Biochemical characterization of FANCD2 and FANCM in the Fanconi Anaemia pathway

Abstract : The maintenance of genome stability is a challenge for all living organisms. DNA is regularly subjected to chemical alterations by both endogenous and exogenous DNA damaging agents. If left unrepaired, these lesions will create mutations or lead to chromosomal instability. DNA crosslinking agents probably bring about the most toxic lesions. By linking covalently the two strands of DNA, crosslinking agents will impede essential cellular processes such as replication and transcription. Cells from Fanconi anaemia patients are extremely sensitive to these agents. Fanconi anaemia (FA) is a rare chromosomal instability disorder that leads to developmental defects, pancytopenia and cancer susceptibility. FA is a genetically heterogeneous disease with thirteen complementation groups identified. Proteins encoded by the FA genes work together in the FA pathway. Eight of these proteins form the FA core complex (FANC-A, B, C,E, F, G, L and -M), whose integrity is required to monoubiquitinate FANCD2 and FANCI in response to DNA damage. The hypersensitivity of FA cells to crosslinking agents, which perturb the progression of replication forks, has led to the hypothesis that FA proteins play a crucial role in the response to replication stress. However, at the molecular level, the functions of the FA pathway remain largely unknown. Our efforts were first focused on the characterization of FANCD2, "the key effector of the FA pathway". Using different substrates, we found that in vitro, purified hFANCD2 preferentially binds single strand DNA and double strand DNA extremities. Concomitantly, FANCM was identified as a new component of the FA core complex. Moreover FANCM was shown to have specific branch migration activities and probably a role as a "landing platform" on DNA for the other components of the core complex. By using FANCM mutants carrying deletions within the internal domain, we investigated the role of FANCM as a DNA anchor protein for the core complex. We observed that indeed, a specific part of the internal domain of FANCM interacts with components of the core complex. Finally, in collaboration with Weidong Wang's lab we characterized two new components of the FA pathway: FAAP10 and FAAP16. As a heterodimer these two proteins show affinity for dsDNA, and anneal complementary oligonucleotides in vitro. Moreover these proteins can associate with FANCM via a part of its internal domain. We find that FANCM, FAAP 10 and FAAP 16 can co-exist on the branch point of replication and recombination intermediates, and that FAAP10 and FAAP16 stimulate replication fork reversal by FANCM. These results suggest that FANCM may function as a landing platform for the core complex. After loading on DNA, the core complex can activate FANCD2 through monoubiquitination leading to its recruitment to the site of damage. Since ssDNA and double strand breaks are intermediates that are generated as a consequence of collapsed replication forks, FANCD2 by binding to ds DNA ends and ssDNA could protect such structures from the recombination repair machinery and prevent unscheduled recombination events. Alternatively, FANCD2 could avoid nucleases from gaining access to collapsed forks, preserving the DNA in state that can be used as a starting point for resumption of DNA synthesis. The overall comprehension of the FA pathway is far from been complete. Our results unravel new aspects of Fanconi Anaemia, which hopefully in the near future will address keys questions leading to a better understanding of the fascinating Fanconi Anaemia. Résumé : Le maintien de l'intégrité du génome est fondamentale chez tous les organismes vivants. L'ADN est constamment altéré par des composés aussi bien endogènes qu'exogènes. Si ces altérations ne sont pas réparées, elles peuvent conduire à l'apparition de mutations, ainsi qu'à une instabilité génomique accrue. Les lésions les plus sévères qui peuvent survenir sur l'ADN, sont les pontages inter caténaires. Des agents pontants en liant de façon covalente les deux brins d'ADN, vont empêcher le déroulement normal de processus cellulaires essentiels tels que la réplication ou la transcription. La compréhension des mécanismes permettant à la cellule de tolérer et réparer ces lésions est primordiale, notamment dans le cas des patients atteints de l'anémie de Fanconi qui présentent une très grande sensibilité à ces composés pontants. L'anémie de Fanconi est une maladie génétique rare appartenant à un groupe de pathologies associées à une grande instabilité chromosomique. Les patients atteints de l'anémie de Fanconi présentent des malformations du squelette, une pancytopénie et une forte propension à la survenue de cancer. L'anémie de Fanconi est génétiquement très hétérogène. À ce jour, 13 gènes codant pour 13 protéines FANC différentes ont été identifiés. Huit de ces protéines fonctionnent ensemble au sein d'un complexe (nommé le complexe FANC) ayant pour but de monoubiquitiner FANCD2 et FANCI en réponse à la formation de lésions sur l'ADN. L'extrême sensibilité des cellules de patients atteints de l'anémie de Fanconi à ces agents pontant l'ADN suggère l'implication des protéines FANC dans la réponse cellulaire suite à une stress réplicatif. Cependant, le rôle moléculaire exact de ces protéines demeure encore inconnu. Après purification, nous avons observé que FANCD2 était capable de lier l'ADN simple brin, ainsi que les extrémités d'ADN in vitro. Dans le même temps, FANCM fut identifié comme appartenant au complexe FANC. FANCM est décrit comme une translocase capable de promouvoir le déplacement de point de jonction dans des structures d'ADN spécifiques in vitro. De plus, en se liant à l'ADN, FANCM peut agir comme une plateforme pour les autres protéines FANC, leur permettant ainsi d'être adressées à l'ADN. En créant des protéines FANCM recombinantes ayant des délétions dans le domaine interne, nous avons pu observer que certaines protéines du complexe FANC se fixent à des sites spécifiques sur le domaine interne de FANCM. Enfin, au travers d'une collaboration, nous avons été amenés à caractériser deux nouvelles protéines appartenant au complexe FANC : FAAP 10 et FAAP16. Elles s'associent à FANCM par l'intermédiaire du domaine interne, et forment ainsi un hétérotrimére. La présence de FAAP10 et FAAP16 n'affecte pas la liaison de FANCM à l'ADN, mais semble potentialiser son activité de régression in vitro. FANCM semble donc fonctionner comme une plateforme pour les autres composants du complexe FANC. Ces derniers, une fois liés à l'ADN permettent la monoubiquitination de FANCD2 et son recrutement au site lésé de l'ADN. FANCD2 en se liant de façon préférentielle à l'ADN simple brin et aux extrémités d'ADN qui sont générés lors de l'arrêt et du démantèlement d'une fourche de réplication, pourrait protéger ces même fourches de réplication arrêtées, d'évènements de recombinaison aléatoires. Nos résultats apportent de nouveaux éléments concernant les mécanismes moléculaires de l'anémie de Fanconi. Enfin, l'étude de l'anémie de Fanconi permet aussi de mieux comprendre les mécanismes mis en place par la cellule pour tolérer des lésions survenant lors de la réplication.

Neurophysiological effects of vasopressin and oxytocin in the central amygdala of the rat : a potential mechanism for the opposite modulation of anxiety and fear behavior

Abstract The amygdala is a group of nuclei in the temporal lobe of the brain that plays a crucial role in anxiety and fear behavior. Sensory information converges in the basolateral and lateral nuclei of the amygdala, which have been the first regions in the brain where the acquisition of new (fear) memories has been associated with long term changes in synaptic transmission. These nuclei, in turn, project to the central nucleus of the amygdala. The central amygdala, through its extensive projections to numerous nuclei in the midbrain and brainstem, plays a pivotal role in the orchestration of the rapid autonomic and endocrine fear responses. In the central amygdala a large number of neuropeptides and receptors is expressed, among which high levels of vasopressin and oxytocin receptors. Local injections of these peptides into the amygdala modulate several aspects of the autonomic fear reaction. Interestingly, their effects are opposing: vasopressin tends to enhance the fear reactions, whereas oxytocin has anxiolytic effects. In order to investigate the neurophysiological mechanisms that could underlie this opposing modulation of the fear behavior, we studied the effects of vasopressin and oxytocin on the neuronal activity in an acute brain slice preparation of the rat central amygdala. We first assessed the effects of vasopressin and oxytocin on the spontaneous activity of central amygdala neurons. Extracellular single unit recordings revealed two major populations of neurons: a majority of neurons was excited by vasopressin and inhibited by oxytocin, whereas other neurons were only excited by oxytocin receptor activation. The inhibitory effect of oxytocin could be reduced by the block of GABAergic transmission, whereas the excitatory effects of vasopressin and oxytocin were not affected. In a second step we identified the cellular mechanisms for the excitatory effects of both peptides as well as the morphological and biochemical mechanisms underlying the opposing effects, by using sharp electrode recordings together with intracellular labelings. We revealed that oxytocin-excited neurons are localized in the lateral part (CeL) whereas vasopressin excited cells are found in the medial part of the central amygdala (CeM). The tracing of the neuronal morphology showed that the axon collaterals of the oxytocin-excited neurons project from the CeL, far into the CeM. Combined immunohistochemical stainings indicated that these projections are GABAergic. In the third set of experiments we investigated the synaptic interactions between the two identified cell populations. Whole-cell patch-clamp recordings in the CeM revealed that the inhibitory effect of oxytocin was caused by the massive increase of inhibitory GABAergic currents, which was induced by the activation of CeL neurons. Finally, the effects of vasopressin and oxytocin on evoked activity were investigated. We found on the one hand, that the probability of evoking action potentials in the CeM by stimulating the basolateral amygdala afferents was enhanced under vasopressin, whereas it decreased under oxytocin. On the other hand, the impact of cortical afferents stimulation on the CeL neurons was enhanced by oxytocin application. Taken together, these findings have allowed us to develop a model, in which the opposing behavioral effects of vasopressin and oxytocin are caused by a selective activation of two distinct populations of neurons in the GABAergic network of the central amygdala. Our model could help to develop new anxiolytic treatments, which modulate simultaneously both receptor systems. By acting on a GABAergic network, such treatments can further be tuned by combinations with classical benzodiazepines. Résumé: L'amygdale est un groupe de noyaux cérébraux localisés dans le lobe temporal. Elle joue un rôle essentiel dans les comportements liés à la peur et l'anxiété. L'information issue des aires sensorielles converge vers les noyaux amygdaliens latéraux et basolatéraux, qui sont les projections vers différents noyaux du tronc cérébral et de l'hypothalamus, joue un rôle clef premières régions dans lesquelles il a été démontré que l'acquisition d'une nouvelle mémoire (de peur) était associée à des changements à long terme de la transmission synaptique. Ces noyaux envoient leurs projections sur l'amygdale centrale, qui à travers ses propres dans l'orchestration des réponses autonomes et endocrines de peur. Le contrôle de l'activité neuronale dans l'amygdale centrale module fortement la réaction de peur. Ainsi, un grand nombre de neuropeptides sont spécifiquement exprimés dans l'amygdale centrale et un bon nombre d'entre eux interfère dans la réaction de peur et d'anxiété. Chez les rats, une forte concentration de récepteurs à l'ocytocine et à la vasopressine est exprimée dans le noyau central, et l'injection de ces peptides dans l'amygdale influence différents aspects de la réaction viscérale associée à la peur. Il est intéressant de constater que ces peptides exercent des effets opposés. Ainsi, la vasopressine augmente la réaction de peur alors que l'ocytocine a un effet anxiolytique. Afin d'investiguer les mécanismes neurophysiologiques responsables de ces effets opposés, nous avons étudié l'effet de la vasopressine et de l'ocytocine sur l'activité neuronale de préparations de tranches de cerveau de rats contenant entre autres de l'amygdale centrale. Tout d'abord, notre intérêt s'est porté sur les effets de ces deux neuropeptides sur l'activité spontanée dans l'amygdale centrale. Des enregistrements extracellulaires ont révélé différentes populations de neurones ; une majorité était excitée par la vasopressine et inhibée par l'ocytocine ; d'autres étaient seulement excités par l'activation du récepteur à l'ocytocine. L'effet inhibiteur de l'ocytocine a pu être réduit par l'inhibition de la transmission GABAergique, alors que ses effets excitateurs n'étaient pas affectés. Dans un deuxième temps, nous avons identifié les mécanismes cellulaires responsables de l'effet excitateur de ces deux peptides et analysé les caractéristiques morphologiques et biochimiques des neurones affectés. Des enregistrements intracellulaires ont permis de localiser les neurones excités par l'ocytocine dans la partie latérale de l'amygdale centrale (CeL), et ceux excités par la vasopressine dans sa partie médiale (CeM). Le traçage morphologique des neurones a révélé que les collatérales axonales des cellules excitées par l'ocytocine projetaient du CeL loin dans le CeM. De plus, des colorations immuno-histochimiques ont révélé que ces projections étaient GABAergiques. Dans un troisième temps, nous avons étudié les interactions synaptiques entre ces deux populations de cellules. Les enregistrements en whole-cell patch-clamp dans le CeM ont démontré que les effets inhibiteurs de l'ocytocine résultaient de l'augmentation massive des courants GABAergique résultant de l'activation des neurones dans le CeL. Finalement, les effets de l'ocytocine et de la vasopressine sur l'activité évoquée ont été étudiés. Nous avons pu montrer que la probabilité d'évoquer un potentiel d'action dans le CeM, par stimulation de l'amygdale basolatérale, était augmentée sous l'effet de la vasopressine et diminuée sous l'action de l'ocytocine. Par contre, l'impact de la stimulation des afférences corticales sur les neurones du CeL était augmenté par l'application de l'ocytocine. L'ensemble de ces résultats nous a permis de développer un modèle dans lequel les effets comportementaux opposés de la vasopressine et de l'ocytocine sont causés par une activation sélective des deux différentes populations de neurones dans un réseau GABAergique. Un tel modèle pourrait mener au développement de nouveaux traitements anxiolytiques en modulant l'activité des deux récepteurs simultanément. En agissant sur un réseau GABAergique, les effets d'un tel traitement pourraient être rendus encore plus sélectifs en association avec des benzodiazépines classiques.

Constraints on Miocene oceanography and climate in the Western and Central Paratethys: O-, Sr-, and Nd-isotope compositions of marine fish and mammal remains

The Paratethys evolved as a marginal sea during the Alpine-Himalayan orogeny in the Oligo-Miocene. Sediments from the northern Alpine Molasse Basin, the Vienna, and the Pannonian Basins located in the western and central part of the Paratethys thus provide unique information on regional changes in climate and oceanography during a period of active Alpine uplift Oxygen isotope compositions of well-preserved phosphatic fossils recovered from the sediments support deposition under sub-tropical to warm-temperate climate with water temperatures of 14 to 28 degrees C for the Miocene. delta(18)O values of fossil shark teeth are similar to those reported for other Miocene marine sections and, using the best available estimates of their biostratigraphic age, show a variation until the end of the Badenian similar to that reported for composite global record. The (87)Sr/(86)Sr isotope ratios of the fossils follow the global Miocene seawater trend, albeit with a much larger scatter. The deviations of (87)Sr/(86)Sr in the samples from the well-constrained seawater curve are interpreted as due to local input of terrestrially-derived Sr. Contribution of local sources is also reflected in the epsilon(Nd) values, consistent with input from ancient crystalline rocks (e.g., Bohemian Massif and/or Mesozoic sediments with epsilon(Nd) < -9. On the other hand, there is evidence for input from areas with Neogene volcanism as suggested by samples with elevated epsilon(Nd) values >-7. Excluding samples showing local influence on the water column, an average epsilon(Nd) value of -7.9 +/- 0.5 may be inferred for the Miocene Paratethys. This value is indistinguishable from the epsilon(Nd) value of the contemporaneous Indian Ocean, supporting a dominant role of this ocean in the Western and Central Paratethys. (C) 2008 Elsevier B.V. All rights reserved.

Reactions and enzymes in the metabolism of drugs and other xenobiotics.

In this article, we offer an overview of the compared quantitative importance of biotransformation reactions in the metabolism of drugs and other xenobiotics, based on a meta-analysis of current research interests. Also, we assess the relative significance the enzyme (super)families or categories catalysing these reactions. We put the facts unveiled by the analysis into a drug discovery context and draw some implications. The results confirm the primary role of cytochrome P450-catalysed oxidations and UDP-glucuronosyl-catalysed glucuronidations, but they also document the marked significance of several other reactions. Thus, there is a need for several drug discovery scientists to better grasp the variety of drug metabolism reactions and enzymes and their consequences.

Médecine interne et gériatrie en soins aigus: mariage de raison [General internal medicine and geriatrics in the acute care setting: opportunities for collaboration]

The health care system faces a difficult challenge as a result of the demographic evolution. The acute hospital is especially challenged by the steady increase of the elderly population. The collaboration between internal medicine and geriatrics in this setting could prove useful to facilitate the adaptation of the acute care setting and greatly enhance education of the health care professionals. Internists and geriatricians can benefit from each other skills and competencies to develop together new models of acute care that would better match the elderly population needs.

CCL21 is sufficient to mediate DC migration, maturation and function in the absence of CCL19.

Mice deficient in CCR7 signals show severe defects in lymphoid tissue architecture and immune response. These defects are due to impaired attraction of CCR7+ DC and CCR7+ T cells into the T zones of secondary lymphoid organs and altered DC maturation. It is currently unclear which CCR7 ligand mediates these processes in vivo as CCL19 and CCL21 show an overlapping expression pattern and blocking experiments have given contradictory results. In this study, we addressed this question using CCL19-deficient mice expressing various levels of CCL21. Complete deficiency of CCL19 and CCL21 but not CCL19 alone was found to be associated with abnormal frequencies and localization of DC in naïve LN. Similarly, CCL19 was not required for DC migration from the skin, full DC maturation and efficient T-cell priming. Our findings suggest that CCL21 is the critical CCR7 ligand regulating DC homeostasis and function in vivo with CCL19 being redundant for these processes.

Immunomarquage des collagènes I, III et IV, de la laminine et de la fibronectine dans la capsule cristallinienne humaine [Immunolabelling of collagen types I, III and IV, laminin and fibronectin in the human lens capsule].

PURPOSE: To localize collagen types I, III, and IV, laminin and fibronectin in the anterior human lens capsule. MATERIAL AND METHODS: Twenty-one anterior capsules were sampled by capsulorhexis during extracapsular cataract extraction (mean age 71.5). All capsules were labelled by an immunostaining specific for each antibodies. Immunostaining of four capsules was revealed with immunoperoxydase and seventeen using indirect immunofluorescence. RESULTS: Labelling of collagen types I and III was observed throughout the entire thickness of the capsule for each technique, the strongest labelling was found in the base of the epithelial cells with immunofluorescence. Collagen type IV was observed at the base of the epithelial cells whichever technique was used. Laminin could be detected in the inner layer of the capsule, using immunoperoxydase or immunofluorescence. No specific labelling was found for fibronectin using the two techniques. CONCLUSIONS: Different kinds of collagens have been found in capsules, more particularly the type III. The latter does not appear on other ocular basement membrane. Because of this uneven distribution in the capsule's thickness, each collagen might have a specific function.

Morbillivirus glycoprotein expression induces ER stress, alters Ca2+ homeostasis and results in the release of vasostatin.

Although the pathology of Morbillivirus in the central nervous system (CNS) is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV) that we inoculated into two different cell systems: a monkey cell line (Vero) and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H) markedly accumulated in the endoplasmic reticulum (ER). This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT), another ER resident chaperon critically involved in the response to misfolded proteins and in Ca(2+) homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca(2+) homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses.

Transsexualisme: enjeux et spécificités liés à la prise en charge d'une demande de réassignation sexuelle [Gender identity disorder: challenges and specificity in the treatment of requests for sexual reassignment].

Gender identity disorder is defined as a permanent desire to relieve one's own sexual features to acquire the sexual features and line to life of the opposite sex. The diagnosis is based on the psychiatric evaluation and treatment on an interdisciplinary approach by endocrinologists, surgeons and psychiatrists, and can be conceptualized into distinct phases: diagnostic evaluation, real life experience, hormonal treatment and surgery. Multiples challenges have to be faced, especially by the psychiatrist who follows the patient during the whole process.

Phylogeography and dispersal in the velvet gecko (Oedura lesueurii), and potential implications for conservation of an endangered snake (Hoplocephalus bungaroides).

ABSTRACT: BACKGROUND: To conserve critically endangered predators, we also need to conserve the prey species upon which they depend. Velvet geckos (Oedura lesueurii) are a primary prey for the endangered broad-headed snake (Hoplocephalus bungaroides), which is restricted to sandstone habitats in southeastern Australia. We sequenced the ND2 gene from 179 velvet geckos, to clarify the lizards' phylogeographic history and landscape genetics. We also analysed 260 records from a longterm (3-year) capture-mark-recapture program at three sites, to evaluate dispersal rates of geckos as a function of locality, sex and body size. RESULTS: The genetic analyses revealed three ancient lineages in the north, south and centre of the species' current range. Estimates of gene flow suggest low dispersal rates, constrained by the availability of contiguous rocky habitat. Mark-recapture records confirm that these lizards are highly sedentary, with most animals moving < 30 m from their original capture site even over multi-year periods. CONCLUSION: The low vagility of these lizards suggests that they will be slow to colonise vacant habitat patches; and hence, efforts to restore degraded habitats for broad-headed snakes may need to include translocation of lizards.