Priority organic pollutants in the urban water cycle (Toulouse, France).

Application of the European Water Framework Directive requires Member States to have better understanding of the quality of surface waters in order to improve knowledge of priority pollutants. Xenobiotics in urban receiving waters are an emerging concern. This study proposes a screening campaign of nine molecular species of xenobiotics in a separated sewer system. Five sites were investigated over one year in Toulouse (France) using quantitative monitoring. For each sample, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, nonylphenols, diethelhexylphthalate, linear alkylbenzene sulphonates, methyl tert-butylether, total hydrocarbons, estradiol and ethinylestradiol were analysed. Ground, rain and roof collected water concentrations are similar to treated wastewater levels. Run-off water was the most polluted of the five types investigated, discharged into the aquatic environment. The wastewater treatment plant reduced xenobiotic concentrations by 66% before discharge into the environment. Regarding environmental quality standards, observed concentrations in waters were in compliance with standards. The results show that xenobiotic concentrations are variable over time and space in all urban water compartments.

Impact of carwash discharge on stormwater quality (Toulouse, France).

The contribution of discharge from carwashes to pollutant levels in stormwater was evaluated. Five carwashes and two outlets in the city of Toulouse (France) were selected. Water samples were collected from December 2006 to December 2007. Concentrations and loadings of classical water quality parameters (conductivity, pH, turbidity, chemical oxygen demand, nitrogen, phosphorus, ammonium, nitrate, suspended solid and volatile suspended solid) and five groups of organic compounds (polycyclic aromatic hydrocarbons, polychlorinated biphenyls, lauryl alkyl benzene sulphonates, methyl tert-butyl ether and total hydrocarbons) were determined. The results suggest that the wastewater derived from carwashes was negligible compared to the volume and flow rates within the stormwater network. However, high concentrations of polycyclic aromatic hydrocarbons, phosphorus and lauryl alkyl benzene sulphonates in liquid waste from carwashes, and the impact of these pollutants on stormwater quality could not be neglected.

Lactobacillus plantarum 299v surface-bound GAPDH: a new insight into enzyme cell walls location.

The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

MCF-7 cell cycle arrested at G1 through ursolic acid, and increased reduction of tetrazolium salts.

The effect of ursolic acid on the proliferation of MCF-7 human breast tumor cells was studied. During investigations of the anti-proliferative effects of this triterpene, we observed a clear difference between MTT colorimetric assay and direct cell counting, particularly 24 h after drug treatment. The MTT assay showed a stimulation of formazan production in the first 24 h exposure of cells to drug. The maximum stimulation was obtained with 15 and 20 microM of ursolic acid (about 30 - 40% of increase with respect to control); however, the number of cells was not increased as revealed by direct cell counting. Ursolic acid is a potent inhibitor of MCF-7 cell proliferation. This triterpene exhibits both cytostatic and cytotoxic activity. It exerts an early cytostatic effect at G1 followed by cell death. Cell cycle analysis is performed by propidium iodide staining and flow cytometry technique. These results suggest that alterations in cell cycle phase redistribution of MCF-7 human breast cancer, by ursolic acid, may significantly influence MTT reduction to formazan.

Associated effects of divergent selection for residual feed consumption on reproduction, sperm characteristics, and mitochondria of spermatozoa.

Eighteen generations of divergent selection for residual feed intake have been completed in two Rhode Island Red lines of domestic fowl. The high intake R+ line and the low intake R- line cocks used to sire Generation 19 of the selection experiment have been compared for associated responses on fertility, hatching, and sperm quality. Evaluations of sperm samples were based on volume, cell concentration, biochemical parameters (pH, uric acid and protein concentrations), and motility and morphology of spermatozoa. Finally, individual spermatozoa were analyzed by flow-cytometry (FCM) using Rhodamine 123 (Rh123) and nonyl-acrydine-orange (NAO) specific fluorochromes to assess, respectively, overall mitochondrial activity and overall mitochondrial content. Hatchability of incubated eggs was 20 points higher for the R- line, mainly because unfertilized eggs were only 6 vs 30% in the R+ line. Early embryo mortality was also twice as high in the R+ line (21%). The ratio of Rh123 to NAO fluorescence was identical for both lines. This result suggests that there was no difference in the energy producing potential of the individual mitochondria. Therefore, the difference seen for both dyes between the two lines might be attributed to a difference in the quantity of mitochondrial inner membranes present in the cell (with 17% less for the R+ line). In the R+ line, the poor performance at fertilization and during early embryonic development was associated with lower production of motile spermatozoa, possibly in relation to a lower quantity of mitochondria in spermatozoa from R+ cocks. Although the female contribution to the differences between lines was not explored separately, results suggest that selection for residual feed intake may have altered some cellular function related to the production of energy in the R+ line.

The influence of biosolids treatment files on the mobility of metal trace elements.

The production of sludge in France is estimated to be about 900,000 metric tons dry matter per year and 60% of this is recycled onto agricultural land. At present, the long term future of this procedure is open to question and among the different arguments being put forward are the levels of metallic trace elements and the risk of accumulation in soils. This study presents the behaviour of metallic trace elements in sludges from three different treatment procedures: thickened liquid sludges, dewatered sludges and dried sludges. These biosolids are mixed with a clay soil and then placed in a temperature and humidity controlled glasshouse. Several containers are seeded with ryegrass and compared with controls. For the three harvests, covering all the amendments studied (including non-amended soil), the differences are not really representative. Absorption by the ryegrass is low in all cases. For the cadmium, the chromium, the nickel and the lead, the roots are 5 to 10 times more concentrated than the leaves. The majority of these elements stay absorbed in the roots, regardless of the amendment used. The addition of the sludges has considerably reduced the uptake of water in ryegrass throughout its growth cycle. Quite apart from their fertilizing qualities, wastewater treatment plant sludges could offer important implications for irrigation.

Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases.

Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated.

Impact of dry weather discharges on annual pollution from a separate storm sewer in Toulouse, France.

The city of Toulouse with its separate sewer system is ideal for studying stormwater. However, during dry weather, the storm sewer also discharges water into the environment, and it is the impact of these discharges on annual pollution from storm sewer that is the object of this study. Samples have been taken from the outlets of two storm drains located in heavily and moderately urbanized areas. Sampling has been undertaken during wet weather and during dry weather between January 2010 and February 2011. Three dry weather and two wet weather samples have been taken every three months and from each outlet. The overall pollution parameters have been analyzed (chemical oxygen demand, biological oxygen demand, total nitrogen, ammonium, nitrate, total phosphorus, suspended solid matter, volatile suspended matter, pH, conductivity, turbidity). Characterization has been completed by analysis of trace organic compounds: polycyclic aromatic hydrocarbons, total hydrocarbons, methyl tert-butyl ether, diethylhexylphthalate, nonylphenols, hormones (estradiol, ethinylestradiol). For certain parameters, the results obtained did not conform to legislative requirements concerning discharge into the natural environment. Correlations between these parameters have been studied, and identified between several of them using principal component analysis. The most important correlation observed was between conductivity and concentration in total phosphorus for one of the outlet. Results showed that dry weather had an impact on annual pollution load from separate storm sewer and that level of urbanization was also a factor. The effect of season has been studied but no significant impact was found.

Nucleic acid specificity of an acridine derivative permits its use for flow cytometric analysis of the cell cycle.

3-amino-6-methoxy-9-(2-hydroxyethylamine) acridine (AMHA) is an acridine derivative, which is easily excited in near ultraviolet and which emits a bright green fluorescence. The dye was preferentially incorporated into nucleic structures as attested by microscopic and cytometric analyses after RNase and DNase treatments. The affinity for RNA seemed low and similar to that observed for propidium iodide. AMHA was quickly accumulated in fixed cells, and in appropriate concentrations (10-50 microM) was a DNA- and RNA-specific dye. AMHA probably exhibits an adenine-thymine specificity, as suggested by its quenching after bromodeoxyuridine uptake: the fluorescence quenching was similar to that obtained for Hoechst 33258. After cell treatment by RNase and in the presence of MgCl2, AMHA staining allowed flow cytometric analysis of the cell-cycle distribution. The resulting histograms were similar to those obtained with propidium iodide (CV near 3.5%, and similar cell cycle distribution). Thus, AMHA is a suitable fluorescent dye for efficient analysis of the cell cycle by flow cytometry.

3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: a new fluorescent dye to detect Mycoplasma contamination in cell cultures.

A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.

Membrane permeabilization of Listeria monocytogenes and mitochondria by the bacteriocin mesentericin Y105.

Mesentericin Y105, a bacteriocin produced by a Leuconostoc mesenteroides strain, dissipates the plasma membrane potential of Listeria monocytogenes and inhibits the transport of leucine and glutamic acid. It also induces an efflux of preaccumulated amino acids from cells. In addition, the bacteriocin uncouples mitochondria by increasing state 4 respiration and decreasing state 3 respiration. The bacteriocin inhibits ATP synthase and adenine nucleotide translocase of the organelle while the affinity of ADP for its carrier is not modified. The results suggest that mesentericin Y105 acts by inducing, directly or indirectly, pore formation in the energy-transducing membranes, especially those of its natural target.