Performance of colorimetric methods for the analysis of low levels of manganese in water.

The present paper describes a laboratory investigation conducted with the main aim to compare two colorimetric methods (1-(2-pyridylazo)-2-naphthol (PAN) from HACH® and porphyrin ligand (T-(4CP)P)) with ICP analyses for the evaluation of low concentrations of manganese in water. The colorimetric porphyrin method was found to provide reliable results compared to those obtained by inductively coupled plasma - optical emission spectroscopy (ICP-OES) even at low Mn2+ concentrations (<10 µg L-1). The presence of MnO2 particles results in an overestimation of the dissolved Mn2+ if unfiltered samples are analyzed by ICP and PAN methods. The presence of particles does not significantly impact sample analysis by porphyrin (T-(4CP)P) colorimetry. Although ICP-OES is still the analytical approach of reference, the porphyrin colorimetric method displays a high detection range and precision and could be a suitable on-site alternative to the current widespread use of the PAN method. In all cases, for both colorimetric methods, the use of Rochelle salt is recommended to alleviate calcium cation interference.

HBZ-mediated shift of JunD from growth suppressor to tumor promoter in leukemic cells by inhibition of ribosomal protein S25 expression.

Human T-cell leukemia virus type 1 (HTLV-1) basic-leucine zipper (bZIP) factor (HBZ) is a key player in proliferation and transformation of HTLV-1-infected cells, thus contributing to adult T-cell leukemia (ATL) development. HBZ deregulates gene expression within the host cell by interacting with several cellular partners. Through its C-terminal ZIP domain, HBZ is able to contact and activate JunD, a transcription factor of the AP-1 family. JunD mRNA is intronless but can generate two protein isoforms by alternative translation initiation: JunD full-length and Δ JunD, an N-terminal truncated form unresponsive to the tumor suppressor menin. Using various cell lines and primary T-lymphocytes, we show that after serum deprivation HBZ induces the expression of Δ JunD isoform. We demonstrate that, unlike JunD, Δ JunD induces proliferation and transformation of cells. To decipher the mechanisms for Δ JunD production, we looked into the translational machinery and observed that HBZ induces nuclear retention of RPS25 mRNA and loss of RPS25 protein expression, a component of the small ribosomal subunit. Therefore, HBZ bypasses translational control of JunD uORF and favors the expression of Δ JunD. In conclusion, we provide strong evidences that HBZ induces Δ JunD expression through alteration of the cellular translational machinery and that the truncated isoform Δ JunD has a central role in the oncogenic process leading to ATL.

MFSD2a, the Syncytin-2 receptor, is important for trophoblast fusion.

The syncytiotrophoblast is formed at the placental periphery through cytotrophoblast fusion, which depends on Human Endogenous Retrovirus-encoded Envelope proteins Syncytin-1 and Syncytin-2. In the current study, the role of Major Facilitator Superfamily Domain Containing 2A (MFSD2a), the Syncytin-2 receptor, in trophoblast fusion and its expression in normal vs. pre-eclampsia placentas were studied. Forskolin-induced fusion of BeWo cells first parallelled an increase in MFSD2a expression. The MFSD2a signal localized in the cytoplasm and at the plasma membrane. Knockdown of MFSD2a expression confirmed its importance in BeWo fusion. Furthermore, reduced MFSD2a expression was noted in severe pre-eclamptic placentas. These data thus support the importance of MFSD2a in trophoblast fusion and placenta development.

Gamma irradiation: a method to produce an abiotic control for biological activated carbon.

The aim of this paper was to investigate the feasibility of using gamma irradiation to inhibit the microbial activity of biological powder activated carbon (PAC) without impacting its adsorptive properties. First of all, the range of dose of gamma rays required to produce abiotic PAC was selected on the basis of heterotrophic plate counts (HPC) inactivation and methylene blue (MB) adsorption kinetics. Doses inferior to 10 kGy were not sufficient to inhibit the culture of heterotrophic bacteria. On the other hand, doses superior to 15 kGy were demonstrated to affect the adsorption rate of MB. Consequently, a dose comprised between 10 and 15 kGy was selected for further investigation. In order to validate the adequacy of the range of dose (i.e. 10-15 kGy), adsorption characteristics were tested by monitoring the removal kinetics of refractory dissolved organic carbon (RDOC). No significant differences were observed between irradiated and non-irradiated biological PAC for the adsorption of RDOC. Irradiated, non-irradiated and virgin PAC were also evaluated in terms of abundance of viable (using the LIVE/DEAD BacLight method) bacteria and in terms of heterotrophic biomass activity. The results of the BacLight method demonstrated that attachment of the biofilm on the PAC was not impacted by the irradiation and heterotrophic activity measurements demonstrated that the latter could be radically reduced in the range of dose selected. In conclusion, when using a proper dose, the gamma irradiation of colonized activated carbon drastically reduced the heterotrophic activity on activated carbon without significantly impacting its adsorptive behaviour.

Using a novel sol-gel stir bar sorptive extraction method for the analysis of steroid hormones in water by laser diode thermal desorption/atmospheric chemical ionization tandem mass spectrometry.

A new coating material was used for a stir bar sorptive extraction (SBSE) method coupled to a high throughput sample analysis technique. This allowed for a simple procedure for fast determinations of eight steroid hormones (estriol, estradiol, ethynylestradiol, estrone, progesterone, medroxyprogesterone, levonorgestrel, northindrone) in water. Sample pre-treatment was performed using an in-house SBSE method based on a polydimethylsiloxane/phenyltrimethylsiloxane/ß-cyclodextrin sol-gel material. The analytes were desorbed by liquid extraction prior to their analysis by laser diode thermal desorption/atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS). Several parameters, including ionic strength, volume and time of extraction as well as volume and time of desorption, were investigated to maximize extraction efficiency by SBSE in aqueous solutions. The in-house stir bar showed good reproducibility and could be used for at least 50 extractions without affecting analytical performance. The recoveries of the spiked steroid hormones ranged from 55% to 96% in all water matrices studied (HPLC grade water, tap water and raw wastewater). Only one compound showed poor recovery values (<2% for estriol) in all matrices. The method detection limits (MDLs) in real matrices were within the range of 0.1-0.3 µg L(-1) except for estriol at 48 µg L(-1). The extraction performance of the in-house SBSE for the eight selected hormones was also compared with that of a commercially-available stir bar coated with polydimethylsiloxane (PDMS). This novel stir bar coating could prove to be useful method for the detection and quantification of trace levels of steroid hormones.

The endogenous retroviral envelope protein syncytin-1 inhibits LPS/PHA-stimulated cytokine responses in human blood and is sorted into placental exosomes.

OBJECTIVES: To examine whether syncytin-1 has immune regulatory functions and is carried by human placental exosomes. Further, to examine whether corticotropin-releasing hormone (CRH) can induce the production of syncytin-1. STUDY DESIGN: Human placental exosomes were isolated from placental explant, primary trophoblast and BeWo cell cultures. The presence of exosomes was confirmed by transmission electron microscopy and western blotting. Exosomal protein was probed with 3 separate antibodies targeting syncytin-1. Syncytin-1 immunosuppression was tested, using either a syncytin-1 recombinant ectodomain protein or a synthetic peptide with the human syncytin-1 immunosuppressive domain sequence, in an in vitro human blood culture system immune challenged with LPS or PHA. The inhibition of cytokine production by syncytin-1 was determined by ELISA of TNF-α, IFN-γ and CXCL10. BeWo cells were stimulated with CRH or vehicle for 24 h. mRNA and Protein was extracted from the cells for real-time PCR and western blotting analysis while exosomes were extracted from conditioned media for analysis by western blotting. RESULTS: Protein expression of syncytin-1 was detected in exosomes isolated from placental explants, primary trophoblast and BeWo cell cultures. Syncytin-1 recombinant ectodomain was also shown to inhibit the production of the Th1 cytokines TNF-α and IFN-γ as well as the chemokine, CXCL10 in human blood cells. Finally, this study showed that syncytin-1 can be stimulated by CRH. CONCLUSIONS: The presence of syncytin-1 in placental exosomes provides a mechanism for syncytin-1 to reach and interact with target cells of the maternal immune system and represents a novel mechanism of endogenous retroviral mediated immunosuppression that may be relevant for maternal immune tolerance.

Comparison of two online flocculation monitoring techniques for predicting turbidity removal by granular media filtration.

Particulate matter removal in drinking water treatment via direct granular filtration requires specific flocculation conditions (a process typically termed 'high energy flocculation'). Predicting filtered water turbidity based on flocculated water characteristics remains difficult. This study has sought to establish a relationship between filtered water turbidity and the flocculated water characteristics. Flocculation oflow-turbidity raw water was evaluated online using a Photometric Dispersion Analyser (PDA) and a Dynamic Particle Analyser in a modified jar test followed by a bench-scale anthracite filter. Coagulants used were alum, PASS100 and ferric sulphate, in addition to a polydiallyldimethylammonium chloride (polyDADMAC) cationic polymer. They were dosed in warm and cold waters, and flocculated with intensities (G) from 0 to 100 s(-1). Of the two instruments selected to analyse flocculation performance, the Dynamic Particle Analyser was shown to be the most sensitive, detecting small changes in floc growth kinetics and even floc growth under low flocculation conditions which remained undetected by the PDA. Floc size was shown to be insufficient in predicting particulate matter removal by direct granular filtration as measured by turbidity, although a threshold d(v) value (50 microm) could be identified for the test conditions evaluated in this project, above which turbidity was systematically lower than 0.2 NTU.

Preliminary study on the occurrence and risk arising from bacteria internalized in zooplankton in drinking water.

In this study, an environmental sampling campaign was conducted to detect internalized E. coli and C. jejuni bacteria in zooplankton and amoebae samples collected at various stages of three water treatment plants in Amsterdam, the Netherlands. Eight sampling locations were selected and sampling was performed twice, at a two-week interval, at each location. Chlorination was used to inactivate free (external) bacteria in the concentrated zooplankton samples and sonication was used to disrupt zooplankton organisms in order to release and recover internalized bacteria. Zooplankton enumeration was performed by microscopy. No internalized E. coli or C. jejuni bacteria were recovered from all of the samples analyzed. The occurrence of internalized E. coli or C. jejuni bacteria in drinking water was estimated to be lower than one internalized bacteria in 105 zooplankton organisms, as derived from the detection limit of the sampling campaign. By using the QMRA approach and the Beta-Poisson model, a risk of infection of less than 9.2E-6 and 5.9E-5 was estimated for internalized E. coli and C. jejuni in drinking water, respectively. This study remains preliminary due to the limited number of samples taken at each location.

Alteration of calcium homeostasis in primary preeclamptic syncytiotrophoblasts: effect on calcium exchange in placenta.

Preeclampsia (PE) is characterized by maternal hypertension, proteinuria, oedema and, in 30% of cases, by intrauterine growth retardation. Causes are still unknown; however, epidemiological and clinical studies have suggested alterations in maternal calcium metabolism. We suggested that in PE, calcium transport by the syncytiotrophoblast (ST) is disturbed. From total placental tissues, we studied the expression of: calcium channels (TRPV5, TRPV6 [transient receptor potential vanilloid]), calcium binding proteins (CaBP-9K, CaBP-28K), plasma membrane calcium ATPase (PMCA)1,2,3,4 pumps, ATP synthase, genes implicated in Ca(2+) release [inositol-1,4,5-triphosphate receptor (IP3R)1,2,3; Ryanodine receptor (RyR)1,2,3] and replenishment (SERCA1,2,3 [sarcoendoplasmic reticulum Ca(2+) ATPases]) from endoplasmic reticulum, channels implicated in mitochondrial Ca(2+) accumulation (VDAC1,2,3 [voltage-dependent anion channels]) and a marker of oxidative stress (hOGG1 [Human 8-oxoguanine-DNA glycosylase 1]), as well as the influence of these variations on calcium transport in primary ST cultures. The mRNA and protein levels were thereby examined by real-time PCR and Western blot analysis, respectively, in two different groups of pregnant women with similar gestational age: a normal group (n= 16) and a PE group (n= 8), diagnosed by a clinician. Our study showed a significant decrease in calcium transport by the ST cultured from preeclamptic placentas. We found a significant (P < 0.05) decrease in mRNA levels of TRPV5, TRPV6, CaBP-9K, CaBP-28K, PMCA1, PMCA4, ATP synthase, IP3R1, IP3R2, RyR1, RyR2 and RyR3 in PE group compared to normal one. We also noted a significant decrease in protein levels of TRPV5, TRPV6, CaBP-9K, CaBP-28K and PMCA1/4 in PE group. In contrast, SERCA1, SERCA2, SERCA3, VDAC3 and hOGG1 mRNA expressions were significantly increased in PE placentas. Calcium homeostasis and transport through placenta is compromised in preeclamptic pregnancies and it appears to be affected by a lack of ATP and an excess of oxidative stress.

Ozone oxidation of pharmaceuticals, endocrine disruptors and pesticides during drinking water treatment.

This study investigates the oxidation of pharmaceuticals, endocrine disrupting compounds and pesticides during ozonation applied in drinking water treatment. In the first step, second-order rate constants for the reactions of selected compounds with molecular ozone (k(O3)) were determined in bench-scale experiments at pH 8.10: caffeine (650+/-22M(-1)s(-1)), progesterone (601+/-9M(-1)s(-1)), medroxyprogesterone (558+/-9M(-1)s(-1)), norethindrone (2215+/-76M(-1)s(-1)) and levonorgestrel (1427+/-62M(-1)s(-1)). Compared to phenolic estrogens (estrone, 17beta-estradiol, estriol and 17alpha-ethinylestradiol), the selected progestogen endocrine disruptors reacted far slower with ozone. In the second part of the study, bench-scale experiments were conducted with surface waters spiked with 16 target compounds to assess their oxidative removal using ozone and determine if bench-scale results would accurately predict full-scale removal data. Overall, the data provided evidence that ozone is effective for removing trace organic contaminants from water with ozone doses typically applied in drinking water treatment. Ozonation removed over 80% of caffeine, pharmaceuticals and endocrine disruptors within the CT value of about 2 mg min L(-1). As expected, pesticides were found to be the most recalcitrant compounds to oxidize. Caffeine can be used as an indicator compound to gauge the efficacy of ozone treatment.

Induction of trophoblast cell fusion by a protein tyrosine phosphatase inhibitor.

In the placenta, trophoblast cell fusion leads to the formation of the syncytiotrophoblast, which plays an essential role in the diffusion of nutrients and hormones from the mother to the fetus. Different protein tyrosine kinases are involved in the modulation of this biological process. Herein, we investigated the impact of a protein tyrosine phosphatase (PTP) inhibitor (bpV[pic]) on trophoblast fusion. Upon bpV[pic] or forskolin treatment, primary and BeWo trophoblastic cells demonstrated higher cAMP levels and a more potent induction of cell fusion, while non-fusogenic JEG-3 cells were non-responsive to these treatments. RT-PCR analyses on stimulated BeWo cells or primary cytotrophoblast cells demonstrated an increase in syncytin-1 mRNA levels, which was more pronounced upon forskolin/bpV[pic] treatment. Using the luciferase gene upstream of the syncytin-1 promoter, similar results were obtained in BeWo cells after stimulation. These results demonstrate that PTPs act negatively on cell fusion in human trophoblasts and could control the timing of syncytiotrophoblast formation during placenta development.

The impact of selected water quality parameters on the inactivation of Bacillus subtilis spores by monochloramine and ozone.

Selected water quality parameters-pH, dissolved organic carbon, turbidity (NTU), and temperature-were tested for their potential effects on ozone and monochloramine inactivation of Bacillus subtilis spores. In oxidant demand-free phosphate-buffer, temperature had the strongest influence on inactivation kinetics when using ozone, pH had a smaller but significant impact on B. subtilis spore inactivation with both monochloramine and ozone. Where monochloramine was applied, modeling and experimental measurements confirmed that dichloramine levels were too low to produce significant inactivation effects under these experimental conditions. It was demonstrated that oxidant demand-free phosphate buffer may not be an adequate environmental analogue for inactivation responses in natural waters.

Regulation of procollagen I (alpha1) by interleukin-4 in human bronchial fibroblasts: a possible role in airway remodelling in asthma.

BACKGROUND: In bronchial mucosa, T cells are in close association with fibroblasts. This cell contact raises the possibility of cross-talk between the two cell types through cytokines, such as interleukin-4 (IL-4). OBJECTIVE: We postulated that IL-4 may modulate collagen synthesis and degradation in the fibroblasts of asthmatics. METHODS: Bronchial fibroblasts from asthmatics (BAF) and normal controls (BNF) were stimulated with IL-4. Procollagen I gene expression and protein production were measured by real-time PCR, RT-PCR, and radioimmunoassay. The effect of IL-4 on the regulation of procollagen I (alpha1) promoter was studied through transient cell transfections. The implication of Sp1 and AP-1 in regulating IL-4-induced procollagen I (alpha1) production was determined. The effect of IL-4 on metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) production and gene expression was evaluated. RESULTS: Following IL-4 stimulation, there was a significant increase in the expression of mRNA of procollagen I (alpha1) by human bronchial fibroblasts of asthmatics and controls. IL-4 has a dose-response effect on mRNA, with a maximal effect at 5 ng/mL, as determined by real-time PCR. The maximal increase in procollagen I (alpha1) was observed at 6 h after IL-4 stimulation in both BNF and BAF. BAFs have a greater increase in the procollagen I (alpha1)/beta2 microglobulin ratio after 6 h of IL-4 stimulation (4.1 x 10-2+/-0.03 to 20.8 x 10-2+/-0.1) compared with BNF (2.9 x 10-2+/-0.006 to 9.2 x 10-2+/-0.08) (P=0.001). In transient transfection experiments, IL-4 increased promoter activity by threefold in BAF and BNF. Sp1 was up-regulated after IL-4 stimulation and AP-1 was down-regulated as shown by electrophoretic mobility shift assay. IL-4 decreased MMP-2 protein and mRNA levels, and did not alter TIMP-2 production. CONCLUSIONS: IL-4 positively regulates procollagen I (alpha1) transcription by direct promoter activation and increases the TIMP-2/MMP-2 ratio, thereby supporting the profibrotic effect of this cytokine. Thus, this study emphasizes that IL-4 may be considered as a link between inflammation and collagen deposition observed in asthmatic airways.

Evaluation of Bacillus subtilis and coliphage MS2 as indicators of advanced water treatment efficiency.

The assessment of water treatment facilities for their efficiency using alternate indicators is of paramount importance. Current methods for assessing efficiency are limited by the specific characteristics of the microorganisms, such as their different sensitivities to disinfectants. A pilot study was carried out to compare different treatment scenarios for the future upgrade of the Sergio Cuevas Water Treatment plant (the largest in the Caribbean) in San Juan, Puerto Rico. The treatment units under investigation included a coagulation-flocculation-sedimentation unit, dual-media filters, micro-filtration units, intermediate ozone injection and contact columns as well as a biological filtration unit. The plant was challenged at different stages of treatment with Bacillus subtilis spores and MS2 coliphages in an attempt to test them as possible alternate indicators of treatment plant performance. These organisms were chosen because of their resistance to disinfection and desiccation, their low analysis costs and ease of detection. The removal of spores and coliphages by each treatment unit tested was calculated by seeding a known concentration (5-7 log10) of spores and coliphages and following the removal or disinfection rates. The seeded indicators were detected using traditional culture techniques. Ballasted clarification was shown to be highly efficient at removing 99.1% (approximately 3 log10) of the spores and 85.1% (approximately 0.86 log10) of MS2. Ozone treatment inactivated 80.37% (approximately 1.4 log10) spores and 99.95% (approximately 3.07 log10) coliphages. The coliphage inactivation rate obtained confirmed data obtained by previous studies indicating that MS2 was less resistant to ozonation than B subtilis spores. The membrane technology had the best efficiency in terms of physical removal of spores achieving over 99.9% (> 3 log10) removal. Coliphage removal mechanisms remain to be determined and will be a future focus of the study. Preliminary results indicate that aerobic spores and coliphages may be useful as indicators to determine the efficiency of different drinking water treatment technologies.

Negative regulation of the NFAT1 factor by CD45: implication in HIV-1 long terminal repeat activation.

HIV-1 gene regulation is greatly dependent on the presence of the -104/-81 enhancer region which is regulated by both NF-kappaB and NFAT transcription factors. We have found that a greater induction in HIV-1 long terminal repeat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulation of a CD45-deficient cell line (J45.01) in comparison to the parental Jurkat cells. Unlike NF-kappaB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transcriptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-induced NFAT activation, NFAT translocation and calcium influx peaked at similar time points for both Jurkat and J45.01 cell lines. The NFAT-dependent promoters from the IL-2 and TNF-alpha genes were also more potently activated by PMA/Iono in J45.01 cells. Interestingly, higher levels of intracellular calcium were consistently demonstrated in PMA/Iono-induced CD45-deficient cell lines (J45.01 and HPB45.0). Furthermore, PMA/Iono induction of calcium mobilization in both Jurkat and J45.01 cell lines was observed to be EGTA-sensitive. Mechanistic studies revealed that CD3zeta and ZAP-70 were more heavily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was present at higher levels in J45.01 nuclear extracts and that the NFAT1 member was predominant. In conclusion, our results indicate that NFAT activation by stimuli acting in a more distal fashion from the TCR-mediated signaling pathway can be down-regulated by CD45 and that this CD45-dependent regulation in turn affects HIV-1 long terminal repeat activation.

Neuraminidase from a bacterial source enhances both HIV-1-mediated syncytium formation and the virus binding/entry process.

Neuraminidases, also termed sialidases, which catalyze the removal of sialic acid residues from various glycoconjugates, have been previously reported to modulate HIV-1 replication. Given that some of the known opportunistic microbes found in patients infected with HIV-1 harbor neuraminidase (NA) activity, we speculated that pathogen-derived NA might be envisaged as an important factor in the pathogenesis of this retroviral infection. In the present study, we have monitored the putative modulation of HIV-1-mediated syncytium formation and virus replication by highly purified bacterial-derived NA from Arthrobacter ureafaciens. Taking advantage of a luciferase-based syncytium quantitative assay, we demonstrate here that the level of HIV-1-mediated syncytium formation is enhanced in the presence of NA and that it necessitates interaction between gp120 and CD4/chemokine coreceptor. By using pseudotyped recombinant luciferase-encoding HIV-1 particles, we found that NA treatment of human CD4-positive target cells (i.e., T lymphoid, monocytoid, and peripheral blood mononuclear cells) significantly augmented single-round infection by T- and macrophage-tropic isolates of HIV-1. The observed increase in HIV-1 infection was linked with an enhancement in the initial steps of the virus replicative cycle as monitored by viral binding and entry assays. Interestingly, NA treatment also enhances infectivity of HIV-1 pseudotypes with envelope glycoprotein from the amphotropic murine leukemia virus or the vesicular stomatitis virus. Taken together, our results provide useful information regarding the possible contribution of microbial agents carrying NA activity to HIV-1 pathogenesis.

Inhibition of HIV-1-mediated syncytium formation and virus replication by the lipophosphoglycan from Leishmania donovani is due to an effect on early events in the virus life cycle.

Previous findings have indicated that the major surface molecule of Leishmania, lipophosphoglycan (LPG), could abrogate HIV-1-induced syncytium formation and virus replication. In the present work, we were interested in characterizing this inhibitory process. Data from a new luciferase-based semiquantitative assay for syncytium formation, relying on the coincubation of a T-cell line containing an HIV-1 LTR-driven luciferase construct with a cell line chronically infected with HIV-1, confirmed that LPG was indeed a strong inhibitor of HIV-1-dependent syncytium formation and that this inhibition was dose-dependent. As determined by flow cytometric analyses, this inhibition was not apparently due to downregulation of CD4, CXCR4 or LFA-1, three distinct surface glycoproteins known to be important in HIV-1 mediated syncytium formation. Furthermore, LPG did not seem to affect signal transduction pathways in T cells as judged by measurement of HIV-1 LTR-driven reporter gene activity upon treatment with different stimuli. However, pretreatment of either of the cell lines used in the assay with LPG led to a significant decrease of virus-mediated syncytium formation, which was further accentuated when both cell lines were pretreated. LPG inhibition of HIV-1 replication was next assessed. When measuring either infection with luciferase-encoding recombinant HIV-1 particles or multinucleated giant cell formation following an acute virus infection, we again observed that LPG was efficient at blocking HIV-1 replication. Specific assays probing different steps of viral entry demonstrated that attachment was not hindered by LPG but that viral entry was modulated, suggesting that LPG targets a postbinding step. Hence, incorporation of LPG into a target cell membrane could influence its fluidity and diminish both the virus-cell and cell-to-cell fusion processes initiated by HIV-1.

Regulation of nuclear factor of activated T cells by phosphotyrosyl-specific phosphatase activity: a positive effect on HIV-1 long terminal repeat-driven transcription and a possible implication of SHP-1.

Although protein tyrosine phosphatase (PTP) inhibitors used in combination with other stimuli can induce interleukin 2 (IL-2) production in T cells, a direct implication of nuclear factor of activated T cells (NFAT) has not yet been demonstrated. This study reports that exposure of leukemic T cells and human peripheral blood mononuclear cells to bis-peroxovanadium (bpV) PTP inhibitors markedly induce activation and nuclear translocation of NFAT. NFAT activation by bpV was inhibited by the immunosuppressive drugs FK506 and cyclosporin A, as well as by a specific peptide inhibitor of NFAT activation. Mobility shift assays showed specific induction of the NFAT1 member by bpV molecules. The bpV-mediated NFAT activation was observed to be important for the up-regulation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) and the IL-2 promoter; NFAT1 was demonstrated to be particularly important in bpV-dependent positive action on HIV-1 LTR transcription. The active participation of p56(lck), ZAP-70, p21(ras), and calcium in the bpV-mediated signaling cascade leading to NFAT activation was confirmed, using deficient cell lines and dominant-negative mutants. Finally, overexpression of wild-type SHP-1 resulted in a greatly diminished activation of NFAT by bpV, suggesting an involvement of SHP-1 in the regulation of NFAT activation. These data were confirmed by constitutive NFAT translocation observed in Jurkat cells stably expressing a dominant-negative version of SHP-1. The study proposes that PTP activity attenuates constitutive kinase activities that otherwise would lead to constant NFAT activation and that this activation is participating in HIV-1 LTR stimulation by PTP inhibition.

Presence of new alternative exons in human and mouse Fli-1 genes.

The mouse Fli-1 proto-oncogene is activated by proviral integration of four murine leukemia retroviruses and its human counterpart is translocated (11,22) in Ewing tumors. We have identified two alternative exons 1 by RACE analysis from a human neuroectodermal tumor. Exons 1a and 1b are located respectively 1.3 and 2.5 kb upstream from the published exon 1. Translation of these alternative messengers is predicted to generate very similar proteins. The sequence upstream from exon 1b showed functional promoter activity. Exon 1b was not conserved in the mouse but was detected in every analyzed human cell, whereas exon 1a was present only in a subset of them and also in various mouse cell lines. These results suggest that both mouse and human Fli-1 gene expression might be under the control of several independent promoter regions.

Occurrence of pathogenic microorganisms in the Saint Lawrence River (Canada) and comparison of health risks for populations using it as their source of drinking water.

A 300-km portion of the Saint Lawrence hydrological basin in the province of Québec (Canada) and 45 water treatment plants were studied. River water used by drinking water treatment plants was analyzed (6-L sample volumes) to determine the level of occurrence of bacterial indicators (total coliforms, fecal coliforms, and Clostridium perfringens) and pathogens (Giardia lamblia, Cryptosporidium, human enteric viruses). Pathogens and bacterial indicators were found at all sites at a wide range of values. Logistic regression analysis revealed significant correlations between the bacterial indicators and the pathogens. Physicochemical and treatment practices data were collected from most water treatment plants and used to estimate the level of removal of pathogens achieved under cold (0 degree C-4 degrees C) and warm (20 degrees C-25 degrees C) water temperature conditions. The calculated removal values were then used to estimate the annual risk of Giardia infection using mathematical models and to compare the sites. The estimated range of probability of infection ranged from 0.75 to less than 0.0001 for the populations exposed. Given the numerous assumptions made, the model probably overestimated the annual risk, but it provided comparative data of the efficacy of the water treatment plants and thereby contributes to the protection of public health.