Comparison of two filtration-elution procedures to improve the standard methods ISO 10705-1 & 2 for bacteriophage detection in groundwater, surface water and finished water samples.

AIM: To select a reliable method for bacteriophage concentration prior detection by culture from surface water, groundwater and drinking water to enhance the sensitivity of the standard methods ISO 10705-1 & 2. METHODS AND RESULTS: Artificially contaminated (groundwater and drinking water) and naturally contaminated (surface water) 1-litre samples were processed for bacteriophages detection. The spiked samples were inoculated with about 150 PFU of F-specific RNA bacteriophages and somatic coliphages using wastewater. Bacteriophage detection in the water samples was achieved using the standard method without and with a concentration step (electropositive Anodisc membrane or a pretreated electronegative Micro Filtration membrane, MF). For artificially contaminated matrices (drinking and ground waters), recovery rates using the concentration step were superior to 70% whilst analyses without concentration step mainly led to false negative results. Besides, the MF membrane presented higher performances compared with the Anodisc membrane. CONCLUSION: The concentration of a large volume of water (up to one litre) on a filter membrane avoids false negative results obtained by direct analysis as it allows detecting low number of bacteriophages in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of concentration step before applying the standard method could be useful to enhance the reliability of bacteriophages monitoring in water samples as bio-indicators to highlight faecal pollution.

Adenovirus, MS2 and PhiX174 interactions with drinking water biofilms developed on PVC, cement and cast iron.

Biofilms colonizing pipe surfaces of drinking water distribution systems could provide habitat and shelter for pathogenic viruses present in the water phase. This study aims (i) to develop a method to detect viral particles present in a drinking water biofilm and (ii) to study viral interactions with drinking water biofilms. A pilot scale system was used to develop drinking water biofilms on 3 materials (7 cm(2) discs): PVC, cast iron and cement. Biofilms were inoculated with viral model including MS2, PhiX174 or adenovirus. Five techniques were tested to recover virus from biofilms. The most efficient uses beef extract and glycine at pH = 9. After sonication and centrifugation, the pH of the supernatant is neutralized prior to viral analysis. The calculated recovery rates varied from 29.3 to 74.6% depending on the virus (MS2 or PhiX174) and the material. Applying this protocol, the interactions of virus models (MS2 and adenovirus) with drinking water biofilms were compared. Our results show that adsorption of viruses to biofilms depends on their isoelectric points, the disc material and the hydrodynamic conditions. Applying hydrodynamic conditions similar to those existing in drinking water networks resulted in a viral adsorption corresponding to less than 1% of the initial viral load.

Laser scanning detection of FISH-labelled Escherichia coli from water samples.

The development of rapid and accurate methods for the detection and quantification of bacteria without cultivation is of increasing importance for water monitoring. The aim of this study was to develop a solid phase cytometry detection method for DVC-FISH labelled Escherichia coli cells. In order to allow Stomatic detection with ChemScan RDI, the fluorescein-tyramide was combined with an oligonucleotide probe directly labelled with horseradish peroxidase to increase the fluorescence intensity. The method developed was tested for the enumeration of pure cultures, for GAC-filtered and drinking water samples. The method, which appeared to be equivalent to the culture method, was less sensitive than the DVC-FISH method followed by microscopic analysis. Research is underway to further optimise the labelling conditions.

[Effect of PML and PML-RAR on the transactivation properties and subcellular localization of steroid hormone receptors]. / Effet de PML et PML-RAR sur les propriétés transactivatrices et la localisation sub-cellulaire des récepteurs stéroïdiens.

PML is a protein involved in the t (15, 17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML since when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. Use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR, which is not localized in nuclear bodies, also enhanced the transactivating activity of PR but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by the retinoic acid.

The Ernst Schering Poster Award. Intracellular traffic of steroid hormone receptors.

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. It is a complex signal. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. The same phenomenon exists for estradiol and glucocorticoid receptors. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement to the cytoplasm is not understood. We have grafted different nuclear localization signals (NLSs) onto beta-galactosidase and have studied the traffic of this protein using heterokaryons and microinjection experiments. We have demonstrated that the same NLSs are involved in both the inward and the outward movement of proteins through the nuclear membrane. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally. Using these techniques, we have shown that the two major antiprogestins, RU486 and ZK98299, act at the same distal level of hormone action.

Effect of PML and PML-RAR on the transactivation properties and subcellular distribution of steroid hormone receptors.

PML (promyelocytic leukemia) is a protein involved in the t (15;17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML, because when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. The use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys-rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR which is not localized in nuclear bodies, also enhanced the transactivating activity of PR, but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by retinoic acid.

Nuclear localization signals also mediate the outward movement of proteins from the nucleus.

Several nuclear proteins, including steroid hormone receptors, have been shown to shuttle continuously between the nucleus and the cytoplasm. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement into the cytoplasm is not understood. We have grafted the nuclear localization signals of the progesterone receptor or the simian virus 40 large tumor antigen onto beta-galactosidase. These additions were shown to impart to the protein the ability to shuttle between the nucleus and the cytoplasm. Microinjected proteins devoid of a nuclear localization signal were unable to exit from the nucleus. The same nuclear localization signals are thus involved in both the inward and the outward movement of proteins through the nuclear membrane. We also show that although the nuclear import requires energy, the nuclear export does not. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally.

Cytoplasmic-nuclear trafficking of progesterone receptor. In vivo study of the mechanism of action of antiprogestins.

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between the nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. Preliminary evidence suggests that the same mechanism exists for estradiol and glucocorticoid receptors. Experiments designed to study the traffic of steroid hormone receptors have been applied to the determination of the molecular mechanism of action of antisteroids. Using these techniques, we have shown that two major antiprogestins, RU486 and ZK98299, act at the same point in the cell as the hormone.

In vivo evidence against the existence of antiprogestins disrupting receptor binding to DNA.

The binding of a steroid hormone to its receptor elicits a sequence of events: activation of the receptor (probably through dissociation from a complex of heat shock proteins), dimerization, binding to hormone responsive elements, and finally modulation of gene transcription. RU 486, the first antiprogestin studied, has been shown to act at the last step of this sequence: provoking an inefficient binding of the receptor to hormone responsive elements. Recently, based on in vitro studies, it has been proposed that ZK 98299 was the prototype of a second class of antiprogestins that were supposed to act through disruption of the binding to DNA. We have devised methods allowing us to study the various steps of agonist or antagonist action in vivo. We show here that RU 486 and ZK 98299 have the same effects on receptor activation, dimerization, and binding to hormone responsive elements; differences in their action are explained by the 10-fold difference in their affinity for the receptor (ZK 98299 having the lower affinity).