[Towards better management for sexual offenders: Presentation and conclusions of a public hearing concerning prevention, assessment, and care]. / Audition publique auteurs de violences sexuelles : prévention, évaluation, prise en charge des 14 et 15 juin 2018 : perspectives et préconisations.

In France, since the law of June 17, 1998, sexual offenders may be convicted to ambulatory mandatory care, articulated with the justice. Twenty years after the implementation of this law, while social and technological developments have redefined certain aspects of delinquency, reference documents and practice guidelines remain to be updated. This is why the professionals of the main structures and associations dealing with perpetrators of sexual violence organized a public hearing under the sponsorship of the French Federation of Resource Centers for Sexual Violence Perpetrators (FFCRIAVS) according to the methodology and with the accompaniment of the High Authority of Health. This article presents the global methodology of the public hearing "Sexual Offenders: Prevention, Evaluation and Care" which was conducted on June 14 and 15, 2018. Thirty-three experts replied to27 questions and presented their conclusions to an Audition Committee and an audience of 200 persons representative of the civil and professional society. After a public debate, the hearing committee prepared a report in which they proposed propositions in order to better care for sexual offenders.

[Smoking cessation: How can we involve patients better in treatment choice?] / Arrêt du tabac : comment mieux impliquer le patient dans le choix du traitement.

Counseling and pharmacotherapy are smoking cessation interventions whose effectiveness has been widely demonstrated. Different pharmacologic treatment options exist with similar efficacy : notably nicotine replacement therapy, varenicline and bupropion. Providers can promote therapeutic adherence and the chances of successful quitting by involving patients in the choice of medication and incorporating their preferences in the decision process. The concept of shared decision-making is based on an exchange between doctor and patient in medical situations with several reasonable options. Decision aids support this approach by facilitating the transmission of information, communication and patient involvement. Despite opportunities for shared decision-making during the smoking cessation process, few decision aids are available. This article summarizes current knowledge in this field and its application to the process of smoking cessation. Shared decision making in smoking cessation is illustrated by a decision aid created to facilitate the choice between different smoking cessation medications.

Multiple signals regulate GAL transcription in yeast.

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.

Phosphorylation of Ga14p at a single C-terminal residue is necessary for galactose-inducible transcription.

Gal4p regulates expression of genes necessary for galactose catabolism in Saccharomyces cerevisiae. We have previously shown that phosphorylation of Gal4p requires both its DNA binding and transcriptional-activation functions and have suggested that phosphorylation occurs as a consequence of interaction with general transcription factors. In this study, we show that phosphorylation occurs rapidly on a limited fraction of overexpressed Gal4p present in a sodium dodecyl sulfate-extractable subcellular fraction while a significant fraction remains stably unphosphorylated. Taken together with our previous observations, we conclude that Gal4p is phosphorylated only if it becomes localized to the nucleus and is capable of both DNA binding and transcriptional activation. We demonstrate that Gal4p is multiply phosphorylated at both the C and N termini, and we identify the precise locations of three sites of phosphorylation at serines 691, 696, and 699. Of these sites, only serine 699 must be phosphorylated for galactose-inducible transcription to occur. Mutation of S-699 to alanine significantly impairs GAL induction by galactose in GAL80+ cells but does not affect transcriptional activation by Gal4p in gal80- cells. In gal80- cells, Gal4p phosphorylation, including that of serine 699, is stimulated by the presence of both galactose and glucose, indicating that phosphorylation at this site is not specifically activated by galactose. Serine 699 phosphorylation requires Gal4p's DNA binding function and is influenced by the function of the RNA polymerase II holoenzyme component Gal11p. These results suggest that a phosphorylation on Gal4p, likely resulting from interaction with the holoenzyme, modulates the induction process by regulating interaction between Gal4p and Gal80p.

A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

Proteins encoded by oncogenes such as v-fps/fes, v-src, v-yes, v-abl, and v-fgr are cytoplasmic protein tyrosine kinases which, unlike transmembrane receptors, are localized to the inside of the cell. These proteins possess two contiguous regions of sequence identity: a C-terminal catalytic domain of 260 residues with homology to other tyrosine-specific and serine-threonine-specific protein kinases, and a unique domain of approximately 100 residues which is located N terminal to the kinase region and is absent from kinases that span the plasma membrane. In-frame linker insertion mutations in Fujinami avian sarcoma virus which introduced dipeptide insertions into the most stringently conserved segment of this N-terminal domain in P130gag-fps impaired the ability of Fujinami avian sarcoma virus to transform rat-2 cells. The P130gag-fps proteins encoded by these transformation-defective mutants were deficient in protein-tyrosine kinase activity in rat cells. However v-fps polypeptides derived from the mutant Fujinami avian sarcoma virus genomes and expressed in Escherichia coli as trpE-v-fps fusion proteins displayed essentially wild-type enzymatic activity, even though they contained the mutated sites. Deletion of the N-terminal domain from wild-type and mutant v-fps bacterial proteins had little effect on autophosphorylating activity. The conserved N-terminal domain of P130gag-fps is therefore not required for catalytic activity, but can profoundly influence the adjacent kinase region. The presence of this noncatalytic domain in all known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues for an important biological function. The relative inactivity of the mutant proteins in rat-2 cells compared with bacteria suggests that the noncatalytic domain may direct specific interactions of the enzymatic region with cellular components that regulate or mediate tyrosine kinase function.

The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function.

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.

Two regulators of Ste12p inhibit pheromone-responsive transcription by separate mechanisms.

The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. Ste12p is negatively regulated by two inhibitor proteins, Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216-688)] from a GAL promoter causes FUS1 induction in a strain expressing wild-type STE12, suggesting that this region can cause the activation of endogenous Ste12p. Residues 262 to 594 are sufficient to cause STE12-dependent FUS1 induction when overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p in yeast extracts. In contrast, recombinant glutathione S-transferase-Dig2p binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but not DIG1, from a GAL promoter inhibits transcriptional activation by an Ste12p DBD-VP16 fusion. Furthermore, disruption of dig1, but not dig2, causes elevated transcriptional activation by a LexA-Ste12p(216-688) fusion. Ste12p has multiple regions within the C terminus (flanking residue 474) that can promote multimerization in vitro, and we demonstrate that these interactions can contribute to the activation of endogenous Ste12p by overproduced C-terminal fragments. These results demonstrate that Dig1p and Dig2p do not function by redundant mechanisms but rather inhibit pheromone-responsive transcription through interactions with separate regions of Ste12p.

Catalytic and non-catalytic domains of the Fujinami sarcoma virus P130gag-fps protein-tyrosine kinase distinguished by the expression of v-fps polypeptides in Escherichia coli.

While protein-tyrosine kinases share a region of sequence identity corresponding to their kinase domains, the specific elements essential for catalysis, substrate binding and substrate specificity are largely undefined. The P130gag-fps transforming protein of Fujinami avian sarcoma virus is a cytoplasmic tyrosine kinase with a complex structure that includes a C-terminal kinase domain. To identify the precise N-terminal border of the v-fps catalytic region and to assess its interactions with non-catalytic domains, C-terminal v-fps polypeptide fragments of decreasing size were expressed in E. coli as trpE-v-fps hybrid proteins. All such polypeptides containing 263 or more residues derived from the C-terminus of P130gag-fps (i.e. residues 920-1182) were enzymatically active as tyrosine kinases. They autophosphorylated at physiological sites in vivo and phosphorylated exogenous substrates such as enolase and poly(glu,tyr) at tyrosine in vitro. Deletion of a further five amino acids from P130gag-fps residues 920-925 abolished all enzymatic activity. This deletion coincides with the predicted N-terminus of the v-fps ATP-binding site at residue 922. These data indicate that the N-terminal border of the ATP-binding site defines the start of the minimal v-fps tyrosine kinase catalytic domain, and show that this minimal domain is competent to bind substrates. More N-terminal non-catalytic sequences appear to functionally interact with the catalytic domain.

Ras-responsiveness of the HIV-1 LTR requires RBF-1 and RBF-2 binding sites.

Lentiviruses characteristically form latent integrated proviruses whose transcription can be induced by cell regulatory signals. The HIV-1 LTR responds to multiple signals, including the Ras pathway. We report here that Ras-responsive HIV-1 transcription requires two previously undescribed factors, RBF-1 and RBF-2 in Jurkat T cells. RBF-1 binds to Ets-like motifs located between nucleotides -151 and -142, and within the NF-kappaB binding sites, but is distinct from Ets-1 or Elf-1. RBF-2 binds the HIV-1 LTR at nucleotides -131 and -121 and immediately 3' of the TATA box. Both RBF-1 and RBF-2 contain DNA binding subunits of relative molecular weight 100 kilodaltons. Mutation of the RBF-1 and RBF-2 binding elements (RBEs) prevents Ras stimulation of HIV-1 LTR-directed transcription. These data define a mechanism for Ras responsiveness of HIV-1 transcription that involves the previously uncharacterized factors RBF-1 and RBF-2.

v-fps protein-tyrosine kinase coordinately enhances the malignancy and growth factor responsiveness of pre-neoplastic lung fibroblasts.

The v-fps oncoprotein was expressed in a pre-neoplastic, growth factor-dependent Chinese hamster lung fibroblast line (CCL39) to study its effect on growth controls and on the induction of malignancy. Two transfectants were characterized which expressed low (39FPS-8) or high (51FPS-6) levels of P130gag-f ps protein-tyrosine kinase activity. 39FPS-8 cells still arrested in quiescence when deprived of growth factors, but developed an increased sensitivity to the mitogenic actions of epidermal growth factor (20-fold) and alpha-thrombin (50-fold), although not to insulin. In contrast, 51FPS-6 cells completely escaped growth controls, divided in serum-free medium, and were insensitive to further growth factor stimulation. Both transfectants produced rapidly growing tumors in nude mice that formed pulmonary metastases from a subcutaneous site, unlike the parental cells which are non-metastatic. 51FPS-6 cells were comparatively more efficient than 39FPS-8 cells in colonizing the lungs after intravenous inoculation. The v-fps tyrosine kinase therefore induces a partial to complete relaxation of growth factor-mediated controls on the CCL39 cell cycle, with the extent of factor independence reflecting the amount of P130gag-f ps synthesized. This reduction in growth factor requirements correlates with the capacity of v-fps to confer the attributes of metastatic tumors upon preneoplastic CCL39 fibroblasts. We speculate that increased sensitivity to growth factor stimulation represents a common mechanism by which tumor cells acquire metastatic properties.

Mutational analysis of a phosphotransfer motif essential for v-fps tyrosine kinase activity.

The catalytic domains of protein-tyrosine kinases such as the P130gag-fps oncoprotein contain the sequence HRDLAARN, followed thirteen residues C-terminal by DFG (P130gag-fps residues 1041-1048 and 1061-1063). These residues define a structural motif conserved among eucaryotic protein kinases (-RD----N, DFG) and shared with several procaryotic 3'aminoglycoside phosphotransferases (H-D----N, D-G). Functional analysis of mutant v-fps proteins employing bacterial and mammalian expression systems indicated that this motif is critical for P130gag-fps kinase activity and oncogenicity. In particular, conservative substitutions of the two invariant aspartates (asp1043, asp1061) with glutamate or asparagine completely eliminated enzymatic activity, suggesting that these residues are essential for catalysis. In contrast, substitution of arg1042 with glutamate decreased but did not eliminate v-fps kinase activity in bacteria. The effects of these and other amino acid substitutions within the phosphotransfer motif and at the nearby autophosphorylation site (tyr1073) of P130gag-fps indicate that these conserved residues are intrinsically essential to the execution or regulation of catalytic activity, and suggest that tight spatial constraints operate within the active centre of the v-fps tyrosine kinase domain.

[Periodic health examination: a collaboration between an academic institution, a private firm and general practitioners]. / Collaboration médecins praticiens et entreprise privée pour une consultation préventive "Bilan de santé": une expérience romande.

A Swiss private company decided to launch a healthy lifestyle-oriented medical visit for its employees. Participating general practitioners had prior training through a one-day course. A brief physical examination, cholesterol and glucose analyses were included in the consultation. Half of the employees participated in the project. Health habits were similar to the general Swiss population. Four months later, 61% reported having changed at least one lifestyle health habit in order to improve their health. Most of participants and practitioners were satisfied with this type of consultation, that confirms the interest and the feasibility of such a project.

GAL4 fusion vectors for expression in yeast or mammalian cells.

We describe two sets of vectors, one for yeast (pY1, pY2 and pY3) and one for mammalian cells (pM1, pM2, and pM3), that simplify the production of fusion proteins containing the DNA-binding domain of GAL4. This protein fragment, consisting of GAL4 amino acid (aa) residues 1-147, binds to a specific 17-bp nucleotide sequence, but is incapable of activating transcription unless fused to a protein that can contribute an activating function. Genetic strategies exploiting this property of GAL4 (aa 1-147) have been developed to characterize transcription factor functional domains, protein-protein interactions, and site-specific proteolysis. The vectors we describe allow fusion to the C terminus of GAL4 (aa 1-147) in any reading frame, and thus facilitate these experimental strategies.

Inactivation of a yeast transactivator by the fused HIV-1 proteinase: a simple assay for inhibitors of the viral enzyme activity.

The human immunodeficiency virus type 1 (HIV-1) proteinase (PR) and its flanking sequences have been fused in frame between the DNA-binding domain and the transcription-activation domain of the yeast protein, GAL4. As has been shown before with the 3C proteinase of Coxsackie virus B3 (CVB3) [Das Mahapatra et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4159-4162], the GAL4::PR fusion protein retains its GAL4 function, providing the PR is inactive. When PR is active, its autocatalytic activity in the hybrid protein is shown to inactivate the transactivation function of GAL4. This provides a simple assay to monitor PR activity. A dose-dependent effect of a potent PR-specific inhibitor is demonstrated in this system and illustrates the sensitivity of the assay. The assay is used for high throughput screening to identify novel inhibitors of the viral PR, and provides a method to generate and analyze mutants and revertants of the PR.

Streptococcal meningitis: effect of CSF filtration on inflammation and neuronal damage.

The effect of CSF filtration on inflammation and neuronal damage was studied in experimental Streptococcus pneumoniae meningitis. New Zealand white rabbits received either antibiotic therapy alone (ceftriaxone i.v., 20 mg/kg bolus, 10 mg/kg maintenance dose; n = 10) or ceftriaxone plus CSF filtration (n = 11) 12 h after intracisternal infection. Immediately after the onset of antibiotic therapy 300 microliters cisternal CSF was removed, passed through a miniaturized CSF-1 filter at a constant flow of 20 microliters/min, and then reinjected. This procedure was repeated six times at intervals of 20 min. Antibiosis plus CSF filtration caused a transient reduction in CSF bacterial titers and leukocyte counts compared with antibiosis alone (P = 0.04 and 0.02 5 h after initiation of therapy). CSF lipoteichoic acid concentrations were not reduced. The concentration of neuron-specific enolase in CSF and the density of apoptotic neurons in the dentate gyrus were almost equal 12 h after the onset of treatment. Adjuvant CSF filtration accelerated the elimination of viable bacteria from CSF in comparison to antibiotic treatment alone. Parameters of neuronal destruction, however, were not reduced.

RK bacterial test for independently measuring chemical toxicity and mutagenicity: short-term forward selection assay.

A short-term bacterial assay system for determining the mutagenic potential of environmental substances was developed and validated. Genotoxic activity was demonstrated for selected substances from 10 categories of chemical agents. The RK test results were obtained with one Escherichia coli assay strain that was transiently exposed to, and then removed from the test substance prior to the selection step for mutant cells. The RK test employs a hitherto unused short-term assay technique for selecting forward mutations in the wild-type selector strain cells. The cells of the selector strain are killed upon shifting to 42 degrees C as a consequence of thermal derepression and subsequent expression of the replication genes from an integrated 10-kilobase fragment of phage lambda. Cells that acquire mutations in the responsible killing genes are detected by their colony-forming ability at 42 degrees C. A substance is determined to be genotoxic if it is capable of increasing the forward mutation frequency for appearance of these mutant cells. Toxicity of the agent is independently evaluated by examining its effect on the viability of the selector strain at 30 degrees C, when the viral replication genes remain repressed. The flexible assay protocol enables determination of the effect of pH on mutagenic activity, the requirement for metabolic activation, and assays of nearly insoluble or highly toxic substances.

GAL4 is regulated by a glucose-responsive functional domain.

The Saccharomyces cerevisiae transcriptional activator GAL4 is regulated by the presence of available carbon sources. Galactose induces activity by inhibiting the negative regulator GAL80, while glucose, the preferred carbon source, antagonizes GAL4 function by several mechanisms. In the present study we present evidence that one mechanisms for inhibition of GAL transcription by glucose involves direct inhibition of the GAL4 protein. We demonstrate that a large, previously uncharacterized, central region of GAL4 contains at least three 'inhibitory domains' and a 'glucose response domain' (GRD). Deletion of the entire central region eliminates direct inhibition of GAL4 by glucose, and furthermore, fusion of the central region to a heterologous transcriptional activator confers inhibition by glucose. The central region inhibitory domains constitutively inhibit transcriptional activation when the GRD is absent. Direct inhibition of GAL4 activity can be detected within 30 min following glucose addition and may represent an early mechanism promoting a switch from galactose to glucose utilization. A model for the regulatory role of the central region is presented, involving interaction with an additional protein that inhibits GAL4 activity when glucose is present.