A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.     

Two antagonistic gustatory receptor neurons responding to sweet-salty and bitter taste in Drosophila

In Drosophila, gustatory receptor neurons (GRNs) occur within hair-like structures called sensilla. Most taste sensilla house four GRNs, which have been named according to their preferred sensitivity to basic stimuli: water (W cell), sugars (S cell), salt at low concentration (L1 cell), and salt at high concentration (L2 cell). Labellar taste sensilla are classified into three types, l-, s-, and i-type, according to their length and location. Of these, l- and s-type labellar sensilla possess these four cells, but most i-type sensilla house only two GRNs. In i-type sensilla, we demonstrate here that the first GRN responds to sugar and to low concentrations of salt (10-50 mM NaCl). The second GRN detects a range of bitter compounds, among which strychnine is the most potent; and also to salt at high concentrations (over 400 mM NaCl). Neither type of GRN responds to water. The detection of feeding stimulants in i-type sensilla appears to be performed by one GRN with the combined properties of S+L1 cells, while the other GRN detects feeding inhibitors in a similar manner to bitter-sensitive L2 cells on the legs. These sensilla thus house two GRNs having an antagonistic effect on behavior, suggesting that the expression of taste receptors is segregated across them accordingly.     

Duplicate sfrp1 genes in zebrafish: sfrp1a is dynamically expressed in the developing central nervous system, gut and lateral line

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.     

A pheromone to behave, a pheromone to learn: the rabbit mammary pheromone

Birth is part of a continuum and is a major developmental change. Newborns need to adapt rapidly to the environment in terms of physiology and behaviour, and ability to locate the maternal source of milk is vital. Mechanisms have evolved resulting in the emission of olfactory cues by the mother and the processing of these cues by the young. Here, we focus on some sensory, cognitive and behavioural strategies developed by the European rabbit (Oryctolagus cuniculus) that optimize the early development of offspring. In this species, chemosensory communication between the mother and young plays a critical role in eliciting adaptive neonatal responses. In particular, lactating females release a molecule, the mammary pheromone, which has several functional impacts. It triggers orocephalic responses involved in the quick localization of nipples and sucking. Moreover, this unconditioned signal promotes rapid appetitive learning of novel odorants, acting as a potent organizer of neonatal cognition. The mammary-pheromone-induced odour memory requires consolidation/reconsolidation processes to be maintained in the long term. Finally, as this mode of conditioning also promotes learning of mixtures of odorants, it supports investigations related to the capacity of neonatal olfaction to extract biological value from the complex environment.     

Design, solid-phase synthesis, and biological evaluation of novel 1,5-diarylpyrrole-3-carboxamides as carbonic anhydrase IX inhibitors

Following previous studies we herein report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 1,5-diarylpyrrole-3-carboxamides prepared by a solid-phase strategy involving a PS(HOBt) resin. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. This study revealed that the 3-position of the pyrrole was opened to the solvent, so we introduced an amino side-chain, protonated at physiological pH both to enhance the aqueous solubility and to decrease the cell membrane penetration. This strategy consisted of preparing membrane-impermeant inhibitors that may selectively target the tumor-associated hCA IX. Physico-chemical characterizations including aqueous solubility and lipophilic parameters are described. Pharmacological studies revealed high hCA IX inhibitory potency in the nanomolar range. Some compounds are selective for hCA IX displaying hCA I/hCA IX and hCA II/hCA IX ratios higher than 20 and 5, respectively.     

Enhanced Gene Silencing in Cells Cured of Persistent Virus Infection by RNA Interference

We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a natural biological phenomenon regulating a wide range of cellular pathways (8, 20). RNAi-based therapies with siRNAs or small hairpin RNAs (shRNAs) have been developed against several viral infections, and a reduction of the viral yield by several orders of magnitude has frequently been obtained (4, 9). However, virus clearance from cells and the complete cure of persistent virus infections have only rarely been reported (24, 25). We have developed several models of persistent virus infection by using poliovirus (PV), a positive-strand RNA virus of the Picornaviridae family (5, 7, 16, 21). We previously studied the effects of antiviral siRNAs applied months after the infection of HEp-2 cells with a persistent PV mutant (7, 25). We used a mixture (“the Mix”) of two synthetic siRNAs targeting the viral RNA genome in the 5′ noncoding (NC) region and the 3D polymerase (3D^pol) (siRNA-5′NC and siRNA-3D^pol, respectively; synthesized by Sigma-Proligo). When repeated transfections with the Mix were performed in persistently PV-infected cultures, most cultures stopped producing virus (25). Here, we investigate the important issue of changes in RNAi efficacy following siRNA treatment, 2 to 5 months after the cure. The efficiency of gene silencing in cells was stable during this period.We used the HEp-Q4 and -Q5 cell lines, which were cured of persistent PV infection after transfections with the Mix (25). The cured cells and their parental cell line, HEp-2, had similar growth rates (data not shown). To compare PV silencing efficiencies in the three cell lines, they were transfected either with the Mix or with an irrelevant siRNA (siRNA-IRR) in the presence of Lipofectamine 2000 (Invitrogen) in 24-well plates as previously described (25). Treated and mock-treated cells were infected 16 h posttransfection with PV strain Sabin 3, at a multiplicity of infection (MOI) of 1 50% infectious dose (ID₅₀) per cell. The viral progeny was titrated 24 h postinfection, as previously described (16). HEp-Q4 and HEp-Q5 were permissive to PV infection, although viral yields were about 1 log lower in these cells than in HEp-2 cells (Fig. ​(Fig.1A).1A). Virus silencing was observed in all three cell lines treated with the Mix; however, silencing was significantly more efficient in HEp-Q4 (≈2.2 times more efficient; P = 0.013, Student''s t test) and HEp-Q5 (≈5.6 times more efficient; P = 0.015) than in HEp-2 cells (Fig. 1A and B). Similar results were obtained with an shRNA (Thermo Scientific) targeting the same region as the siRNA-5′NC (data not shown).Open in a separate windowFIG. 1.Efficiency of enterovirus silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A) Yield of progeny virus produced by cells infected at an MOI of 1 ID₅₀, 16 h posttransfection with the antiviral Mix containing two anti-PV siRNAs (20 pmol), the irrelevant siRNA-IRR (20 pmol), or no siRNA. Samples were harvested 24 h postinfection. Each bar represents the mean value ± SEM of six infected cultures from three independent experiments. (B to E) For each cell line, silencing efficiency is expressed as the ratio of infectious virus yield (titer in ID₅₀/ml) in the presence of the irrelevant siRNA-IRR to infectious virus yield (titer in ID₅₀/ml) in the presence of the antiviral siRNAs in cured cells, normalized with respect to the silencing efficiency in HEp-2 cells. S2, PV strain Sabin 2. (F) GFP silencing efficiency for each cell line is expressed as a ratio [1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] in cured cells, normalized with respect to the efficiency of silencing in HEp-2 cells. Each bar represents the mean value ± SEM of at least four cultures from two independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.We investigated whether the differences in silencing efficacies between the three cell lines were due to differences in siRNA transfection efficiency by transfecting HEp-2, HEp-Q4, and HEp-Q5 cells with fluorescein isothiocyanate-conjugated siRNA (siRNA-FITC; 20 pmol/well; Cell Signaling) and testing them between 4 and 48 h posttransfection. The fluorescence of transfected cells was measured with a FACScan flow cytometer (Becton Dickinson), and data were analyzed with CellQuest software (Becton Dickinson). The percentages of siRNA-FITC-positive cells were similar for all cell types (Fig. ​(Fig.2A).2A). The mean fluorescence per positive cell and the percentage of cells displaying fluorescence peaked 16 and 24 h posttransfection, respectively, and decreased thereafter (Fig. ​(Fig.2).2). These findings suggest both that the presence of siRNAs in cells was similarly transient in the three cell types, as previously reported (27), and that the high silencing efficiencies in cured cells were not a consequence of higher transfection efficiencies. All subsequent experiments were performed between 16 and 40 h posttransfection.Open in a separate windowFIG. 2.Transfection efficiencies of fluorescein-conjugated siRNAs in HEp-2, HEp-Q4, and HEp-Q5 cells. A fluorescent siRNA-FITC (20 pmol) was used to transfect each of the three cell lines in the presence of Lipofectamine 2000. Fluorescent cells were analyzed 4 to 48 h posttransfection by using a FACScan flow cytometer (Becton Dickinson). The percentage of fluorescent cells (A) and the mean fluorescence per positive cell, in arbitrary units (B), are shown. Each bar represents the mean value ± SEM. (C) Representative FACS plots (cell granularity versus cell size), showing the similarities between the three cell populations.Fluorescence-activated cell sorting (FACS) plots for granularity versus cell size were very similar for the three cell lines (Fig. ​(Fig.2C),2C), as were those for cell numbers versus fluorescence (not shown), suggesting highly related cell populations. Although highly probable, it remains to be confirmed that the cured cells originated from a subpopulation of HEp-2 cells.Virus silencing was also investigated in cured cells infected with Sabin 2 or coxsackievirus A17 (CAV17) strain 67591 (22) or in cells transfected with Sabin 2 RNA. The experimental conditions used for Sabin 2 and CAV17 were identical to those for Sabin 3, except that only the 3D polymerase was targeted by siRNAs. Sabin 2 RNA (1 μg) was prepared as previously described (12) and used with siRNA-3D^pol (20 pmol/well) for the cotransfection of cells in the presence of Lipofectamine 2000. Virus yields were determined 7.5 h after transfection. In all cases, virus silencing was more effective in HEp-Q4 and -Q5 cells than in HEp-2 cells (Fig. 1C to E). Additional experiments were performed with a PV replicon encoding the green fluorescent protein (GFP), PV-eGFP (28) (2 μg/well), which was used with siRNA-eGFP (20 pmol/well; Ambion) for cotransfection. GFP fluorescence was measured by flow cytometry, 16 h after transfection. As for PV, a higher silencing efficiency was observed in cured cells than in HEp-2 cells (Fig. ​(Fig.1F1F).We then investigated whether the lower level of viral multiplication in HEp-Q4 and -Q5 cells in the absence of siRNAs involved an entry or postentry step. We quantified the expression of the PV receptor (CD155) at the surface of cells. We used flow cytometry after indirect immunofluorescence labeling with anti-CD155 antibodies, as previously described (16). More than 98.4% ± 2% (mean ± standard error of the mean [SEM]) of cured cells, like HEp-2 cells, tested positive for CD155 (data not shown). In the absence of siRNAs, a decrease in viral replication was also observed in HEp-Q4 and -Q5 cells infected with the Sabin 2 PV strain in cells, in which the early stages of the viral cycle were bypassed by transfection with Sabin 2 RNA, and in cells infected with the CAV17 virus, which uses a cell receptor other than CD155 (12) (data not shown). Together, these results suggest that PV multiplication is reduced at a postentry step, probably at replication, in cured cells.We investigated whether PV silencing was also enhanced in other HEp-derived cells in which Sabin 3 PV multiplication was reduced by using HEp-S31 (cl18) cells that had been cured of persistent PV infection by growth at a supraoptimal temperature rather than by RNAi (2). PV yield was ≈1.6 logs lower in HEp-S31 (cl18) cells than in HEp-2 cells (data not shown). Sabin 3 PV silencing in HEp-S31 (cl18) cells was 1.7 ± 0.9 times more effective (mean of six experiments) than that in HEp-2 cells (relative efficacy of 1) (data not shown), but this difference was not significant. However, these results do not exclude the possibility that reduced PV replication facilitates PV silencing by the Mix in cured cells. We therefore pursued our work with a different virus.We investigated whether the high silencing efficiency in HEp-Q4 and -Q5 cells was specific to enteroviruses by using a measles virus expressing GFP, MV-eGFP (26), and siRNA-eGFP to silence GFP expression. Cells were transfected with either siRNA-eGFP or siRNA-IRR, infected with MV-eGFP (1 ID₅₀ per cell, 16 h posttransfection), and the GFP silencing efficiency was determined 40 h posttransfection by flow cytometry. For each cell line, silencing efficiency was expressed as a percentage {[1 − (percentage of siRNA-eGFP-transfected cells expressing GFP)/(percentage of siRNA-IRR-transfected cells expressing GFP)] × 100}. GFP silencing was significantly stronger in HEp-Q4 cells (≈14%; P = 0.048) and HEp-Q5 cells (≈17%; P = 0.010) than in HEp-2 cells (Fig. ​(Fig.3A).3A). There was no significant difference in the silencing efficiency of GFP between HEp-Q4 and -Q5 cells (Fig. ​(Fig.3A).3A). The anti-PV Mix did not silence GFP expression (data not shown), indicating that the silencing of GFP was not due to anti-PV siRNAs persisting in cured cells months after the initial treatment.Open in a separate windowFIG. 3.Efficiency of GFP and luciferase silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A and B) GFP silencing, expressed as a percentage calculated for each cell line as follows: {[1 − (GFP expression in the presence of siRNA-eGFP)/(GFP expression in the presence of the irrelevant siRNA-IRR)] × 100}. (A) Cells were infected 16 h posttransfection with a measles virus encoding eGFP (MV-eGFP [26]) at an MOI of 1 ID₅₀/cell, and fluorescent cells were analyzed 24 h after infection (40 h posttransfection). Each bar represents the mean value ± SEM of three independent experiments. (B) Cells were cotransfected with pEGFP-C1 and siRNA-eGFP or siRNA-IRR and analyzed 40 h later. Each bar represents the mean value ± SEM of four independent experiments. (C) Luciferase silencing efficiency for each cell line, expressed as the ratio of luciferase activity in the presence of the irrelevant siRNA-IRR to luciferase activity in the presence of the specific siRNAs in cured cells, normalized with respect to silencing efficiency in HEp-2 cells. Relative efficiencies are shown as in Fig. ​Fig.11 for luciferase, because the enzymatic reaction amplified the signal. Each bar represents the mean value ± SEM of triplicates from three independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.To test whether the high silencing efficiency in HEp-Q4 and -Q5 cells was dependent on viral infection, plasmid vectors pEGFP-C1 (Clontech Laboratories) and pRL-CMV (Promega) were used to generate GFP (6) and Renilla luciferase (18), respectively. These plasmids do not replicate in human cells. Cells (10⁶) were cotransfected with pEGFP-C1 (1 μg) and siRNAs (20 pmol) in the presence of Lipofectamine 2000, as recommended by the manufacturer. GFP fluorescence was analyzed by flow cytometry 40 h posttransfection. Silencing efficiencies were expressed as a percentage {[1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] × 100)}. Mean silencing efficiency was significantly higher in HEp-Q4 (≈15%; P = 0.003) and HEp-Q5 (≈15%; P = 0.002) cells than in HEp-2 cells (Fig. ​(Fig.3B).3B). The efficiency with which the GFP encoded by pEGFP-C1 was silenced was similar in HEp-Q4 and -Q5 cells.The efficacy of siRNAs was then assessed with pRL-CMV, which encodes the Renilla luciferase and Silencer Renilla luciferase (AM4630; Ambion). Cells (10⁶) were cotransfected with the plasmid (100 ng) and either specific or irrelevant siRNA (7 pmol) in the presence of Lipofectamine 2000. Luciferase assays were performed with a Dual-Glo luciferase assay system (Promega), as recommended by the manufacturer at 40 h posttransfection, and luminescence was measured with a luminometer (Centro LB960; Berthold). The results of the sensitive luciferase assays confirmed that the relative efficiency of silencing was significantly higher in cured than in parental cells (Fig. ​(Fig.3C).3C). By contrast, results obtained in HEp-S31 (cl18) cells, cured without siRNAs, were not significantly different from those obtained in control HEp-2 cells (data not shown), strongly suggesting that the treatment of HEp-Q4 and -Q5 cells with specific siRNAs selected cells in which siRNAs mediated silencing more efficiently than in parental cells.The difference in silencing efficiency between cured and HEp-2 cells may be due to differences in the abundance and/or efficacy of cellular factors involved in gene silencing. Some major actors of the RNAi pathway, particularly those associated with the RNA-induced silencing complex (RISC), have been identified (3, 10, 13, 19). The active endonucleolytic core of the RISC includes the guide strand of the siRNA and a slicer protein called Argonaute 2 (Ago2) (17). We used Western blotting to study Ago-2 and other factors contributing to the function of RISC (3, 10, 11, 14, 19, 23): the endonuclease Dicer, the transactivation response RNA binding protein (TRBP), the protein activator of double-stranded RNA-dependent protein kinase (PACT), and the RNA helicase A (RHA) (Fig. ​(Fig.4).4). Exportin 5, which plays a role upstream from the dicing process in the export of small RNA precursors (29), was included as a control.Open in a separate windowFIG. 4.Comparative analysis of proteins involved in RNAi in HEp-2, HEp-Q4, and HEp-Q5 cell lines. Whole-cell lysates were tested for Exportin 5 (A), Dicer (B), Ago-2 (C), the helicase RHA (D), TRBP (E to H) and PACT (I) by Western blotting with the corresponding specific antibodies. Blots were subsequently stripped and reprobed with antiactin antibodies to confirm equal protein loading. (E and F) TRBP levels in HEp-Q4 and HEp-Q5 cells were determined by densitometry and are plotted in arbitrary units, as ratios relative to the level of actin and to the level of TRBP in HEp-2 cells. In panel F the symbols correspond to TRBP levels determined in nine different experiments. (G) TRBP levels in HEp-2 cells transfected with pcDNA-TRBP (14) and in cells cotransfected with pcDNA-TRBP and siRNA-TRBP. (H) TRBP levels were compared in human IMR5 cells, HEpS31 (cl18) cells previously cured of persistent PV infection by growth at a supraoptimal temperature, and the control HEp-2 cell line. TRBP/actin densitometry and PACT/actin densitometry results are indicated in arbitrary units in the histograms below the corresponding Western blot results shown in panels H and I.Proteins (30 to 50 μg) from each cell line were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20% Tricine gels; Invitrogen) and transferred to nitrocellulose membranes (Amersham Biosciences) as previously described (1). The membranes were incubated with one of the following primary antibodies (1): anti-Ago2 monoclonal antibody (MAb; Abcam), anti-RHA MAb (Abcam), and anti-TRBP2 MAb (Santa Cruz Biotechnology); rabbit antibodies against Dicer (Santa Cruz Biotechnology); anti-PACT MAb (Santa Cruz Biotechnology), and anti-Exportin 5 MAb (Abcam). The antiactin MAb (AC-40; Sigma-Aldrich) was used to check for equal protein loading. Membranes were then washed and treated with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 2 h at room temperature. Protein bands were detected with an enhanced chemiluminescence detection kit (ECL⁺; Amersham Biosciences) and a G:box (Syngene).Exportin 5, Dicer, Ago-2, and RHA were similarly abundant in all three cell lines (Fig. 4A to D), suggesting that quantitative differences in protein levels were unlikely to be responsible for the enhanced silencing in HEp-Q4 and -Q5 cells. There was significantly more TRBP in HEp-Q4 (≈21%; P = 0.026) and HEp-Q5 (≈28%; P = 0.016) cells than in HEp-2 cells, as indicated by the results of nine experiments (Fig. 4E and F). The specificity of the anti-TRBP antibody was checked on extracts of HEp-2 cells transfected with a plasmid encoding TRBP, pcDNA-TRBP (14), with and without silencing by siRNA-TRBP (Fig. ​(Fig.4G).4G). GFP silencing was not enhanced in HEp-2 cells overproducing TRBP, and it was not decreased by downregulating TRBP gene expression with siRNA-TRBP (data not shown). These results suggest that the high levels of TRBP in the cured cell lines are not the cause of the enhanced silencing in these cells.There was less TRBP protein in HEp-S31 (cl18) cells (2) than in HEp-2 and other control cells (IMR5) (Fig. ​(Fig.4H),4H), indicating that high levels of TRBP are not necessarily selected in cells persistently infected with PV. PACT was slightly downregulated in the cured cells (Fig. ​(Fig.4I).4I). Moreover, PACT is unlikely to be involved in the enhanced silencing in cured cells, because we used synthetic siRNAs and PACT functions principally during siRNA production by Dicer (14). We did not investigate the activities or subcellular distributions of the various factors involved in RNAi in the three cell lines, and they may differ. It is also possible that other factors, not tested here, contribute to the efficacy of siRNAs in cured cells. The molecular details of the mechanism involved remain to be determined.Overall, our results suggest that both a decrease in viral replication and the enhancement of gene silencing contributed to the mechanism by which cells persistently infected with poliovirus were cured by RNAi. Our results also indicate that cells displaying enhanced silencing may be selected during treatment with siRNAs. This may result in profound changes to cell phenotype, because RNAi plays an essential role in the regulation of cellular gene expression (15).     

Phosphoinositide 3-kinase �� regulates membrane fission of Golgi carriers for selective cytokine secretion

Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.     

The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues

In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176–183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Δ5- and Δ6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3.