Jean-Claude Nicolas

Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor.

The trafficking of the androgen receptor (AR) in transfected cells was studied using a green fluorescent protein (GFP)-AR chimera. The reporter molecule GFP enabled the localization of AR in living cells with a good spatial and temporal resolution. After the construction of GFP-AR and verification of the size of the fusion protein produced, we demonstrated that GFP-AR conserves the functional properties of the AR: GFP-AR had the same androgen-binding affinity as AR, and GFP-AR efficiently transactivated an androgen-responsive gene in response to synthetic androgen at 30 degrees C. The fusion protein was first detected throughout the cytoplasm without hormone, fluorescence becoming nuclear rapidly after androgen incubation. This hormone dependence of AR trafficking was confirmed by the use of the mutant GFP-AR-del4, which lacked the androgen-binding function. The mutant was localized in the cytoplasm in the absence of hormone, but the distribution was not modified by androgen incubation. An ACAS 570 scanning laser cytometer was used to quantify fluorescence in a single living cell, first without and then with hormone. Different hormones and antihormones were tested to determine the dynamics of GFP-AR translocation into the nucleus. All the drugs used were able to induce nuclear translocation, and steady state level was rapidly attained within 1 h. The ratio of receptors in cytoplasmic and nuclear compartments was related to both affinity and concentration of ligand. The data from this follow-up study demonstrated for the first time the intracellular dynamics of the hormone-dependent trafficking of AR in a single living cell.

Phenylphenols, biphenols, bisphenol-A and 4-tert-octylphenol exhibit α and β estrogen activities and antiandrogen activity in reporter cell lines

We previously demonstrated the interactions of different chemical compounds with estrogen receptors ERα and ERβ and the androgen receptor (AR) using different reporter cell lines. In this study, we characterize the ERα, ERβ and AR activity of different biphenyls using the same tools. We provide evidence that several phenyl derivatives present both estrogenic and antiandrogenic activity. The extent of hydroxylation and the position of the hydroxyl function were important in determining their estrogenicity and antiandrogenicity. Of the tested compounds, bisphenol-A and 4,4′ biphenol had very high estrogenic activity, although it was lower than that of the strong estrogenic alkylphenol, 4-tert-octylphenol. Bisphenol-A and 4,4′ biphenol were able to activate ERs at concentrations lower than 1 μM, whereas the other compounds only activated at concentrations above 1 μM. Interestingly, 4,4′ biphenol was a better agonist for ERβ than for ERα. No androgenic activity was detected for any of these compounds. Bisphenol-A, 3-OH phenylphenol, 4-OH phenylphenol and 4,4′ biphenol exhibited antiandrogenic activity close to that of 4-tert-octylphenol (IC50≈5 μM). In whole cell binding assays, these compounds displaced [3H] R1881 with Ki=10 μM. Although these Ki values seem high in comparison with that of hydroxyflutamide (0.4 μM), one must keep in mind that environmental chemicals can accumulate in adipose tissues for several years. In conclusion, these environmental chemicals may have a negative impact on androgen action during fetal and post-natal life.

Quantification of Human Cytomegalovirus DNA by Real-Time PCR

ABSTRACT A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).

Quantification of Human Immunodeficiency Virus Type 1 Proviral Load by a TaqMan Real-Time PCR Assay

ABSTRACT Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log10. Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 106 PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 106 PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy (P = 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5′ end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4+ cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.

Estrogenic Activity of Cosmetic Components in Reporter Cell Lines: Parabens, UV Screens, and Musks

In this work, the estrogenic effects of three classes of substances included in cosmetic formulations—parabens, ultraviolet (UV) screens, and musk fragrances—were studied. Their estrogenic activity was measured with the use of three reporter cell lines: HELN, HELN ERα, and HELN ERβ. These three cell lines allowed for the measurement of estrogenic activity toward estrogen receptors α and β (ERα and ERβ), while taking nonspecific interactions into account. Eight of the 15 substances tested showed specific estrogenic activity with the following degree of potency on ERα: butylparaben > propylparaben > homosalate = octyl-dimethyl-PABA = 4-methyl-benzylidenecamphor = octyl-methoxycinnamate > ethylparaben = galaxolide. Among these active substances, parabens activated ERα and ERβ similarly, UV screens activated ERα moderately and had almost no effect on ERβ, and fragrances did not activate ERβ. Methylparaben, ethylparaben, musk moskene, celestolide, and cashmeran did not activate estrogenic responses up to 10−5 M. Musk ketone and benzophenone-3 were not considered estrogenic at 10−5 M. We thank S. Bintein and the MATE, France (Ministère de l’Aménagement du Territoire et de l’Environnement), for financial support. M. Manley, professional translator, improved the English.

Specific recognition of androgens by their nuclear receptor: a structure-function study

Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17β substituent in the steroid D-ring. To better understand the specific recognition of ligands by the human androgen receptor (hAR), a homology model of the ligand-binding domain (LBD) was constructed based on the progesterone receptor LBD crystal structure. Several mutants of residues potentially involved in the specific recognition of ligands in the hAR were constructed and tested for their ability to bind agonists. Their transactivation capacity in response to agonist (R1881) and antagonists (cyproterone acetate, hydroxyflutamide, and ICI 176344) was also measured. Substitution of His874 by alanine, only marginally impairs the ligand-binding and transactivation capacity of the hAR receptor. In contrast, mutations of Thr877 and, to a greater extent, Asn705 perturb ligand recognition, alter transactivation efficiency, and broaden receptor specificity. Interestingly, the N705A mutant acquires progesterone receptor (PR) properties for agonist ligands but, unlike wild type AR and PR, loses the capacity to repress transactivation with nonsteroidal antagonists. Models of the hAR·LBD complexes with several ligands are presented, which suggests new directions for drug design.

Quantitation of HTLV-I proviral load by a TaqMan real-time PCR assay

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood mononuclear cells (PBMCs). The HTLV-I copy number was referred to the actual amount of cellular DNA by means of the quantitation of the albumin gene. Ten copies of HTLV-I DNA could be detected with 100% sensitivity, and the assay had a wide range of at least 5 log(10). Intra- and inter-assay reproducibility was evaluated using independent extractions of PBMCs from an HTLV-I-infected patient (coefficients of variation, 24 and 7% respectively). The performance of this TaqMan PCR assay, coupled with its high throughput, thus allows reliable routine follow-up of HTLV-I proviral load in infected patients. Preliminary results using clinical samples indicate a higher proviral load in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers, and also suggest the usefulness of this quantitative measurement to assess the etiological link between HTLV-I and adult T-cell leukaemia/lymphoma-like syndromes.

Virus Diversity in a Winter Epidemic of Acute Diarrhea in France

ABSTRACT In France, an epidemic peak of acute diarrhea is observed each winter. Previous results suggested a viral etiology for these winter epidemics. We investigated the role of enteric viruses in acute diarrhea and their molecular diversity. One hundred sixty-one patients with acute diarrhea and 45 healthy patients (controls) from the general population were given a standardized questionnaire between December 1998 and May 1999. Stool specimens were screened for group A and C rotaviruses, human caliciviruses, astroviruses, and adenovirus types 40 and 41 by reverse transcription-PCR and/or enzyme immunoassay. Virologic analysis was positive for 63 cases (39%). Caliciviruses and group A rotaviruses were the most frequent (19 and 17% of cases, respectively). Two control stool specimens were found positive for group A rotavirus, and one was found positive for astrovirus. Molecular characterization of the strains disclosed a cocirculation of P[8],G1, P[8],G4, and P[4],G2 rotaviruses; type 1, 2, 3, 4, and 8 astroviruses; and Sapporo-like and Norwalk-like human caliciviruses. These four types of viruses accounted for an attributable risk of acute diarrhea of 34.7% for the general population, under the assumption of a causal role of these viruses.

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

Phylogenetic Analyses Indicate an Atypical Nurse-to-Patient Transmission of Human Immunodeficiency Virus Type 1

ABSTRACT A human immunodeficiency virus (HIV)-negative patient with no risk factor experienced HIV type 1 (HIV-1) primary infection 4 weeks after being hospitalized for surgery. Among the medical staff, only two night shift nurses were identified as HIV-1 seropositive. No exposure to blood was evidenced. To test the hypothesis of a possible nurse-to-patient transmission, phylogenetic analyses were conducted using two HIV-1 genomic regions (pol reverse transcriptase [RT] and env C2C4), each compared with reference strains and large local control sets (57 RT and 41 C2C4 local controls). Extensive analyses using multiple methodologies allowed us to test the robustness of phylogeny inference and to assess transmission hypotheses. Results allow us to unambiguously exclude one HIV-positive nurse and strongly suggest the other HIV-positive nurse as the source of infection of the patient.