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.

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.

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.

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.

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.

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.

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.

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.

The GATA-1 and Spi-1 transcriptional factors bind to a GATA/EBS dual element in the Fli-1 exon 1.

Fli-1 is a proto-oncogene which is rearranged in tumors induced by three different retroviruses, Cas-Br-E, F-MuLV, and 10A1. This gene is a member of the Ets gene family, a class of transcription factors that recognize and bind to a DNA motif known as the Ets binding site (EBS). Our laboratory has previously cloned and characterized the promoter region of both human and mouse Fli-1 genes. We had then identified several regulatory elements conserved between the two species. Two of them, an exon 1 GATA/EBS dual element and an EBS element located in the 5' end of intron 1, were analysed in the present study. EMSA analysis performed with nuclear extracts from different cell lines showed that the EBS element in intron 1 (EBSi) was bound by one potential Ets-related ubiquitous factor. The GATA/EBS element was bound by several factors that seemed Ets-related, one of which was found to be specifically expressed in hematopoietic cells. the GATA/EBS dual element was thus chosen for further analysis. A human Fli-1-derived genomic fragment containing the GATA/EBS led to enhanced transcription when positioned upstream of the SV40 promoter in the erythroleukemic HEL cell line. In addition, an increasing number of GATA/EBS oligonucleotides upstream of this same promoter resulted in a copy number-dependent increase in luciferase activity which was greatly reduced when the EBS consensus sequence was mutated. One of the factors binding to the GATA/EBS region was identified to be Spi-1 by supershift analysis and was also shown to bind to the EBS element of the human Ets-2 gene. Supershift analysis also demonstrated the binding of the GATA-1 factor to the GATA/EBS dual element. Our results suggest that Spi-1 and GATA-1 might play a key role in the regulation of Fli-1.

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.

Characterization of the human and mouse Fli-1 promoter regions.

Overexpression of the Fli-1 gene has been shown to be involved in retrovirus-induced mouse tumors. Cloning of the 5' flanking sequence of the mouse and human Fli-1 exon 1 was performed. At least two major transcription initiation sites were localized respectively at 143 and 114 nucleotides upstream of the previously defined mouse Fli-1 cDNA 5' end. The sequences flanking the CAP sites show good conservation between human and mouse (94%). The promoter region contains a potential TATA box lying 30 bp from one of the major identified CAP sites. Several conserved elements, such as GATA, EBS, GC rich, AP-2, AP-3 elements and a repetition of GA were observed next to the two major CAP sites. Furthermore, this latter was shown to form a H-DNA structure in vitro by S1 nuclease sensitivity experiments. The highly conserved 5' non-translated region of exon 1 is predicted to form a very stable hairpin structure which could regulate the Fli-1 expression at the post-transcriptional level. In Cas-Br-E-induced tumors, all the proviruses are found clustered within 35 nucleotides directly upstream the Fli-1 ATG start codon, thus deleting the hairpin structure from the transcript. Promoter activity was tested using the CAT reporter gene transfected in mouse and human erythroid cell lines. No promoter activity could be detected with various mouse Fli-1 promoter-CAT constructs containing 600 bp of the 5' flanking region, the complete exon 1, the 5' end of intron 1 and/or retroviral LTR sequence. Constructions of the human homologue containing nearly 1.5 kbp of Fli-1 5' flanking region was also inactive in transfected cells. These results suggest that multiple levels of regulation might control the Fli-1 expression.

Expression and DNA rearrangement of proto-oncogenes in Cas-Br-E-induced non-T-, non-B-cell leukemias.

The Cas-Br-E murine leukemia virus is a non-defective retrovirus that induces non-T-, non-B-cell leukemias in susceptible NIH/Swiss mice. A collection of tumors was examined for genomic DNA structure and RNA expression of known or putative proto-oncogenes and one tumor-suppressor gene, with the aim of identifying genes involved in Cas-Br-E-induced non-T-, non-B-cell leukemogenesis. Fli-1, p53, and Evi-1 were found to be rearranged in 72%, 23%, and 18% of the tumors, respectively, whereas no DNA alteration were detected for c-myc, c-myb, Pim-1, Evi-2, and EpoR genes. Evi-1 rearrangements are rarely associated with p53 or Fli-1 alterations. However, rearrangements of these last two genes are very often associated within the same tumor. Moreover, patterns of coordinated expression of critical cell growth-regulating genes are consistently associated with specific tumor types. These data suggest that Cas-Br-E can induce two types of hematopoietic neoplasias by different mechanisms.

LIVE/DEAD BacLight : application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water.

A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit (BacLight) was applied to estimate both viable and total counts of bacteria in drinking water. BacLight is composed of two nucleic acid-binding stains: SYTO 9 and propidium iodide. SYTO 9 penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal incubation conditions were found to be 15 to 20 min, at room temperature in the dark. Total (red + green) and viable (green) cells can hence be counted simultaneously. Factors affecting the staining procedure were tested (addition of glutaraldehyde, staining time, chlorine impact). In the absence of stress, BacLight viable counts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. BacLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, growth rate or temperature) induced a decrease in viability that was more pronounced for CTC and plate counts than for BacLight viable counts.

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.

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.

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.

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.

Triplex-forming oligonucleotides with unexpected affinity for a nontargeted GA repeat sequence.

We examined the affinity and the specificity of triplex formation for different purine ODNs directed against two portions of a purine sequence derived from the mouse fli-1 gene. As expected, the ODNs antiparallel to the purine strand of their target can form triplex DNA. One parallel ODN showed binding to its target sequence. We explain this unusual binding by an interaction of the ODN with a GA repetition present in the sequence. We further examined the interaction of this ODN with a target composed of 14 GA repetitions. Unexpectedly, one ODN shows higher affinity for a partially complementary GA target relative to its completely complementary target. For another ODN, the binding to the GA target is weaker and might involve skipping of bases in a way that resembles alternate strand triplex formation.

Binding of HIV-1 to its receptor induces tyrosine phosphorylation of several CD4-associated proteins, including the phosphatidylinositol 3-kinase.

Cell surface CD4 molecules are known to be important in several physiological responses of T lymphocytes. The use of human immunodeficiency virus (HIV) particles or purified gp120 molecules as CD4 cross-linking agents has been shown to result in a cascade of intracellular biochemical events. In addition, we and other have provided evidence suggesting that virus-mediated CD4 multimerization can lead to modulation of HIV-1 long terminal repeat-dependent activity and virus production. We were thus interested in measuring the effect of HIV-1 particles on intracellular tyrosine-phosphorylation levels, mostly of CD4-associated proteins. Using the T cell line CEM-T4, we observed that HIV-1 induces an increase in tyrosine phosphorylation of four major proteins physically complexed to the CD4 molecule. Immunoblot analysis permitted the identification of two of these proteins, p56lck and phosphatidylinositol 3-kinase (PI 3-kinase) p85 alpha. No concomitant variation in the level of these two CD4-associated proteins was observed after HIV-1-induced CD4 cross-linking. To our knowledge, this is the first report linking HIV-1-mediated CD4 multimerization to an increase in tyrosine phosphorylation of the PI 3-kinase complex. The four CD4-associated molecules described in this report are most likely implicated in virus-induced CD4-linked signaling events.