounds of interest indicated that extraction procedure used in this study gave low recoveries of estrogen-like xenobiotics, leading to probably under-estimated MCF-7 responses. Surprisingly, a full scan mode has revealed an unexpected compound in the sample of BKME which was: 7-isopropyl-1,1,4a-trimethyl-1,2,3,4a,9,10,10a-octahydrophenanthrene, a product of pulp mill manufacture. In parallel to in vitro, in vivo assessment of estrogenic impact of effluent was followed on the gudgeon (Gobio gobio). Samples were achieved during 2 different periods of the reproductive cycle, resting period (RP) and pre-spawning period (pSP). Unspecific physiological parameters to estrogenic exposure (gonadosomatic index and systematic testis cell counting) displayed no significant differences related to endocrine disruption of the reproductive tract, only differences were correlated with the reproductive state of fish (RP versus pSP). Concerning the potent biomarker of estrogen exposure, vitellogenin (vtg), only basal induction was revealed but not related to estrogenic exposure. Nevertheless, vtg over-expression was found for male fish presenting a feminization of the reproductive tract captured downstream the STP station of Wégnez in the Vesdre River. Intersexuality, another indicator of the estrogenicity impact in fish, was observed in every site. Actually, ovotestis was systematically formed by protoplasmic oocyte observed in low percentage in every group analysed (impacted and references). Moreover, in fish captured in Wégnez, oocyte diameter was significantly higher compared to the other groups. In this study, only moderate to none impact in population of gudgeon was noticed. Moreover, in this case no discrepancy between in vitro and vivo was viewed although both approaches revealed gaps in monitoring effluent incidence into the environment. We should remain careful in the interpretation when only partial approaches are used in order to characterize impact in the aquatic milieu. [less ▲]

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See detailRemoval of Androgens and Estrogens from Water by Reactive Materials
Kai, Cai; Phillips, D. H.; Elliott, Chris et al

in 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE 2010) (2010)

Nowadays, endocrine disruptor compounds in the water system have become a concern due to the risk of contamination to wild life and humans even at the nanogram level. Excess estrogens and androgens are a ... [more ▼]

Nowadays, endocrine disruptor compounds in the water system have become a concern due to the risk of contamination to wild life and humans even at the nanogram level. Excess estrogens and androgens are a major contributor group of endocrine compounds. Statistical surveys have shown that dairy farms contribute to over 90% of the total estrogens in the UK and US. An analytical system is being developed to assess the efficiency of reactive materials to remove target hormonal contaminants from dairy farm effluent. This can be achieved using reporter gene assays (RGAs) to detect low level steroid hormones. A preliminary study comparing the efficiency of granular activated carbon, zero-valent iron, and organoclay was carried out using bench-scale evaluations in negative control HPLC water. Their potential ability to remove testosterone and 17- beta -estradiol spiked at reported environmental levels was measured by androgenic and estrogenic luminescent reporter assay respectively. [less ▲]

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See detailEvaluation of the endorcine disrupting chemicals contamination in the North Sea porpoise population using in vitro assays
Didimo Imazaki, Pedro Henrique ULg; Brose, François ULg; Van Der Heiden, Edwige ULg et al

Poster (2009)

During the last decades, the production endocrine disrupting chemicals reached such levels that they are now spread all over nature. They are known to be very slowly degraded, decreasing the environmental ... [more ▼]

During the last decades, the production endocrine disrupting chemicals reached such levels that they are now spread all over nature. They are known to be very slowly degraded, decreasing the environmental quality and posing ecological risks. Marine mammals inhabiting polluted environments accumulate high levels of these chemicals, so they can be considered good indicators of marine pollution. Thirteen major organochloride pollutants were chosen to make subject of this study: o,p’-DDD; p,p’-DDD; p,p’-DDE; o,p’-DDT; p,p’-DDT; HCB; α-HCH; β-HCH; γ-HCH; δ-HCH; PCB 138; PCB 153 and PCB 180. All these chemicals will have their endocrine disrupting effects characterized individually and collectively by report gene expression assays. The MCF7-ERE cells used in these assays were produced in the Molecular Biology and Genetic Engineering Laboratory of the University of Liège and are originated from a human mammary carcinoma. They carry a gene expressing the synthesis of luciferase and controlled by oestrogen receptors. Then, the thirteen chemicals will be searched in the blubber tissues of porpoises by the use of gas chromatography-mass spectrometry. The blubber samples will also have their endocrine disrupting effect characterized. For the moment, the estrogenic effects of the HCH isomers were put in evidence. The next steps of this work must be soon accomplished. It is expected a great level of these compounds to be found in the samples. On the other hand, their endocrine disrupting effects and how they act in agonist, antagonist and synergist ways are subject that still needs to be cleared. [less ▲]

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See detailEvaluation of the contamination of North Sea Porpoises (Phocoena phocoena) by endocrine disrupting chemicals using CG-MS and in vitro assays
Didimo Imazaki, Pedro Henrique ULg; Brose, François ULg; Remacle, Anne-Sophie ULg et al

Poster (2009)

During the last decades, the production of endocrine disrupting chemicals reached such elevated levels that they are now spread all over the environment. Endocrine disrupting chemicals are known to be ... [more ▼]

During the last decades, the production of endocrine disrupting chemicals reached such elevated levels that they are now spread all over the environment. Endocrine disrupting chemicals are known to be very slowly degraded, decreasing the environmental quality and causing ecological risks. Marine mammals inhabiting polluted environments accumulate high quantities of these chemicals, and are good indicators of marine pollution Thirteen major organochloride pollutants (known from literature to contaminate north sea porpoise) were chosen in this study – o,p’-DDD; p,p’-DDD; p,p’-DDE; o,p’-DDT; p,p’-DDT; HCB; α-HCH; β-HCH; γ-HCH; δ-HCH; PCB 138; PCB 153 and PCB 180. The thirteen chemicals were tested individually and in mixtures by the use of report gene expression assays. The MCF7-ERE cells used in the assays were originated from MCF7 human mammary tumor cells transfected with the ERE-luciferase reporter system. Preliminary results suggest that o,p’-DDD; p,p’-DDD; o,p’-DDT; HCB; β-HCH; δ-HCH; PCB 138 and PCB 180 are able to stimulate the luciferase expression of MCF7-ERE cells when they are present individually or in mixtures. No synergism was observed within mixtures. When MCF7-ERE cells were exposed to the chemicals and 17β-oestradiol simultaneously, p,p’-DDE; α-HCH; PCB 138 and PCB 180 could raise the luciferase expression in comparison to the exposition of 17β-oestradiol alone; on the other hand, o,p’-DDD; p,p’-DDT; HCB; β-HCH; γ-HCH and δ-HCH seem to inhibit the luciferase expression at low doses. In the next step, the pollutants will be searched and quantified in the blubber tissue of porpoises by use of gas chromatography-mass spectrometry. The blubber tissue samples will also have their endocrine disrupting effects characterized. This work is ongoing and It is expected a relevant level of these compounds to be found in the samples. Conversely, their endocrine disrupting effects and how they act in agonist, antagonist ways are subject that still needs to be studied. [less ▲]

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See detailCYP1A1 and CYP3A4 modulation by dietary flavonoids in human intestinal Caco-2 cells.
Sergent, Thérèse; Dupont, Isabelle; Van Der Heiden, Edwige ULg et al

in Toxicology Letters (2009), 191

Flavonoids have been proposed to exert beneficial effects in a multitude of disease states. However, evidence of potential toxic actions has also emerged. Since large doses of flavonoids can be ... [more ▼]

Flavonoids have been proposed to exert beneficial effects in a multitude of disease states. However, evidence of potential toxic actions has also emerged. Since large doses of flavonoids can be encountered in the intestine simultaneously with ingested drugs and pollutants, this study aimed at investigating nine individual flavonoid compounds and their interactions with the major intestinal isoforms of cytochrome P450, i.e. CYPs 1A1 and 3A4, using human intestinal Caco-2 cells cultivated in a serum-free medium. Genistein, quercetin and chrysin provoked a dose-dependent inducing effect on the CYP1A1 activity, measured with the EROD assay. However, they did not affect the CYP1A1 mRNA expression, suggesting they are not aryl hydrocarbon receptor-ligands in intestinal cells and act at a post-transcriptional level. Chrysin, at 50muM, was detected as a potent inhibitor of the TCDD-induced CYP1A1 activity, leading the activity to ca. 10% of the TCDD-control value (n=3), this effect involving, at least partly, direct interactions at the enzyme level. Quercetin was also shown to significantly inhibit the constitutive CYP3A4 activity, measured by the 6beta-(OH)-testosterone assay, and to impair its induction by 1,25-vitamin D(3). Chrysin, quercetin and genistein, were detected as significant inhibitors of the 1,25-vitamin D(3)-induced CYP3A4 activity. In vivo, these effects could result in reduced activation of procarcinogens and/or in drug bioavailability limitation. They underline the importance of intestinal studies to assess food safety and health risks linked to the ingestion of flavonoid-enriched supplements or functional foods. [less ▲]

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See detailCYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells-Comparison with other conazole pesticides
Sergent, Thérèse; Dupont, Isabelle; Jassogne, Coralie et al

in Toxicology Letters (2009), 184(3), 159-168

Imazalil (IMA) is a widely used imidazole-antifungal pesticide and. therefore. a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ... [more ▼]

Imazalil (IMA) is a widely used imidazole-antifungal pesticide and. therefore. a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ingested drugs and pollutants, we have investigated the effects of IMA, at realistic intestinal concentrations, on xenobiotic-metabolizing enzymes and efflux pumps by using Caco-2 cells, as a validated in vitro model of the human intestinal absorptive epithelium. For comparison, other conazole fungicides, i.e. ketoconazole, propiconazole and tebuconazole. were also studied. IMA induced cytochrome P450 (CYP) 1A1 activity to the same extent as benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in a dose-and time-dependent manner. Cell-free aryl hydrocarbon receptor (AhR) binding assay and reporter gene assay suggested that IMA is not an AhR-ligand, implying that IMA-mediated induction should involve an AhR-independent pathway. Moreover, IMA strongly inhibited the CYP3A4 activity in 1,25-vitamin D-3-induced Caco-2 cells. The other fungicides had weak or nil effects on CYP activities. Study of the apical efflux pump activities revealed that ketoconazole inhibited both P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP-2) or breast cancer resistance protein (BCRP), whereas IMA and other fungicides did not. Our results imply that coingestion of IMA-contaminated food and CYP3A4- or CYP1A1-metabolizable drugs or chemicals could lead to drug bioavailability modulation or toxicological interactions, with possible adverse effects for human health. (C) 2008 Elsevier Ireland Ltd. All rights reserved. [less ▲]

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