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International audience; Although involved in processes leading to the emergence and development of hormone-dependent breast cancers, the estrogen receptor alpha (ERalpha) also prevents transformed cells from progressing toward a more aggressive phenotype. The transcriptional activity of ERalpha is mediated through two transactivation functions, called activation function 1 and 2, whose respective involvement varies in a cell-specific manner. Here, we identify the Rho/megakaryoblastic leukemia 1 (MKL1) signaling pathway as a main actor in controlling the cell-specific activity of both transactivation functions of ERalpha. Notably, we show that, when the coregulator MKL1 is sequestered in an inactive form by unpolymerized actin, the transcriptional activity of ERalpha mainly relies on the activation function 1. The activation of MKL1, which results from its dissociation from unpolymerized actin, promoted by the ability of Rho to support polymeric actin accumulation, silences the activation function 1 of ERalpha and allows the receptor to mainly act through its activation function 2. Importantly, this switch in the respective contribution exerted by both transactivation functions is correlated with an impaired ability of ERalpha to efficiently transactivate estrogen-regulated reporter genes. MKL1 is further shown to be present on estrogen-responsive genes in vivo. Interestingly, the Rho/MKL1 signaling pathway is activated during the epithelial-mesenchymal transition. A reduced transactivation efficiency of ERalpha, resulting from the activation of this pathway, may therefore suppress the protective role exerted by ERalpha toward tumor progression and invasiveness.

This paper presents the design of the NEDA (Neutron Detector Array) electronics, a first attempt to involve the use of digital electronics in large neutron detector arrays. Starting from the front-end modules attached to the PMTs (PhotoMultiplier Tubes) and ending up with the data processing workstations, a comprehensive electronic system capable of dealing with the acquisition and pre-processing of the neutron array is detailed. Among the electronic modules required, we emphasize the front-end analog processing, the digitalization, digital pre-processing and communications firmware, as well as the integration of the GTS (Global Trigger and Synchronization) system, already used successfully in AGATA (Advanced Gamma Tracking Array). The NEDA array will be available for measurements in 2016.

This paper presents the final design and results of the FADC Mezzanine for the EXOGAM (EXOtic GAMma array spectrometer) and NEDA (Neutron Detector Array) detectors. The measurements performed include those of studying the effective number of bits, the energy resolution using HP-Ge detectors, as well as timing histograms and discrimination performance. Finally, the conclusion shows how a common digitizing device has been integrated in the experimental environment of two very different detectors which combine both low-noise acquisition and fast sampling rates. Not only the integration fulfilled the expected specifications on both systems, but it also showed how a study of synergy between detectors could lead to the reduction of resources and time by applying a common strategy.

International audience; This board will be part of the upgrade for the new electronics for the EXOGAM2 (HP-Ge detector array) and NEDA (BC501A-based neutron detector array), therefore it was necessary to deal with the problem of providing a sampling card with high resolution for new gamma spectroscopy experiments while sampling at very high rates, with a broad bandwidth in order to preserve the shape for further analysis. Pulse shape analysis is of paramount importance in neutron detectors, such as NEDA, based on scintillators that are sensitive to γ-rays as well. High resolution and high speed are often two parameters which conform a trade-off and it is hard to achieve both simultaneously. The aforementioned constraints and the urge of building new sampling electronics to improve the signal analysis in nuclear physics experiments, led to the development of this FADC mezzanine This involves sampling rates up to 250 Msps preserving a high resolution of 11.3 effective bits in order to satisfy the experiment demands. In this work is described the design and the test bench proposed for a proper high speed ADC characterization system and the results obtained up to now.

In nuclear physics experiments, a decision maker named as “trigger” gives a bit pattern which allows the fired detectors identification. As the data acquisition dead time is greater than the time between physical events, timing information is essential. We add to the trigger function a Time to Digital Converter (TDC) in order to make a separation between events. The paper describes the architecture chosen for the TDC and illustrates the contribution of each element to the TDC performance. An eight-bit counter is used for the dynamic range of the TDC (in microsecond) associated to a delay line improving the resolution (in nanosecond). The study shows that exploiting the two system clock states (high and low) allows to reduce the number of delay line cells. The Differential Nonlinearity Measurements are given for different resolutions (1, 2 and 5 ns) and illustrate the clock period, the clock duty cycle and the delay line contributions to the TDC performances.

19th IEEE-NPSS Real Time Conference (RT) -- MAY 26-30, 2014 -- Nara, JAPAN, WOS: 000380588000133, This paper presents the design of the NEDA frontend electronics, a first attempt to involve the use of digital electronics in large neutron detector arrays. Among the electronic modules taking part, we emphasize on the front-end analog processing, the digitalization, digital pre-processing, communications firmware, as well as the integration of the Global Trigger and Synchronization system. The NEDA array will be available for measurements in 2015., Osaka Univ, Res Nucl Phys, IEEE Nucl & Plasma Sci Soc

Calcitonin is a peptide which acts in the brain to modulate behavior and hormone release, possibly through an interaction with serotonergic systems. We investigated the effects of chronic systemic injections of salmon calcitonin on the [3H]-8-OHDPAT binding to 5-HT1A receptors in the frontal cortex and hippocampus in adrenalectomized and intact (non adrenalectomized) rats. The results show that salmon calcitonin increases the maximal density of 5-HT1A binding sites in both structures in adrenalectomized animals (and decreases the affinity in the frontal cortex only). Calcitonin does not alter this binding in intact rats. These results demonstrate the existence of interactions between calcitonin, serotonin and glucocorticoids, and raise the hypothesis of a neurotrophic effect of calcitonin on serotonergic neurons.

Background Testicular Leydig cells use either exogenous or de novo synthesized cholesterol as the substrate for the production of testosterone with hormone stimulation. Although the long-term effect of trophic hormones on Leydig cell cholesterol uptake, storage, and deesterification has been well documented, the early effects of the human choriogonadotropin (hCG) on cell cholesterol/lipid distribution are not yet known. Methods Sections of cells treated with hCG for 15 sec to 30 min were examined by electron microscopy (EM) for the surface density of lipid moieties in the cytoplasm. In addition, the time-dependent distribution of lipids within the cytoplasmic inclusions and the ultimate destination of this substrate were evaluated by EM. The results were analyzed with standard morphometric methods. Results and Conclusion The surface density of cytoplasmic lipid pools increased significantly within the 15 sec following the exposure of cells to hCG, and it tapered off to the control level in the subsequent 30 min. Such a fluctuation in the amount of cytoplasmic lipids may be due to (1) the quantity of released substrate from the reticular compartment or (2) the rate of its transport across the outer mitochondrial membrane to the inner mitochondrial cytochrome P450 side-chain cleavage, where steroidogenesis begins with the conversion of cholesterol to pregnenolone. These two processes were not quantitatively coordinated in the stimulated cell during the intitial 30 min, resulting in a surplus of cytoplasmic lipid pools. To compensate for such uneven metabolic balance, the cell apparently disposed of the excess substrate by a mechanism of molecular regrouping from a micellar configuration to a bilayer structure followed by exocytosis. Anat. Rec. 248:374-379, 1997. © 1997 Wiley-Liss, Inc.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

We examined the topography of the MA-10 Leydig tumor cell mitochondrial peripheral-type benzodiazepine receptor (PBR). In previous studies, the 18 kDa PBR was found to be functionally associated with the voltage-dependent anion channel, located in the junctions between outer and inner membranes. Transmission electron (TEM) and atomic force microscopy (AFM) of immunogold labeled PBR on Leydig cell mitochondrial preparations showed that the 18 kDa PBR protein is organized in clusters of 4-6 molecules. Addition of hCG to Leydig cells induces a rapid, within 30 sec, increase in PBR ligand binding and morphological changes, namely redistribution of PBR molecules in large clusters (7 particles). These hCG-induced changes were inhibited by a cAMP-dependent protein kinase inhibitor and by the benzodiazepine flunitrazepam. AFM further demonstrated the rapid reorganization of the mitochondrial membrane, where the formation of contacts between the outer and the inner mitochondrial membrane may facilitate cholesterol transfer.

2012 18th IEEE-NPSS Real Time Conference, RT 2012 -- 9 June 2012 through 15 June 2012 -- Berkeley, CA -- 95754 This board will be part of the upgrade for the new electronics for the EXOGAM2 (HP-Ge detector array) and NEDA (BC501A-based neutron detector array), therefore it was necessary to deal with the problem of providing a sampling card with high resolution for new gamma spectroscopy experiments while sampling at very high rates, with a broad bandwidth in order to preserve the shape for further analysis. Pulse shape analysis is of paramount importance in neutron detectors, such as NEDA, based on scintillators that are sensitive to ?-rays as well. High resolution and high speed are often two parameters which conform a trade-off and it is hard to achieve both simultaneously. The aforementioned constraints and the urge of building new sampling electronics to improve the signal analysis in nuclear physics experiments, led to the development of this FADC mezzanine This involves sampling rates up to 250 Msps preserving a high resolution of 11.3 effective bits in order to satisfy the experiment demands. In this work is described the design and the test bench proposed for a proper high speed ADC characterization system and the results obtained up to now. © 2012 IEEE.

Native MA-10 mouse Leydig tumor cell mitochondrial preparations were examined by transmission electron (TEM) and atomic force (AFM) microscopic procedures in order to investigate the topography and organization of the peripheral-type benzodiazepine receptor (PBR). Mitochondria were immunolabeled with an anti-PBR antiserum coupled to gold-labeled secondary antibodies. Results obtained indicate that the 18,000 MW PBR protein is organized in clusters of 4-6 molecules. Moreover, on many occasions, the interrelationship among the PBR molecules was found to favor the formation of a single pore. Taking into account recent observations that the 18,000 MW PBR protein is functionally associated with the pore forming 34,000 MW voltage-dependent anion channel (VDAC) these results suggest that (i) the mitochondrial PBR complex could function as a pore, thus allowing the translocation of cholesterol and other molecules to the inner mitochondrial membrane, and (ii) the native receptor is a multimeric complex of an approximate 140,000 MW composed on an average of five 18,000 PBR subunits, one 34,000 VDAC subunit, and associated lipids.

Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein. In digitonin-permeabilized MA-10 Leydig tumor cells incubated with [γ-32P]ATP, phosphorylation of MBR was detectable during treatment of the cells with dibutyryl cAMP. In conlusion, these data show that the MBR protein is an in vitro and in situ substrate of Protein kinase A, but the role of this phosphorylation in the regulation of steroidogenesis remains to be established.

Purpose: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage whose expression is strongly decreased during osteoarthritis (OA) process. Specific proteins (Sp) and Sox proteins have been previously characterized to be major regulators of this gene. Besides, the existence of a link between estrogen deprivation and osteoarthritis in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a critical modulator of cartilaginous matrix homeostasis. The aim of present study was to investigate the molecular mechanisms regulating type II collagen gene expression under the effect of 17β-E2, and to characterize the genomic pathway via estrogen receptors (ER). Methods: Articular chondrocytes were isolated from 3-week old rabbits and incubated for 24 hours with increasing concentrations of 17β-E2 (0 to 10 nM). Nuclear proteins were extracted and submitted to gel retardation assays in order to determine Sp and Sox proteins binding activities on COL2A1 gene. In some experiments, relative expression of Sp1/Sp3 or Sox-9 was inhibited by siRNAs strategies. Moreover, chromatin immunoprecipitation was used to study protein-protein interactions on COL2A1 promoter and enhancer sequences. Results: We have previously shown that 17β-E2 could stimulate type II collagen neosynthesis, protein level and corresponding steady-state levels of COL2A1 mRNAs in primary and in vitro dedifferentiated rabbit articular chondrocytes. 17β-E2 action was mediated, in part, by an ERα/Sp transactivating effect on the -266/-63 bp region of COL2A1 proximal promoter. Our new data showed that 17β-E2 also increases the DNA-binding activities of Sp1/Sp3 and Soxs proteins to the first intron region including COL2A1 specific enhancer. In addition, Sp1, Sp3 and Sox-9 siRNAs prevented hERα66-induced transactivation of COL2A1, suggesting that these factors are required for COL2A1 up-regulation. Moreover, chromatin immunoprecipitation assays indicated that ERα physically interacts in vivo with Sp1, Sp3, Sox-9 and p300 transcription factors, on both COL2A1 promoter and enhancer sequences. Thus, considering that all these transcription factors are known to act in concert, we proposed that 17β-E2 in vivo effect could be mediated by a bridging complex, composed of ERα, Sp1, Sp3, Sox-9 and p300 transcription factors, which could establish a physical link between the two main cisregulatory regions of the gene, and consequently, may facilitate COL2A1 transcription process. Besides, this transcriptional activation of COL2A1 gene requires the ERα transactivation domains, AF-1 and AF-2, as demonstrated by using ERα mutants lacking these functional domains. Conclusions: Understanding the molecular basis for 17β-E2/ERα induction of COL2A1 transcription provides new insights into molecular mechanisms of OA and could be therefore attractive for some applications in the tissue engineering of cartilage.

International audience; Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Poster; International audience

Poster; International audience

International audience; OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

International audience; The estrogen receptor alpha (ER alpha) is key in regulating normal breast development and function and is closely involved in the onset and progress of cancers. ER alpha transcriptional activity is mediated through two activation functions, AF1 and AF2, whose activity is tightly regulated in a cell-specific manner through yet unknown processes. Here, we demonstrate that cell-cell junctions generate cell permissiveness to AF1 through an up-regulation of the activity of an AF1 sub-region termed box 1. Moreover, the loss of E-cadherin expression is shown to silence the AF1 activity of ER alpha, allowing the receptor to mainly act through its AF2. This switch from an AF1 to an AF2 cell permissiveness also consequently results in the attenuation of ER alpha activity. Therefore, a loss of cell-cell junctions, a key process that occurs during the epithelial-mesenchymal transition, should have a broad impact on ER alpha transcriptional functions.

Historically, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis was initially considered as the only physiological and programmed form of cell death. This type of death is recurrently associated with caspases, a family of cysteine proteases activated in apoptotic conditions. However, it is now widely recognized that programmed cell death (PCD) can also occur in the complete absence of caspase activation. The existence of non-caspase PCD pathways was corroborated by the discovery of caspase-independent executioners, such as the mitochondrial protein Apoptosis-Inducing Factor (AIF). Necrosis has often been viewed as an accidental and uncontrolled cell death process. Nevertheless, increasing evidence shows that, like apoptosis, necrosis could be a highly orchestrated type of PCD. Indeed, apoptosis and necrosis present more similarities than it has been originally thought. Here, we summarize the different classifications of PCD and the current knowledge of a necrotic PCD pathway mediated by AIF: alkylating DNA-damage mediated death. We also outline the molecular mechanisms controlling this form of PCD and discuss their potential relevance in physiological and pathological settings. These emerging data on the molecular mechanisms regulating programmed necrosis may certainly have potent therapeutic consequences in treating both apoptotic-resistant tumors and degenerating adult neurons.

Poster

International audience; PURPOSE: Overexpression of PBR has been found in several tumor types including ovarian, colon, breast adenocarcinomas, esophageal cancer. There is evidence suggesting that PBR ligands regulate cell proliferation. However, their action is probably cell-type specific. We decided to evaluate mitotic activity of PBR ligands in some normal and neoplastic cell lines. MATERIAL AND METHODS: The cells were maintained according to standard procedures. Ligand binding assay was performed in cell extract using PK-11195 or Ro-54864 and [N-methyl-3H] Ro-54864 or [N-methyl-3H] PK-11195. Cell proliferation was evaluated using 5-[3H]-thymidine assay. Western Immunoblot assay was conducted using polyclonal anti-PBR antibody. RESULTs: We have found that, macrophages evoked strong binding of both Ro-54864 and PK-11195. This phenomenon was accompanied by drastic decrease in the cell divisions. Similar effect was found only in the case of non-estrogen-dependent breast cancer cells MDA-MB 231. It suggest that PBR-ligand mediated inhibition of mitogenesis may represent a new anticancer strategy in non-estrogen-dependent breast cancer. In respect to macrophages inhibition of the cell division by both PBR ligands may have implication in modulation of inflammatory response. It has been postulated that PBR ligands may have anti-inflammatory activity in rheumatoid arthritis. The presence of peripheral benzodiazepine receptors in chondrocytes, T cells, macrophages and mesenchymal cells suggest that peripheral benzodiazepine receptor ligands may interfere with the cytokine network and thus modulate inflammatory response. CONCLUSIONS: The data suggest that PBR-ligand mediated inhibition of DNA synthesis in non-estrogen dependent breast cancer cells and in macrophages may represent a new therapeutic approach of breast anti-cancer and anti-inflammatory therapy.

International audience; A large area telescope for measurements with radioactive beams in inverse kinematics for transfer reactions or unbound final states is described. The performance and the first beam tests results are described.

NESTER SEP ACC; We have developed a new telescope array, dedicated to the study of direct reactions of exotic nuclei on light targets in inverse kinematics. This device, called MUST2, is briefly described, and the results of the first tests performed with an alpha source and Ni beams at 10 and 75 MeV/u on a CDH target are presented.

11 pages; International audience; The expression of two human estrogen receptor-alpha (hERalpha) isoforms has been characterized within estrogen receptor-alpha-positive breast cancer cell lines such as MCF7: the full-length hERalpha66 and the N terminally deleted hERalpha46, which is devoid of activation function (AF)-1. Although hERalpha66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hERalpha46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hERalpha46 is mainly expressed in the nucleus at relatively low levels, whereas hERalpha66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hERalpha46 accumulating within the nucleus. Although hERalpha46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G(0)/G(1) phases. To gain further details on the influence of hERalpha46 on cell growth, we used PC12 estrogen receptor-alpha-negative cell line, in which stable transfection of hERalpha66 but not hERalpha46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hERalpha46 inhibits the hERalpha66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hERalpha46 antagonizes the proliferative action of hERalpha66 in MCF7 cells in part by inhibiting hERalpha66 AF-1 activity.

ACC SEP NESTER; Over the past four years we have studied (p,p$^'$), (d,p) ,(d,$^3$He) and other reactions using radioactive beams in inverse kinematics to obtain spectroscopic information for nuclei away from the valley of stability After a general overview of the experimental method we will describe our ongoing MUST II development. This is to build a very compact (1000cm$^3$) three stage telescope with an active area of 100cm$^2$ with position resolution of 0.7×0.7 mm$^2$ and time of flight measurement. The mass identification and energy dynamic range is of 0.4 to 80 MeV.A up to alpha particles. The compactness of the array is assured through the use of an ASIC development to measure the time of flight and energy. The large solid angle coverage of 2.6sr and compactness of this array will allow it to be used in particle-gamma coincidence experiments

In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PPactivated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheraltype benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPAR and PPAR/, but not for PPAR. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPAR-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARdependent inhibition of Leydig cell PBR gene expression. (Endocrinology 143: 2571–2583, 2002)

In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PP-activated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheral-type benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPARα and PPARβ/δ, but not for PPARγ. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPARα-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARα-dependent inhibition of Leydig cell PBR gene expression.

In nuclear physics, the interaction between the ion beam and the target produces a a large amount of events. Some of these events have no interest for the studied physical phenomenon ; the useful events are sorted using a trigger. We have studied and realized a new trigger suitable for the GANIL (Grands Accélérateur National d'Ions Lourds) experiments. After an overview of the triggers used up to now at GANIL, we give the main features of the new trigger (GANIL Master Trigger, GMT) such as modularity, universality and versality. After a description of the trigger operating modes, we depict the trigger realization steps. The trigger informs about fired detectors in a beam to target collision but gives no information about timing, nevertheless the timing is a very important piece of information if the trigger analysis duration in several times greater than the beam period. We suggest a timing measurement structure (Time to Digital Converter, TDC) able to eliminate this imprecision. The Dead time, the low level intégration and the consumption contraisnts lead us to prefer a digital architecture based on a digital counter associated with delay lines. Simple equations are given in order to define the operating area of the TDC. This area depends on the clock duty cycle and the delay line taps. Measures of Differential Non Linearity (DNL) for different time résolutions (1 ,2, 5 and 10 ns) allow to establish the limits of this system and to underline some solutions to improve these features., En physique nucléaire, l'interaction faisceau-cible engendre une multitude d'événements qui ne sont pas tous d'intérêt pour le phénomène physique étudié. Il est donc important de disposer d'un système de sélection nommé trigger. Nous avons étudié et réalisé un nouveau trigger adapté aux expériences menées au GANIL. Nous avons présenté un historique des différents triggers utilisés aux GANIL (Grand Accélérateur National d'Ions Lourds) et avons montré la nécessité d'un nouveau trigger modulaire, universel et ouvert. Après une description des différents modes de fonctionnement du nouveau trigger (GANIL Master Trigger, GMT) nous avons décrit les phases de sa conception et de sa réalisation. Le trigger renseigne sur la configuration des détecteurs temporels, or cette information peut être fondamentale dans le cas où la fenêtre d'analyse du trigger couvre plusieurs périodes du faisceau. Nous avons donc proposé une structure de mesure de temps (Time to Digital Converter, TDC) qui permet de lever cette indétermination. Les contraintes de temps mort, d'intégration et de consommation nous ont conduit à proposer une architecture numérique basée sur un compteur associé à une ligne A Retard (LAR). Des calculs simples ont permis de définir la zone de fonctionnement du TDC. Cette zone dépend du rapport cyclique de l'horloge et des retards de la LAR. Des mesures de Non Linéarité Différentielle (NLD) pour des résolutions différentes (1,2,5 et 10 ns) ont permis d'établir les limites de ce système et de mettre en évidence des solutions d'amélioration de ces caractéristiques.

Caseins are a known source of biologically active peptides. In this study, we have shown evidence of a novel anxiolytic activity in a tryptic hydrolysate of bovine α s1-casein. Injection of 3 mg/kg of this hydrolysate significantly reduced the epileptic symptoms caused by pentylenetetrazole in rats. Anxiety reduction was also observed when the hydrolysate was tested in the elevated plus-maze and in the conditioned defensive burying rat models. Peptides isolated from the hydrolysate were examined for their affinity for the γ-amino-butyric acid (GABA) type A receptor. Only one peptide, named α -casozepine, corresponding to the 91–100 fragment from bovine α s1-casein, expressed affinity for GABA A receptor. In vitro, the peptide had 10,000 less affinity for the benzodiazepine site of the GABA A than did diazepam. However, in the conditioned defensive burying paradigm it was 10-fold more efficient than diazepam. The difference observed between the in vitro and in vivo activity of α -casozepine could not been explained by an action via the peripheral-type benzodiazepine receptor; α -casozepine had no affinity for this receptor. The α -casozepine amino acid sequence could be related to the carboxyterminal sequence of the polypeptide diazepam binding inhibitor, an endogenous ligand of the central GABAA and peripheral-type benzodiazepine receptors.

International audience; We report on the new GANIL DAQ system based on VXI-C crates and electronic modules (trigger, converters, scalers...) with a complete software running under LynxOS real-time kernel. The system elements can be used in various configurations depending on the type of experiments undertaken by the physicists. Moreover the older electronic subsystems (VXI-D, CAMAC...) can be easily coupled with the newest ones. The easiness of use and the modularity characterize the hardware equipment and the software application. The real-time software is implemented on one or several VME CPUs based on a PowerPC processor. The number of processes can be distributed on these CPUs and are dedicated to a specific job. The data flow is sent by such a task onto the Ethernet 10/100 network using TCP/IP protocol towards the “tapeserver” alpha workstation. This whole “new generation” DAQ system is operational from several months and many experiments have already used it in different configurations

The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

Eukaryotic steroid hormones, derived from cholesterol, are involved in the maintenance of the organism’s homeostasis, adaptability to the environment, and developmental and reproductive functions. In addition to the well-defined actions in peripheral tissues, steroids have pleiotropic actions on the central nervous system (CNS), where they control a number of neuroendocrine and behavioral functions. Thus, comprehension of the molecular systems underlying the control of steroid hormone biosynthesis is essential for the study and treatment of a multitude of physiological disorders.

To evaluate the role of the mitochondrial peripheral-type benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBR-negative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450.

Glucocorticoid excess has broad pathogenic potential including neurotoxicity, neuroendangerment, and immunosuppression. Glucocorticoid synthesis is regulated by ACTH, which acts by accelerating the transport of the precursor cholesterol to the mitochondria where steroidogenesis begins. Ginkgo biloba is one of the most ancient trees, and extracts from its leaves have been used in traditional medicine. A standardized extract of Ginkgo biloba leaves, termed EGb 761 (EGb), has been shown to have neuroprotective and antistress effects. In vivo treatment of rats with EGb, and its bioactive components ginkgolide A and B, specifically reduces the ligand binding capacity, protein, and messenger RNA expression of the adrenocortical mitochondrial peripheral-type benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased corticosteroid synthesis. As expected, the ginkgolide-induced decrease in glucocorticoid levels resulted in increased ACTH release, which in turn induced the expression of the steroidogenic acute regulatory protein. Because ginkgolides reduced the adrenal PBR expression and corticosterone synthesis despite the presence of high levels of steroidogenic acute regulatory protein, these data demonstrate that PBR is indispensable for normal adrenal function. In addition, these results suggest that manipulation of PBR expression could control circulating glucocorticoid levels, and that the antistress and neuroprotective effects of EGb are caused by to its effect on glucocorticoid biosynthesis.

Trophic hormone regulation of steroid synthesis can be thought of as being either acute, occurring within minutes and resulting in the rapid synthesis of steroids, or chronic, occurring over a long period of time and resulting in continued steroid production. The primary point of control in the acute stimulation of steroidogenesis by hormones involves the first step in this biosynthetic pathway, where cholesterol is converted to pregnenolone by the cholesterol side-chain cleavage cytochrome P-450 (P-450scc) enzyme and auxiliary electron transferring proteins, localized on the inner mitochondrial membranes (1–4). More detailed studies have shown that the reaction catalyzed by P-450scc is not the rate-limiting step in the synthesis of steroid hormones, but rather the transport of the precursor, cholesterol, from intracellular sources to the inner mitochondrial membrane and the subsequent loading of cholesterol in the P-450scc active site (1–4). This hormone-dependent transport mechanism was shown to be mediated by adenosine 3′:5′-cyclic monophosphate (cAMP), to be regulated by a cytoplasmic protein, and to be localized in the mitochondrion where it regulates the intramitochondrial transport of cholesterol (1–4).