The orphan nuclear receptor NR5A2 regulates peri-ovulatory events and their consequent luteinization in mice

Le récepteur nucléaire Nr5a2, également connu sous le nom de liver receptor homolog-1 (Lrh-1), est exprimé au niveau de l’ovaire chez la souris, exclusivement dans les cellules lutéales et de la granulosa. La perturbation de Nr5a2, spécifique aux cellules de la granulosa chez la souris à partir des follicules primaires dans la trajectoire du développement folliculaire a démontré que Nr5a2 est un régulateur clé de l’ovulation et de la fertilité chez la femelle. Notre hypothèse veut que Nr5a2 régule les évènements péri- et post-ovulatoires dans une séquence temporelle lors de la folliculogénèse. Afin d'étudier l’implication de Nr5a2 lors de l’ovulation et de la lutéinisation à différents stades du développement folliculaire, nous avons généré deux modèles de souris knockout spécifiques aux cellules de la granulosa pour Nr5a2: 1) Nr5a2Amhr2-/-, avec une réduction de Nr5a2 à partir des follicules primaires et subséquents; 2) Nr5a2Cyp19-/-, avec une réduction de Nr5a2 débutant au stade antral de développement en progressant. L’absence de Nr5a2 à partir des follicules antraux a résulté en une infertilité chez les femelles Nr5a2Cyp19-/-, de même qu’en des structures non-fonctionnelles similaires aux structures lutéales au niveau des ovaires, en une réduction des niveaux de progestérone synthétisée ainsi qu’en un échec dans le support d’une pseudo-gestation. La synthèse de progestérone a été entravée suite à l’absence de Nr5a2 par l’entremise d’une régulation à la baisse des gènes reliés au transport du cholestérol, Scarb1, StAR et Ldlr, démontré par qPCR. Les complexes cumulus-oocytes des femelles Nr5a2Cyp19-/- immatures super-stimulées ont subi une expansion in vivo, mais l’ovulation a été perturbée, possiblement par une régulation à la baisse du gène du récepteur de la progestérone (Pgr). Un essai d’expansion du cumulus in vitro a démontré une expansion défectueuse du cumulus chez les Nr5a2Amhr2-/-, associée à un dérèglement de la protéine des jonctions communicantes (Gja1; Cx43). Cependant, l’expansion du cumulus chez les Nr5a2Cyp19-/- n’a pas été autant affectée. Des résultats obtenus par qPCR ont démontré une régulation à la baisse dans l’expression des gènes Areg, Ereg, Btc et Tnfaip6 chez les deux modèles de cellules ovariennes knockout à 2h et 4h post hCG. Nous avons observé que 85% des oocytes, chez les deux génotypes mutants, peuvent subir une rupture de la vésicule germinative, confirmant leur capacité de maturation in vivo. La technique d’injection intra-cytoplasmique de spermatozoïdes a prouvé que les oocytes des deux génotypes mutants sont fertilisables et que 70% des embryons résultants ont poursuivi leur développement vers le stade de blastocyste, et ce, indépendamment du génotype. En conclusion, Nr5a2 régule la fertilité chez les femelles tout au long du processus du développement folliculaire. Il a été démontré que Nr5a2 est essentiel à la lutéinisation et que sa perturbation dans les cellules somatiques ovariennes ne compromet pas la capacité des oocytes à être fertilisés. En vue d’ensemble, nous avons fourni une investigation inédite et complète, utilisant de multiples modèles et techniques afin de déterminer les mécanismes par lesquels Nr5a2 régule les importants processus que sont l’expansion du cumulus, l’ovulation ainsi que la formation du corps jaune.

The nuclear pore complex and its transporters : from virus-host interactors to subverting the innate antiviral immunity

Les virus ont besoin d’interagir avec des facteurs cellulaires pour se répliquer et se propager dans les cellules d’hôtes. Une étude de l'interactome des protéines du virus d'hépatite C (VHC) par Germain et al. (2014) a permis d'élucider de nouvelles interactions virus-hôte. L'étude a également démontré que la majorité des facteurs de l'hôte n'avaient pas d'effet sur la réplication du virus. Ces travaux suggèrent que la majorité des protéines ont un rôle dans d'autres processus cellulaires tel que la réponse innée antivirale et ciblées pas le virus dans des mécanismes d'évasion immune. Pour tester cette hypothèse, 132 interactant virus-hôtes ont été sélectionnés et évalués par silençage génique dans un criblage d'ARNi sur la production interferon-beta (IFNB1). Nous avons ainsi observé que les réductions de l'expression de 53 interactants virus-hôte modulent la réponse antivirale innée. Une étude dans les termes de gène d'ontologie (GO) démontre un enrichissement de ces protéines au transport nucléocytoplasmique et au complexe du pore nucléaire. De plus, les gènes associés avec ces termes (CSE1L, KPNB1, RAN, TNPO1 et XPO1) ont été caractérisé comme des interactant de la protéine NS3/4A par Germain et al. (2014), et comme des régulateurs positives de la réponse innée antivirale. Comme le VHC se réplique dans le cytoplasme, nous proposons que ces interactions à des protéines associées avec le noyau confèrent un avantage de réplication et bénéficient au virus en interférant avec des processus cellulaire tel que la réponse innée. Cette réponse innée antivirale requiert la translocation nucléaire des facteurs transcriptionnelles IRF3 et NF-κB p65 pour la production des IFNs de type I. Un essai de microscopie a été développé afin d'évaluer l’effet du silençage de 60 gènes exprimant des protéines associés au complexe du pore nucléaire et au transport nucléocytoplasmique sur la translocation d’IRF3 et NF-κB p65 par un criblage ARNi lors d’une cinétique d'infection virale. En conclusion, l’étude démontre qu’il y a plusieurs protéines qui sont impliqués dans le transport de ces facteurs transcriptionnelles pendant une infection virale et peut affecter la production IFNB1 à différents niveaux de la réponse d'immunité antivirale. L'étude aussi suggère que l'effet de ces facteurs de transport sur la réponse innée est peut être un mécanisme d'évasion par des virus comme VHC.

Hypoxia suppresses glutamate transport in astrocytes

Glutamate uptake by astrocytes is fundamentally important in the regulation of CNS function. Disruption of uptake can lead to excitotoxicity and is implicated in various neurodegenerative processes as well as a consequence of hypoxic/ischemic events. Here, we investigate the effect of hypoxia on activity and expression of the key glutamate transporters excitatory amino acid transporter 1 (EAAT1) [GLAST (glutamate-aspartate transporter)] and EAAT2 [GLT-1 (glutamate transporter 1)]. Electrogenic, Na+-dependent glutamate uptake was monitored via whole-cell patch-clamp recordings from cortical astrocytes. Under hypoxic conditions (2.5 and 1% O2 exposure for 24 h), glutamate uptake was significantly reduced, and pharmacological separation of uptake transporter subtypes suggested that the EAAT2 subtype was preferentially reduced relative to the EAAT1. This suppression was confirmed at the level of EAAT protein expression (via Western blots) and mRNA levels (via real-time PCR). These effects of hypoxia to inhibit glutamate uptake current and EAAT protein levels were not replicated by desferrioxamine, cobalt, FG0041, or FG4496, agents known to mimic effects of hypoxia mediated via the transcriptional regulator, hypoxia-inducible factor (HIF). Furthermore, the effects of hypoxia were not prevented by topotecan, which prevents HIF accumulation. In stark contrast, inhibition of nuclear factor-kappaB (NF-kappaB) with SN50 fully prevented the effects of hypoxia on glutamate uptake and EAAT expression. Our results indicate that prolonged hypoxia can suppress glutamate uptake in astrocytes and that this effect requires activation of NF-kappaB but not of HIF. Suppression of glutamate uptake via this mechanism may be an important contributory factor in hypoxic/ischemic triggered glutamate excitotoxicity.

TNF-alpha mediated transport of NF-kappaB to the nucleus is independent of the cytoskeleton-based transport system in non-neuronal cells

In unstimulated cells, proteins of the nuclear factor kappaB (NF-kappaB) transcription factor family are sequestered in the cytoplasm through interactions with IkappaB inhibitor proteins. Tumor necrosis factor alpha (TNF-alpha) activates the degradation of IkappaB-alpha and the nuclear import of cytoplasmic NF-kappaB. Nuclear localization of numerous cellular proteins is mediated by the ability of the cytoskeleton, usually microtubules, to direct their perinuclear accumulation. In a former study we have shown that activated NF-kappaB rapidly moves from distal processes in neurons towards the nucleus. The fast transport rate suggests the involvement of motor proteins in the transport of NF-kappaB. Here we address the question how NF-kappaB arrives at the nuclear membrane before import in non-neuronal cells, i.e., by diffusion alone or with the help of active transport mechanisms. Using confocal microscopy imaging and analysis of nuclear protein extracts, we show that NF-kappaB movement through the cytoplasm to the nucleus is independent of the cytoskeleton, in the three cell lines investigated here. Additionally we demonstrate that NF-kappaB p65 is not associated with the dynein/dynactin molecular motor complex. We propose that cells utilize two distinct mechanisms of NF-kappaB transport: (1) signaling via diffusion over short distances in non-neuronal cells and (2) transport via motor proteins that move along the cytoskeleton in neuronal processes where the distances between sites of NF-kappaB activation and nucleus can be vast.

Chaotic transport in reversed shear tokamaks

For tokamak models using simplified geometries and reversed shear plasma profiles, we have numerically investigated how the onset of Lagrangian chaos at the plasma edge may affect the plasma confinement in two distinct but closely related problems. Firstly, we have considered the motion of particles in drift waves in the presence of an equilibrium radial electric field with shear. We have shown that the radial particle transport caused by this motion is selective in phase space, being determined by the resonant drift waves and depending on the parameters of both the resonant waves and the electric field profile. Moreover, we have shown that an additional transport barrier may be created at the plasma edge by increasing the electric field. In the second place, we have studied escape patterns and magnetic footprints of chaotic magnetic field lines in the region near a tokamak wall, when there are resonant modes due to the action of an ergodic magnetic limiter. A non-monotonic safety factor profile has been used in the analysis of field line topology in a region of negative magnetic shear. We have observed that, if internal modes are perturbed, the distributions of field line connection lengths and magnetic footprints exhibit spatially localized escape channels. For typical physical parameters of a fusion plasma, the two Lagrangian chaotic processes considered in this work can be effective in usual conditions so as to influence plasma confinement. The reversed shear effects discussed in this work may also contribute to evaluate the transport barrier relevance in advanced confinement scenarios in future tokamak experiments.

A dedicated setup for the measurement of the electron transport parameters in gases at large electric fields

Electron transport parameters are important in several areas ranging from particle detectors to plasma-assisted processing reactors. Nevertheless, especially at high fields strengths and for complex gases, relatively few data are published. A dedicated setup has been developed to measure the electron drift velocity and the first Townsend coefficient in parallel plate geometry. An RPC-like cell has been adopted to reach high field strengths without the risk of destructive sparks. The validation data obtained with pure Nitrogen will be presented and compared to a selection of the available literature and to calculations performed with Magboltz 2 version 8.6. The new data collected in pure Isobutane will then be discussed. This is the first time the electron drift velocity in pure Isobutane is measured well into the saturation region. Good agreement is found with expectations from Magboltz. (C) 2009 Elsevier B.V. All rights reserved.

Sdo1p, the yeast orthologue of Shwachman-Bodian-Diamond syndrome protein, binds RNA and interacts with nuclear rRNA-processing factors

The Shwachman-Bodian-Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre-60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co-immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright (C) 2009 John Wiley & Sons, Ltd.

Metabolism and transport of oxazphosphorines and the clinical implications

The oxazaphosphorines including cyclophosphamide (CPA), ifosfamide (IFO), and trofosfamide represent an important group of therapeutic agents due to their substantial antitumor and immuno-modulating activity. CPA is widely used as an anticancer drug, an immunosuppressant, and for the mobilization of hematopoetic progenitor cells from the bone marrow into peripheral blood prior to bone marrow transplantation for aplastic anemia, leukemia, and other malignancies. New oxazaphosphorines derivatives have been developed in an attempt to improve selectivity and response with reduced toxicity. These derivatives include mafosfamide (NSC 345842), glufosfamide (D19575, β-D-glucosylisophosphoramide mustard), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidine). This review highlights the metabolism and transport of these oxazaphosphorines (mainly CPA and IFO, as these two oxazaphosphorine drugs are the most widely used alkylating agents) and the clinical implications. Both CPA and IFO are prodrugs that require activation by hepatic cytochrome P450 (CYP)-catalyzed 4-hydroxylation, yielding cytotoxic nitrogen mustards capable of reacting with DNA molecules to form crosslinks and lead to cell apoptosis and/or necrosis. Such prodrug activation can be enhanced within tumor cells by the CYP-based gene directed-enzyme prodrug therapy (GDEPT) approach. However, those newly synthesized oxazaphosphorine derivatives such as glufosfamide, NSC 612567 and NSC 613060, do not need hepatic activation. They are activated through other enzymatic and/or non-enzymatic pathways. For example, both NSC 612567 and NSC 613060 can be activated by plain phosphodiesterase (PDEs) in plasma and other tissues or by the high-affinity nuclear 3'-5' exonucleases associated with DNA polymerases, such as DNA polymerases and ε. The alternative CYP-catalyzed inactivation pathway by N-dechloroethylation generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA). Various aldehyde dehydrogenases (ALDHs) and glutathione S-transferases (GSTs) are involved in the detoxification of oxazaphosphorine metabolites. The metabolism of oxazaphosphorines is auto-inducible, with the activation of the orphan nuclear receptor pregnane X receptor (PXR) being the major mechanism. Oxazaphosphorine metabolism is affected by a number of factors associated with the drugs (e.g., dosage, route of administration, chirality, and drug combination) and patients (e.g., age, gender, renal and hepatic function). Several drug transporters, such as breast cancer resistance protein (BCRP), multidrug resistance associated proteins (MRP1, MRP2, and MRP4) are involved in the active uptake and efflux of parental oxazaphosphorines, their cytotoxic mustards and conjugates in hepatocytes and tumor cells. Oxazaphosphorine metabolism and transport have a major impact on pharmacokinetic variability, pharmacokinetic-pharmacodynamic relationship, toxicity, resistance, and drug interactions since the drug-metabolizing enzymes and drug transporters involved are key determinants of the pharmacokinetics and pharmacodynamics of oxazaphosphorines. A better understanding of the factors that affect the metabolism and transport of oxazaphosphorines is important for their optional use in cancer chemotherapy.

Solid state ion transport and phase behaviour in composites of N,N-methyl propylpyrrolidinium tetrafluoroborate and amorphous polyethylene oxide

The binary and ternary addition of 2 wt.% LiBF₄ and 2 wt.% amorphous polyethylene oxide (aPEO) respectively to the plastic crystal forming salt P₁₃BF₄ (where P₁₃⁺=methylpropyl pyrrolidinium cation) was investigated with specific focus on the phase behaviour and evaluation of transport characteristics. Differential scanning calorimetry (DSC), optical thermomicroscopy, solid state nuclear magnetic resonance (NMR), and AC impedance spectroscopy were used to develop an understanding of the conduction process in the pure and mixed systems. The morphology of the ternary compound appeared as hexagonal spherulites upon solidification. Multinuclear NMR Pulsed Field Gradient measurements (¹H,¹⁹F,⁷Li) to probe both cation and anion diffusion coefficients are reported. The anion is shown to be the most diffusive (at 320 K:¹⁹F=2.5×10⁻¹¹ m² s⁻¹; 1H: 1.8×10⁻¹¹ m² s⁻¹; ⁷Li: 1.1×10⁻¹¹ m² s⁻¹) in the ternary compound, with enhanced conductivity (2.7×10⁻⁵ S cm⁻¹ at 310 K) just below the melt.

Transport and phase dynamics of poly(vinyl pyrrolidone) doped plastically crystalline N-methyl-N-propylpyrrolidinium tetrafluoroborate

The plastic crystal phase forming N-methyl-N-propylpyrrolidinium tetrafluoroborate organic salt (P₁₃BF₄) was combined with 2, 5 and 10 wt.% poly(vinyl pyrrolidone) (PVP). The ternary 2 wt.% PVP/2 wt.% LiBF₄/P₁₃BF₄ was also investigated. Thermal analysis, conductivity, optical thermomicroscopy, and Nuclear Magnetic Resonance (¹¹B, ¹⁹F, ¹H, ⁷Li) were used to probe the fundamental transport processes. Both the onset of phase I and the final melting temperature were reduced with increasing additions of PVP. Conductivity in phase I was 2.6 × 10^{− 4} S cm^{− 1} 5.2 × 10^{− 4} S cm^{− 1} 1.1 × 10^{− 4} S cm^{− 1} and 3.9 × 10− 5 S cm− 1 for 0, 2, 5 and 10 wt.%PVP/P₁₃BF₄, respectively. Doping with 2 wt.% LiBF₄ increased the conductivity by up to an order of magnitude in phase II. Further additions of 2 wt.% PVP slightly reduced the conductivity, although it remained higher than for pure P₁₃BF₄.

Structure and transport properties in an N,N-substituted pyrrolidinium tetrafluoroborate plastic crystal system

The structure and transport of N-propyl-N-methylpyrrolidinium tetrafluoroborate (P₁₃BF₄) has been investigated over a wide temperature range in consequence to exhibiting properties suitable for potential solid-state superionic electrolyte applications. Prior to melting, the organic salt, P₁₃BF₄, transforms into a plastic crystal phase. Intrinsic conductivity in this solid, phase I (45–65 °C), is comparable to that in the melt (_~10⁻³ S cm⁻¹). Ionic motion and transport properties were investigated by ¹H and ¹¹B nuclear magnetic resonance (NMR) spectroscopy. Pressure-induced plastic flow in this system may accommodate volume changes in device application and to this extent, X-ray diffraction (XRD) has been used. Scanning electron microscopy (SEM) revealed complex surface morphology and lattice imperfections associated with the strong orientational disorder of the plastic state.

An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copper II

Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copper^II [Cu^II(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson’s disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from Cu^II(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from Cu^II(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with Cu^II(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a Cu^II(atsm)-induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from CuII(atsm). Using these cell culture models our data demonstrate that compromised ETC function, due to the absence of O2 as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of Cu^II(atsm) that increases cellular retention of the Cu.

Probing ion exchange in the triflic acid-guanidinium triflate system: a solid-state nuclear magnetic resonance study

Knowledge of ion exchange and transport behavior in electrolyte materials is crucial for designing and developing novel electrolytes for electrochemical device applications such as fuel cells or batteries. In the present study, we show that, upon the addition of triflic acid (HTf) to the guanidinium triflate (GTf) solid-state matrix, several orders of magnitude enhancement in the proton conductivity can be achieved. The static 1H and 19F solid-state NMR results show that the addition of HTf has no apparent effect on local molecular mobility of the GTf matrix at room temperature. At higher temperatures, however, the HTf exhibits fast ion exchange with the GTf matrix. The exchange rate, as quantified by our continuum T2 fitting analysis, increases with increasing temperature. The activation energy for the chemical exchange process was estimated to be 58.4 kJ/mol. It is anticipated that the solid-state NMR techniques used in this study may be also applied to other organic solid-state electrolyte systems to investigate their ion-exchange processes.

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.