Alternatively, ABCG4 may have differential mechanisms of substrate recognition and transport compared with ABCA1 and ABCG1. PrebHDL formed by ABCA1 contains relatively more PC and less SM compared with HDL formed by ABCG1. The SM-poor preb-HDL could be a good acceptor of cholesterol and SM transported by ABCG1. ABCG4, expressed in the central nervous system, would mediate the efflux of cholesterol to the PC-rich SP600125 apoE-HDL formed by ABCA1. The different substrates of ABCA1, ABCG1, and ABCG4 may be important for the production of HDL composed of various lipids. Furthermore, the contents of cholesterol, PC, and SM in HDL may be regulated by the balanced efflux mediated by ABCA1, ABCG1, and ABCG4. We have shown that lipid effluxes mediated by ABCA1 and ABCG1 are increased and impaired, respectively, by a decrease of cellular SM levels. In this study, we showed that the efflux of cholesterol mediated by ABCG4 is not affected by decreased SM levels. The distinct localizations of ABCA1, ABCG1, and ABCG4 on the plasma membrane may account for the different effects of cellular SM levels on their functions. A membrane environment of lipid rafts that are thick and liquid-ordered might be required for the function of ABCG1. Decreased SM levels disrupt raft domains, where ABCG1 resides, leading to the decreased activity of ABCG1. On the other hand, a decrease of SM level would be favorable for ABCA1 because it enlarges the area where ABCA1 functions. ABCG4 is localized to Brij 96 raft domains and partly to non-raft domains as shown in Fig. 2. This unsettled localization of ABCG4 may explain the observation that decreased SM levels had no effect on its activity. It has been reported that cellular SM content is increased in macrophages treated with acetyl-low density lipoprotein, in macrophages during differentiation from monocyte, in peritoneal macrophages during aging of rats, and in alveolar macrophages during postnatal development. Changes in cellular SM levels may be involved in regulating the function of ABCA1 and ABCG1, and in the development of atherosclerosis. Mendez et al. have reported that apoA-I removes cellular cholesterol from Triton X-100-soluble membranes, and that HDL removes cholesterol from Triton X-100-soluble and -insoluble membranes. These findings are in accord with our result that ABCA1 and ABCG1 were localized to Triton X-100-soluble and insoluble domains, respectively. We suggest that apoA-I removes cholesterol from non-raft domains, where ABCA1 resides, and that HDL removes cholesterol both from raft domains, where ABCG1 resides, and from non-raft domains, possibly by simple diffusion. Because cholesterol, newly synthesized or derived from lipoproteins like LDL, is trafficked to raft domains.

Interestingly, the synchronous synaptic release of GABA from interneurons of SLM promoted CA3 pyramidal cell activation. Thus, the reduction of Cx45 could lead to desynchronization of GABA release from the interneurons, consequently decreasing CA3 pyramidal cell activity. Therefore, downregulation of Cx45 in the SLM during the latent period could indicate a plastic homeostatic change of the coupled network in order to restore its activity after the epileptogenic insult, since interneuron networks within SLM could regulate the input from the entorhinal cortex to the hippocampus. Whether modulation of Cx45 expression in SO and SLM remains at later time points is not known, but it would be worth evaluating the issue. Interestingly, whereas changes in Cx45 distribution were seen in the latent period, alterations in Cx43 distribution through CA3 layers were observed in the acute period. Astroglial networks provide the supply of glucose and lactate necessary for the maintenance of hippocampal synaptic activity in an activitydependent manner, and it has been shown that this metabolic network of astrocytes is mediated by GJ composed by Cx43 and Cx30. Thus, the increase in Cx43 distribution pattern observed in the SP is probably due to the high energetic demand promoted by the sustained epileptic activity. Additionally, the coupled astrocytes could provide an important intercellular pathway for both delivery of metabolites to distant locations during episodes of epileptic discharges and buffering of ions such as potassium. Increase of Cx43 in SP could account for two opposite situations: besides providing distribution of metabolites of neurotransmitters and potassium to distant locations during episodes of epileptic discharges, a Cx43 increase could lead to the formation of GJ hemichannels, a pathway for adenosine release, which could also contribute to the local FG-4592 neuroprotective effects; by allowing the propagation of toxic metabolites and death signals to adjacent cells, the increase in astroglial network coupling could amplify the damage to more distant sites, in addition to the possible contribution to the rapid propagation of the electrical signal. Contrary to the observed in SP, we found decreased labeling of Cx43 in the CA3 SR. It has been demonstrated that the contribution of GJ in potassium buffering in the hippocampus is layer-dependent. By comparing the properties of astrocytes from mice with Cx30/Cx43 deficiencies, those authors found that single astrocytes from SR reach larger areas than those from SLM, indicating the unequal size and orientation of astrocytes from those areas. Additionally, they found no impairment of potassium buffering in SR of transgenic mice, in contrast to the observed in the SLM, evidencing the participation of other elements in the potassium redistribution in the SR. Therefore, the reduction of Cx43 labeling noticed here in the SR is unlikely to have a great impact in spatial buffering of potassium during SE; moreover, as CA3 SR is well supplied by a vascular source, which in turn accounts for the degree of astrocyte coupling, such decrease of Cx43 labeling in this layer during epileptic activity may not have a significant impact in the physiology of CA3 SR cells.

This could also suggest that only homologous chromosomes are replicating at the same, which could be associated with similar homologous chromosome conformation at early meiosis in wheat, as it has previously described. Finally, our results support previous findings on replication during early meiosis in wheat-rye hybrids in the presence and in the absence of the Ph1 locus, explained as an increment in the activation of origins and hence the rate of replication of disperse chromatin in wheat-rye hybrids in the absence of the Ph1 locus. In summary, flow cytometry has been revealed as a suitable tool to detect and quantify DNA replication during early meiosis in wheat. Replication was detected in wheat during premeiosis and early meiosis until the stage of pachytene, when chromosomes are associated in pairs to further recombine and correctly segregate in the gametes. Moreover, flow cytometric results suggested that the Ph1 locus is affecting the rate of replication during early meiosis in wheat, being lower in the presence of the Ph1 locus and consequently, replication during early meiosis lasts longer and finishes later than in the absence of the Ph1 locus. The biological significance of replication at early meiosis and the effect of the Ph1 locus in replication suggest a solid connection between DNA replication and chromosome associations at the onset of meiosis in a polyploid like wheat. Further studies are needed to build upon these results for unravelling the underlying molecular mechanisms. The VGF gene product, and/or its WY 14643 derived peptides, appear to be involved in reproduction since vgf null mice were sexually immature and almost completely infertile. The 66 kDa VGF precursor is composed of 617 or 615 amino acid residues, and gives rise to several low molecular weight VGF peptides which are abundant in multiple brain regions, peripheral neurones, and certain endocrine and neuroendocrine cell populations. Despite their abundance and wide distribution, limited data are available on their role and function/s. Among the VGF peptides with proven biological activity are included TLQP-21, TLQP-62 and the peptides called NERPs. TLQP-21 was shown to act on various mechanisms, including the regulation of energy balance, inflammatory and neuropathic pain, chronic stress, and gastric motility and emptying. With respect to reproduction, induction of VGF mRNA was reported in the pituitary immediately after the estrus, in parallel with a clear-cut decrease in certain VGF peptides, as well as changes in their localisation in gonadotrophs and lactotrophs. A distinct seasonal modulation in cell-type-specific processing of the VGF precursor was revealed in the anterior pituitary of female sheep, while significant upregulation of VGF mRNA was found related to reproductive maturation in baboon ovary. More recently, TLQP-21 was shown to exert a number of actions on the rat reproductive axis. Central administration of TLQP-21 in pubertal and adult male rats induced gonadotrophin secretion via release of gonadotropin-releasing hormone, and stimulated testosterone secretion in vitro in pre-pubertal animals.

We did not explore if BRM, the other ATPase subunit of SWI/SNF complexes could replace BRG1 in those complexes since these two enzymatic subunits are exclusive. It would be interesting if the BAF47 switch in the SWI/SNF and N-CoR-1 complexes could be correlated with involvement of BRM in the same complexes. From a personal communication from S. Albini it appears that BRM could play some specific roles during muscle terminal differentiation. Interestingly, N-CoR-1 has been implicated in muscle mass control and myogenesis. Since BAF47 can participate to different type of complexes, either involved in chromatin remodeling like SWI/SNF or in transcription repression like N-CoR-1, it is likely that this subunit plays a dual and complex roles in the transcriptional control. We were also interested in the role of individual SWI/SNF subunits in the irreversible cell cycle exit, since BRG1 and BAF47 are bona fide tumor suppressor genes. We have shown that these two subunits are present on cyclin D1 promoter in proliferation and upon U0126 differentiation induction. Interestingly, BrdU incorporation experiments indicate that cell cycle exit upon serum deprivation is not total for BRG1 or BAF47-depleted myoblasts, while BAF53a-depleted cells behave as control cells. For BRG1, similar results were reported by de la Serna et al.. Surprisingly, upon GM addition, only BAF47- but not BRG1depleted cells were able to re-enter cell cycle. At a molecular level, cyclin D1 is re-expressed in BAF47-depleted cells. Re-expression of cyclin D1 in the absence of BAF47 is in agreement with a previous study that showed that BAF47 is targeted to cyclin D1 promoter along with HDAC1 to repress its expression. The BAF47 effect on Cyclin D1 repression and cell cycle exit is specific and somehow not directly correlated to BAF47 requirement in myoblast differentiation since BRG1 and BAF53 do not modify permanent cell cycle exit but are, anyhow, essential in myoblast terminal differentiation. The different and complex roles played by SWI/SNF BAFs are far from being totally elucidated. The essential roles of BRG1 and BAF60c in myogenic differentiation have been already clearly demonstrated. Our present study demonstrates that BAF47 and BAF53a are also required for proper myogenic differentiation. We have shown that BAF47 could play a dual role both in permanent cell cycle exit and muscle gene transcription and it participates in different complexes involved in transcription regulation. BAF47 participation to these complexes varies upon differentiation that could represent a fine mechanism to tune transcription. We have also highlighted a specific role for BAF47 compared to BRG1 in the control of the irreversible cell cycle withdrawal, even if both of them are known as tumor suppressor genes. The specificity of BAF47 in cell proliferation control is probably the reason why this tumor suppressor is inactivated in almost all the rhabdomyosarcomas, while the BRG1 inactivation is less frequent. Cisplatin is an effective chemotherapeutic agent that is widely used against several types of solid tumors. However, its clinical use is limited by its potent nephrotoxicity.

The mechanism of RIG-I activation has been widely studied over the past few years. RIG-I preferentially recognizes 59-triphosphorylated blunt ended double-stranded RNA, but it can also bind to long double-stranded RNA without 59ppp. The recognition of an agonist RNA triggers a conformational change, allowing RIG-I to become active thanks to the release of the CARD domains. The free CARDs are then accessible for poly-ubiquitination and recruitment of the adaptor mitochondrial antiviral signal protein. The precise mechanisms of RIG-I activation are still not fully understood. It has been proposed that RIG-I-mediated activation relies on RIG-I oligomerization via dimerization of RIG-I C terminal domain, multiple oligomerization sites within RIG-I, and/or RNA-mediated oligomerization. In the present study, we question the necessity of RIG-I self-oligomerization for signal induction. RIG-I oligomerization, induced by synthetic cognate RNA able to activate RIG-I and as well as activation by measles virus, was analysed by co-immunoprecipitation and a sensitive protein complementation assay. In the absence of convincing evidence of self-oligomerization our data support monomeric RIG-I as being the minimal signal transduction unit. RIG-I oligomerization was proposed to occur during activation by a RNA ligand by two groups in 2007–2008. Since then, the observation of RIG-I oligomerization has progressively become one of the landmarks of RIG-I activation, as many prominent papers in the field tend to report data supporting this idea. However, the biochemical support remains rather poor, and the rationale enigmatic. The RIG-I oligomerization concept originated from in vitro analysis by gel filtration of a mixture of pure RIG-I protein and short 59ppp-RNA. However, a significant shift of the volume of elution observed after chromatography does not necessarily indicate a linear augmentation of mass. Indeed the shape of the molecule can influence its migration properties through the reticulated gel and a conformational change occurs when RIG-I binds an agonist RNA with the tightening of the helicase around the RNA and the release of the CARDs. RIG-I oligomerization has also been observed by band shift in Blue Native Gel electrophoresis. In addition to some reliability concerns depending on the RNA source used to activate RIG-I, a band shift indicates a molecular change and does not necessarily prove oligomerization. The migration properties of a protein can be altered by a small bound RNA that is highly negatively charged and/or by its engagement into a multimolecular complex. In contrast, size-exclusion chromatography on a S200 column coupled to multi-angle laser light scattering analysis of mixtures of pure RIG-I protein with short dsRNA was compatible only with RNA/RIG-I 1:1 monomer complexes. In agreement with our observations, RIG-I and hairpin duplexes of 10, 20 or 30 base pairs with a single 59ppp end form 1:1 complexes as analysed by analytical ultracentrifugation-sedimentation velocity. Accordingly, crystal Dabrafenib structures of RIG-I bound to short RNA shows only monomeric RIG-I:RNA complexes in a 1:1 ratio. Only when dsRNA contains two 59 triphosphate ends, could RIGI:RNA complexes be observed in a 2:1 ratio.