High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes.

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l(-1) and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1(R) allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1(R) and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1(V) or the duplicated ace-1(D) allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.

[The pharmaceutical record in an emergency department: Assessment of its accessibility and its impact on the level of knowledge of the patient's treatment]. / Le dossier pharmaceutique dans un service d'accueil des urgences : évaluation de son accessibilité et de son impact sur le niveau de connaissance du traitement du patient.

Knowledge of the home medication list may impact therapeutic decisions made in the emergency department (ED). In France, the pharmaceutical record (PR) is a shared professional tool arising from the pharmacists lists of all drugs dispensed during the last 4 months. This PR is included in a microchip equipping a "Vitale" card detained by each beneficiary of health insurance benefits. Since 2011, the law authorises experimentally the consultation of the PR by some hospital doctors such as those working in emergency medicine. The purpose of this work is to assess the accessibility to this PR and to verify the hypothesis that its consultation increases the level of information concerning the treatment of patients admitted in an ED. A prospective, single-center, observational study was conducted during a 15-day period on all patients arriving at the Agen hospital emergency department. Of the 1046 patients enrolled in the study, 828 (79 %) presented a "Vitale" card in which a PR furnished with data was found in 45 % of the cases. The only paper source of information available was provided by the PR (25 %), a medical letter (6 %) or a prescription (3 %). A dual reconciliation between 2 of these sources was possible at a rate of about 4 % each whereas only 3 % of patients showed up with the 3 sources of available information. The consultation of PR by the ED staff is significantly possible. It improves quantitatively the level of information and thus optimizes medication assessment, the initial and critical step of the medical management of patients.

Animal models of REM dysfunctions: what they tell us about the cause of narcolepsy and RBD?

Rapid eye movement sleep behavior disorder (RBD) is a parasomnia characterized by the loss of muscle atonia during paradoxical (REM) sleep (PS). Conversely, cataplexy, one of the key symptoms of narcolepsy, is a striking sudden episode of muscle weakness triggered by emotions during wakefulness, and comparable to REM sleep atonia. The neuronal dysfunctions responsible for RBD and cataplexy are not known. In the present review, we present the most recent results on the neuronal network responsible for PS. Based on these results, we propose an updated integrated model of the mechanisms responsible for PS and explore different hypotheses explaining RBD and cataplexy. We propose that RBD is due to a specific degeneration of a subpopulation of PS-on glutamatergic neurons specifically responsible of muscle atonia, localized in the caudal pontine sublaterodorsal tegmental nucleus (SLD). Another possibility is the occurrence in RBD patients of a specific lesion of the glycinergic/GABAergic premotor-neurons localized in the medullary ventral gigantocellular reticular nucleus. Conversely, cataplexy in narcoleptics would be due to the activation during waking of the caudal PS-on SLD neurons responsible for muscle atonia. A direct or indirect pathway activated during positive emotion from the central amygdala to the SLD PS-on neurons would induce such activation. In normal conditions, the activation of SLD neurons would be blocked by the simultaneous excitation by the hypocretins of the PS-off GABAergic neurons localized in the ventrolateral periaqueductal gray and the adjacent deep mesencephalic reticular nucleus gating the activation of the PS-on SLD neurons.

Alternating vigilance states: new insights regarding neuronal networks and mechanisms.

Since the discovery of rapid eye movement (REM) sleep (also known as paradoxical sleep; PS), it is accepted that sleep is an active process. PS is characterized by EEG rhythmic activity resembling that of waking with a disappearance of muscle tone and the occurrence of REMs, in contrast to slow-wave sleep (SWS, also known as non-REM sleep) identified by the presence of delta waves. Here, we review the most recent data on the mechanisms responsible for the genesis of SWS and PS. Based on these data, we propose an updated integrated model of the mechanisms responsible for the sleep-wake cycle. This model introduces for the first time the notion that the entrance and exit of PS are induced by different mechanisms. We hypothesize that the entrance from SWS to PS is due to the intrinsic activation of PS-active GABAergic neurons localized in the posterior hypothalamus (co-containing melanin-concentrating hormone), ventrolateral periaqueductal gray and the dorsal paragigantocellular reticular nucleus. In contrast, the exit from PS is induced by the inhibition of these neurons by a PS-gating system composed of GABAergic neurons localized in the ventrolateral periaqueductal gray and just ventral to it, and waking systems such as the pontine and medullary noradrenergic neurons and the hypothalamic hypocretin neurons. Finally, we review human neurological disorders of the network responsible for sleep and propose hypotheses on the mechanisms responsible for REM behavior disorder and narcolepsy.

A presumptive developmental role for a sea urchin cyclin B splice variant.

We show that a splice variant-derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation.

Nishida procedure associated with botulinum toxin in a long-standing complete bilateral sixth cranial nerve palsy. / Procedimiento de Nishida asociado a toxina botulínica en parálisis completa del sexto par craneal bilateral de larga evolución.

In severe cases of abducens or sixth cranial nerve palsy, transpositions of the superior rectus and inferior rectus into the paralytic lateral rectus have been demonstrated to be useful. Numerous techniques have been described over time to carry out these transpositions, such as the Hummelsheim, O'Connor, Jensen, Foster, or Nishida technique. The first 4 techniques mentioned above have an increased risk of anterior segment ischaemia. The case is presented of a long-standing bilateral sixth cranial nerve palsy secondary to a severe cranial injury. Given the risk of ischaemia of the anterior segment, the Nishida technique was chosen in order to reduce the risk of suffering from this complication. This is combined with botulinum toxin in both middle rectus to try to resolve the muscle contracture associated with the long evolution of the case, obtaining good results at 6, and 12 months after the surgical procedure.

Role of the dorsal paragigantocellular reticular nucleus in paradoxical (rapid eye movement) sleep generation: a combined electrophysiological and anatomical study in the rat.

It is well known that noradrenergic locus coeruleus neurons decrease their activity during slow wave sleep and are quiescent during paradoxical sleep. It was recently proposed that their inactivation during paradoxical sleep is due to a tonic GABAergic inhibition arising from neurons located into the dorsal paragigantocellular reticular nucleus (DPGi). However, the discharge profile of DPGi neurons across the sleep-waking cycle as well as their connections with brain areas involved in paradoxical sleep regulation remain to be described. Here we show, for the first time in the unanesthetized rat that the DPGi contained a subtype of neurons with a tonic and sustained firing activation specifically during paradoxical sleep (PS-on neurons). Noteworthy, their firing rate increase anticipated for few seconds the beginning of the paradoxical sleep bout. By using anterograde tract-tracing, we further showed that the DPGi, in addition to locus coeruleus, directly projected to other areas containing wake-promoting neurons such as the serotonergic neurons of the dorsal raphe nucleus and hypocretinergic neurons of the posterior hypothalamus. Finally, the DPGi sent efferents to the ventrolateral part of the periaqueductal gray matter known to contain paradoxical sleep-suppressing neurons. Taken together, our original results suggest that the PS-on neurons of the DPGi may have their major role in simultaneous inhibitory control over the wake-promoting neurons and the permissive ventrolateral part of the periaqueductal gray matter as a means of influencing vigilance states and especially PS generation.

The satiety molecule nesfatin-1 is co-expressed with melanin concentrating hormone in tuberal hypothalamic neurons of the rat.

Overlapped in the tuberal hypothalamic area (THA), melanin-concentrating hormone (MCH) and hypocretin (Hcrt) neurons contribute to the integrated regulation of food intake, energy regulation and sleep. Recently, physiological role in appetite suppression has been defined for a novel hypothalamic molecule, nesfatin-1. Acute i.c.v. infusion of nesfatin-1 (nesf-1) promotes anorexia whereas chronic treatment reduces body weight in rats. This satiety molecule is expressed in neurons from areas prominently involved in appetite regulation including THA. We therefore sought functionally relevant to determine whether nesf-1 might be a reliable signaling marker for a new cell contingent within THA, in addition to MCH and Hcrt neurons. Thus, we completed a detailed topographical mapping of neurons immunostained for nesf-1 (nesf-1+) together with cell quantification in each discrete nucleus from THA in the rat. We further combined the immunodetection of nesf-1 with that of MCH or Hcrt to assess possible co-expression. More than three quarters of the nesf-1+ neurons were encountered in nuclei from the lateral half of THA. By double immunofluorescent staining, we showed that all neurons immunoreactive for melanin concentrating hormone (MCH+) neurons depicted nesf-1 immunoreactivity and approximately 80% of the nesf-1+ neurons were labeled for MCH. Maximal co-expression rates were observed in the lateral THA containing approximately 86% of the double-labeled neurons plotted in THA. The present data suggest that nesf-1 co-expressed in MCH neurons may play a complex role not only in food intake regulation but also in other essential integrative brain functions involving MCH signaling, ranging from autonomic regulation, stress, mood, cognition to sleep.

The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells.

BACKGROUND: Ras-mediated transformation of mammalian cells has been shown to activate multiple signalling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA have recently been directly implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, we investigated the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant-negative forms in NIH3T3 cells. RESULTS: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. CONCLUSIONS: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

Growth-regulated expression of rhoG, a new member of the ras homolog gene family.

Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.

Kinectin is a key effector of RhoG microtubule-dependent cellular activity.

RhoG is a member of the Rho family of GTPases that activates Rac1 and Cdc42 through a microtubule-dependent pathway. To gain understanding of RhoG downstream signaling, we performed a yeast two-hybrid screen from which we identified kinectin, a 156-kDa protein that binds in vitro to conventional kinesin and enhances microtubule-dependent kinesin ATPase activity. We show that RhoG(GTP) specifically interacts with the central domain of kinectin, which also contains a RhoA binding domain in its C terminus. Interaction was confirmed by coprecipitation of kinectin with active RhoG(G12V) in COS-7 cells. RhoG, kinectin, and kinesin colocalize in REF-52 and COS-7 cells, mainly in the endoplasmic reticulum but also in lysosomes. Kinectin distribution in REF-52 cells is modulated according to endogenous RhoG activity. In addition, by using injection of anti-kinectin antibodies that challenge RhoG-kinectin interaction or by blocking anti-kinesin antibodies, we show that RhoG morphogenic activity relies on kinectin interaction and kinesin activity. Finally, kinectin overexpression elicits Rac1- and Cdc42-dependent cytoskeletal effects and switches cells to a RhoA phenotype when RhoG activity is inhibited or microtubules are disrupted. The functional links among RhoG, kinectin, and kinesin are further supported by time-lapse videomicroscopy of COS-7 cells, which showed that the microtubule-dependent lysosomal transport is facilitated by RhoG activation or kinectin overexpression and is severely stemmed upon RhoG inhibition. These data establish that kinectin is a key mediator of microtubule-dependent RhoG activity and suggest that kinectin also mediates RhoG- and RhoA-dependent antagonistic pathways.

Raf-MEK-Erk cascade in anoikis is controlled by Rac1 and Cdc42 via Akt.

Signals from the extracellular matrix are essential for the survival of many cell types. Dominant-negative mutants of two members of Rho family GTPases, Rac1 and Cdc42, mimic the loss of anchorage in primary mouse fibroblasts and are potent inducers of apoptosis. This pathway of cell death requires the activation of both the p53 tumor suppressor and the extracellular signal-regulated mitogen-activated protein kinases (Erks). Here we characterize the proapoptotic Erk signal and show that it differs from the classically observed survival-promoting one by the intensity of the kinase activation. The disappearance of the GTP-bound forms of Rac1 and Cdc42 gives rise to proapoptotic, moderate activation of the Raf-MEK-Erk cascade via a signaling pathway involving the kinases phosphatidlyinositol 3-kinase and Akt. Moreover, concomitant activation of p53 and inhibition of Akt are both necessary and sufficient to signal anoikis in primary fibroblasts. Our data demonstrate that the GTPases of the Rho family control three major components of cellular signal transduction, namely, p53, Akt, and Erks, which collaborate in the induction of apoptosis due to the loss of anchorage.

Critical activities of Rac1 and Cdc42Hs in skeletal myogenesis: antagonistic effects of JNK and p38 pathways.

The Rho family of GTP-binding proteins plays a critical role in a variety of cellular processes, including cytoskeletal reorganization and activation of kinases such as p38 and C-jun N-terminal kinase (JNK) MAPKs. We report here that dominant negative forms of Rac1 and Cdc42Hs inhibit the expression of the muscle-specific genes myogenin, troponin T, and myosin heavy chain in L6 and C2 myoblasts. Such inhibition correlates with decreased p38 activity. Active RhoA, RhoG, Rac1, and Cdc42Hs also prevent myoblast-to-myotube transition but affect distinct stages: RhoG, Rac1, and Cdc42Hs inhibit the expression of all muscle-specific genes analyzed, whereas active RhoA potentiates their expression but prevents the myoblast fusion process. We further show by two different approaches that the inhibitory effects of active Rac1 and Cdc42Hs are independent of their morphogenic activities. Rather, myogenesis inhibition is mediated by the JNK pathway, which also leads to a cytoplasmic redistribution of Myf5. We propose that although Rho proteins are required for the commitment of myogenesis, they differentially influence this process, positively for RhoA and Rac1/Cdc42Hs through the activation of the SRF and p38 pathways, respectively, and negatively for Rac1/Cdc42Hs through the activation of the JNK pathway.

RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs.

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

Focal choroidal excavation: Clinical findings and complications.

OBJECTIVE: To describe the clinical findings and its complications in 2 patients with focal choroidal excavation (FCE). METHODS: A retrospective case-series including 4 eyes of 2 patients with FCE that underwent a comprehensive ophthalmological examination including slit-lamp examination, colour fundus photography, spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography (FA), and indocyanine green angiography. RESULTS: In the 2 patients, both the anterior and posterior segment evaluations were mostly normal despite the of presence yellowish spots in the macular area of the right eye of patient 1, and of a small yellowish elevated lesion with serous macular detachment in the macular area of the left eye in patient 2. At diagnosis, SD-OCT revealed a conforming FCE in patient 1, and in patient 2, an FCE with perilesional subretinal fluid and a neuroepithelium detachment, suspicious of FCE complicated with central serous retinopathy (CSCR). At one year of follow-up, patient 1 developed choroidal neovascularisation (CNV) over the focal choroidal excavation. FA and indocyanine green angiography examinations revealed areas with hypofluorescence in earlier frames, and a diffuse leakage in late frames. After ranibizumab injections, the SD-OCT of patient 1 revealed no active exudation, while patient 2 showed partial resolution of subretinal fluid. CONCLUSIONS: FCE is a newly described entity of unclear aetiology. It is characterised by a choroidal excavation in eyes, with absence of posterior staphyloma, scleral ectasia, trauma, or retinal disease. Although most lesions remain stable, there could be an association with CRSC or CNV.

Requirements for c-fos mRNA down regulation in growth stimulated murine cells.

The fos proto-oncogene is rapidly and transiently expressed in resting cells exposed to growth stimulation. This gene is down-regulated at least at two levels: transcriptional repression and mRNA degradation. To determine the sequences and the structures involved in mRNA instability, we analyzed in mouse Ltk- cells various fos/beta-globin constructs for their transcriptional activity and the half-lives of the corresponding RNAs. In these cells, rabbit beta-globin genes under the control of a 500 bp fos SRE (serum responsive element)/promoter region are transiently transcribed within 30 min after stimulation. Analysis of the decay kinetics of RNA originating from these constructs led to the following conclusions with respect to the nature of c-fos destabilizer elements: (i) 100 bases from c-fos 3' untranslated region are able to confer instability when inserted into a normally stable beta-globin RNA; (ii) however, the degradation is more rapid when the complete untranslated region is inserted; (iii) rapid mRNA breakdown requires more determinants than two AUUUA motives and is associated with a reduction in size, presumably due to a poly(A) shortening; (iv) remarkably, c-fos destabilizing sequences remain active even when part of the coding sequence.

Characterization of late response genes sequentially expressed during renewed growth of fibroblastic cells.

Many proto-oncogenes are rapidly and transiently activated during the early stages of the cellular transition from a resting G0 state to the DNA synthesis (S) phase. To get better understanding of the gene complexity involved at later stages, we isolated, by cDNA cloning, and identified 17 genes that are activated sequentially during the period of time from proto-oncogene expression to the onset of DNA synthesis in the hamster CCL39 fibroblastic cell line. When protein synthesis is inhibited, induced expression of these genes is unaffected for 10 of them, enhanced for four, in a fashion similar to the immediate-early response genes, and inhibited for three, as observed for delayed early-response genes. In addition to rhoG, a new member of the ras homolog gene family (Vincent et al., 1992), cDNA sequencing indicated that six of them correspond to cytoskeletal proteins (alpha-tubulin, vascular alpha-actin and skeletal gamma-actin), extracellular matrix protein (thrombospondin), secreted protease (plasminogen activator inhibitor-1) and energy-linked transporter (mitochondrial proton/phosphate symporter). This overall survey shows that numerous differentially regulated gene activations are associated with the cell cycle progression, and suggests that proteins involved in cellular reshaping participate actively in the control of cellular growth.

Extinction of rac1 and Cdc42Hs signalling defines a novel p53-dependent apoptotic pathway.

Apoptosis is a normal physiological process which eliminates cells that do not receive adequate extracellular signals. One of the pathways signalling apoptosis is controlled by the small GTPases of the Rho family, also involved in cell proliferation, differentiation and motility. Another major apoptosis signalling pathway involves the p53 tumour suppressor which is activated by a variety of stress and mediates growth arrest or apoptosis in normal cells. We show here that upon detachment from the extracellular matrix, fibroblasts undergo rapid apoptosis that can be rescued by constitutive activation of Rac1 and Cdc42Hs GTPases. Conversely, inhibition of Rac1 and Cdc42Hs efficiently triggers apoptosis in adherent cells. Interestingly, apoptosis is not observed in p53-/- cells either cultured in suspension or inhibited for Rac1 and Cdc42Hs activity. Moreover, Rac1 and Cdc42Hs extinction in normal cells activates endogenous p53. Using specific inhibitors of MAPK pathways, we demonstrate that, in our experimental system, p38 signals survival, while ERK activity is required for apoptosis. Our data constitute the first demonstration that Rac1 and Cdc42Hs control pathways that require simultaneous signalling through MAPK ERK and p53 to induce apoptosis.

The gene for a new brain specific RhoA exchange factor maps to the highly unstable chromosomal region 1p36.2-1p36.3.

Guanine nucleotide exchange factors from the Dbl family are proto-oncogenic proteins that activate small GTPases of the Rho family. Here we report the characterization of GEF720, a novel Dbl-like protein related to p115Rho-GEF. GEF720 activated RhoA both in our recently developed Yeast Exchange Assay and in biochemical in vitro exchange assays. GEF720 induced RhoA dependent assembly of actin stress fibers in REF52 fibroblastic cells. In NIH3T3 cells this Dbl-like protein elicited formation of transformation foci with a morphology similar to RhoA-V14 induced foci. In the PC12 neuron-like cell line, expression of GEF720, whose mRNA is brain specific, inhibited NGF-induced neurite outgrowth. Finally, GEF720 gene is located on human chromosome 1 on band 1p36, between Tumor Protein 73 and Tumor Necrosis Factor Receptor 12, two genes rearranged in many neuroblastoma cell lines. Together, these results show that this new Dbl related protein, GEF720, is an exchange factor that can directly activate RhoA in vivo and is potentially involved in the control of neuronal cell differentiation. GEF720 is also a new candidate gene involved in the progression of neuroblastoma and developmental abnormalities associated with rearrangements in the 1p36 chromosomal region.

Role and origin of the GABAergic innervation of dorsal raphe serotonergic neurons.

Extracellular electrophysiological recordings in freely moving cats have shown that serotonergic neurons from the dorsal raphe nucleus (DRN) fire tonically during wakefulness, decrease their activity during slow wave sleep (SWS), and are nearly quiescent during paradoxical sleep (PS). The mechanisms at the origin of the modulation of activity of these neurons are still unknown. Here, we show in the unanesthetized rat that the iontophoretic application of the GABA(A) antagonist bicuculline on dorsal raphe serotonergic neurons induces a tonic discharge during SWS and PS and an increase of discharge rate during quiet waking. These data strongly suggest that an increase of a GABAergic inhibitory tone present during wakefulness is responsible for the decrease of activity of the dorsal raphe serotonergic cells during slow wave and paradoxical sleep. In addition, by combining retrograde tracing with cholera toxin B subunit and glutamic acid decarboxylase immunohistochemistry, we demonstrate that the GABAergic innervation of the dorsal raphe nucleus arises from multiple distant sources and not only from interneurons as classically accepted. Among these afferents, GABAergic neurons located in the lateral preoptic area and the pontine ventral periaqueductal gray including the DRN itself could be responsible for the reduction of activity of the serotonergic neurons of the dorsal raphe nucleus during slow wave and paradoxical sleep, respectively.