S147A prefers to form heterodimers with Neurogenin 2 or other bHLH partners, suggesting a molecular basis for the switch.A fundamental feature of central nervous system development is that neurons are generated before glia. In the embryonic spinal cord, for example, a group of neuroepithelial stem cells (NSCs) generates motor neurons (MNs), before switching abruptly to oligodendrocyte precursors (OLPs). We asked how transcription factor OLIG2 participates in this MN-OLP fate switch. We found that Serine 147 in the helix-loop-helix domain of OLIG2 was phosphorylated during MN production and dephosphorylated at the onset of OLP genesis. Mutating Serine 147 to Alanine (S147A) abolished MN production without preventing OLP production in transgenic mice, chicks, or cultured P19 cells. We conclude that S147 phosphorylation, possibly by protein kinase A, is required for MN but not OLP genesis and propose that dephosphorylation triggers the MN-OLP switch. Wild-type OLIG2 forms stable homodimers, whereas mutant (unphosphorylated) OLIG2 ► OLIG2(S147) dephosphorylation precedes the motor neuron (MN)-oligodendrocyte (OL) fate switch ► Mutant OLIG2(S147A) can induce OL but not MN fate ► Dephosphorylated S147 favors OLIG2-NGN2 over OLIG2-OLIG2 dimers ► This suggests a sequestration model for the MN-OL fate switch All the neurons and glial cells of the mature central nervous system (CNS) are generated by neuroepithelial stem cells (NSCs) in the ventricular zone (VZ) that surrounds the lumen of the embryonic neural tube . Cell diversification occurs in stages. First, a neurogenic prepattern is laid down in the plane of the VZ, under the action of graded morphogens released from organizing centers within or outside the neural tube. This leads to a mosaic of molecularly distinct progenitor domains, each of which goes on to generate a characteristic subset of neurons and glia. Superimposed on this spatial pattern is a temporal pattern of cell generation from some regions of the VZ. For example, in the developing cerebral cortex, different classes of projection neuron are generated in sequence ; these sSpatial pattern in the ventral half of the developing spinal cord is established largely through the action of Sonic hedgehog (SHH) protein released from the notochord and floor plate at the ventral midline. SHH activates or inhibits different sets of transcription factors at different distances from the floor plate (different concentrations of SHH). Subsequently, cross-repressive interactions among the transcription factors expressed in adjacent regions of the VZ establish sharp boundaries of gene expression in the dorsal-ventral axis, establishing a set of ribbon-like NSC domains that run parallel to one another along the neuraxis. In the ventral half of the cord, these domains are known  as p3, pMN, p2, p1, and p0 . Six addThe pMN domain contributes all of the MNs and ∼80% of the OLPs in the mouse spinal cord . The remThe OLIG gene products are members of a large family of helix-loop-helix (HLH) transcription factors, which also includes proneural proteins Neurogenin1/2 (NGN1/2) and MASH1/ASCL1 as well as cell lineage regulators MYOD and NEUROD. OLIG2 interacts with different protein partners to regulate specific developmental processes. It can form heterodimers with NGN2 to control MN differentiation, and it can bind NKX2.2 to promote OLP generation and/or differentiation . It can Given the central role of OLIG2 in both MN and OL development, we were keen to discover how this one transcription factor can specify two quite different cell types and especially how it participates in the MN-OLP temporal fate switch. We present evidence that OLIG2 controls the switch by reversible phosphorylation on Serine 147 (S147), a predicted protein kinase A (PKA) target; phosphorylation at this site is required for patterning of the ventral neuroepithelium and MN specification, whereas dephosphorylation favors OLP specification. S147 phosphorylation also causes OLIG2 to switch its preferred dimerization partner from OLIG2 (or OLIG1) to NGN2. We propose that this regulated exchange of cofactors is required for and triggers the MN-OLP fate switch.33P]phosphate, and analyzed radiolabeled OLIG2 proteins by immunoprecipitation (IP) with anti-Myc followed by polyacrylamide gel electrophoresis (PAGE). Two major radioactive OLIG2 bands were visible  PAGE (pH gradient in the second dimension) followed by Western blotting (WB) confirmed that OLIG2 was phosphorylated on multiple sites and that most of the phosphates could be removed by calf intestinal alkaline phosphatase (CIAP) (S147AOlig2) and wild-type (WTOlig2) constructs into Cos-7 cells, and visualized OLIG2 proteins by 2D PAGE and WB as before. This revealed an altered phosphorylation pattern of OLIG2S147A (WTOlig2 with a plasmid encoding a dominant-negative form of PKA (dnPKA) strongly reduced the phosphoprotein signal in the low pH range of the 2D gel (the region affected by S147A mutation), implying that OLIG2 can be phosphorylated by PKA on several sites, including S147  C and 1F.e (CIAP)  and NetPe (CIAP)  identifie (CIAP) . Alignmee (CIAP) B. This sIG2S147A D, indicaing S147 E.ph-S147; ph-S147. OLIG2 was expressed more or less equally at all stages examined . This sort of effect is not unique to OLIG2. For example, phosphorylation/dephosphorylation of serine/threonine residues of the bHLH transcription factor HAND1 have been shown to regulate homodimer versus heterodimer formation, phosphorylation favoring heterodimers of HAND1 and E proteins and dephosphorylation favoring HAND1 homodimers (We tried to mimic constitutive phosphorylation by mutating S147 to glutamic acid (E) or aspartic acid (D). However, both OLIG2modimers . Howevermodimers .WT and OLIG2S147A using a mammalian two-hybrid system   B–2E. Thi1 target B and 2C.enhanced D. Furtheenhanced B–2E. In WT and OLIG2S147A by electrophoretic mobility shift assay (EMSA). OLIG2S147A exhibited significantly weaker binding to the HB9/M100 E box , introducing the S147A mutation while simultaneously adding a V5 epitope tag to the C terminus . For some experiments we also bred the PAC transgenics with Olig1/Olig2 double-null mice (Olig2 (see below).In order to study the phenotypic consequences of OLIG2-S147 phosphorylation in vivo, we generated terminus A. Transger study B. Immunoth lines C and 3D.ckground , therebyull mice , which eIrx3 and Pax6 ; in the absence of OLIG2 function, Irx3 and Pax6 are derepressed in pMN, which takes on the character of p2, generating V2 interneurons and astrocytes instead of MNs and OLPs  were observed to adopt a p2 fate (high PAX6 expression)  . These f−/−Olig2 embryos as a consequence of losing the pMN progenitor domain  stage12–14 can induce ectopic production of HB9-positive MNs by 48 hr post-electroporation, mainly in the p2 domain  Myelin basic protein (MBP)-positive OLs formed in these mutant cell cultures as in wild-type cultures, demonstrating that S147 phosphorylation is not absolutely required for OL lineage progression -derived cell line can differentiate into different cell lineages under appropriate inductive conditions . For exaWT or OLIG2S147A. Without RA/SHHAg1.2 induction, both cell lines grew and behaved like the parent P19 line. With RA/SHHAg1.2 induction, the P19-OLIG2WT line produced significantly increased numbers of HB9-positive cells (p < 0.001) and NG2-positive cells (p < 0.05) compared to induced P19 control cells (WT can enhance the output of MNs and OL lineage cells from ES cells (S147A cultures developed a decreased number of HB9-positive cells (p < 0.05) compared with control P19 cells (S147A cells induced with RA/SHHAg1.2 generated many more NG2-positive cells compared to induced P19-OLIG2WT (p < 0.001) or parental P19 (p < 0.001) lines  lines F–6J. The1) lines I and MBP1) lines . These dWe have shown that phosphorylation of OLIG2 on S147 is required for the early functions of OLIG2 in neuroepithelial patterning and MN specification but is subsequently dispensable for OLP specification. We have also shown that S147 is phosphorylated during MN specification in the ventral spinal cord, dephosphorylated at the onset of OLP production, and that that dephosphorylation switches the binding preference of OLIG2 away from OLIG1/2 toward NGN2. We believe that this represents a key part of the regulatory mechanism that operates through OLIG2 to switch NSC fate from MNs to OLPs during ventral spinal cord development. Other phosphorylation events might also be important for the functional regulation of OLIG2, e.g., it was recently shown that casein kinase 2 (CK2)-mediated phosphorylation is required for the oligodendrogenic activity of OLIG2 . Serine S147AOlig2 mice, the pMN domain was transformed mainly to p2, and consequently, MN development was blocked. This does not reflect a global loss of OLIG2 function because expression studies in Cos-7 cells demonstrated that OLIG2S147A is a stable protein that is indistinguishable from OLIG2WT by mobility on sodium dodecyl sulfate (SDS)-PAGE, subcellular localization, or its ability to bind known transcriptional partners such as SOX10 or NKX2.2. Most importantly, OLIG2S147A did not lose its ability to specify OL lineage cells, although fewer OLPs than normal developed in the spinal cords of S147AOlig2 mice, and these were delayed, appearing at E15.5–17.5 instead of E12.5 as in wild-type cord. This fits with the fact that the pMN progenitor domain, which normally produces ∼80% of all OLPs in the cord, is lost in the mutant. The remaining ∼20% of OLPs are produced from more dorsal progenitor domains, which do not depend on the neuroepithelial patterning function of OLIG2  stably transfected with an S147AOlig2 expression vector generated many more OL lineage cells—both NG2+ OLPs and MBP+ OLs—and less HB9+ MNs than did P19 cells stably transfected with WTOlig2. The MBP+ cells that formed in these experiments did not adopt the multiprocess-bearing morphology typical of normal OLs in culture, so other factors in addition to OLIG2 are presumably required for full OL differentiation of P19 cells.Our data reveal that gain or loss of a phosphate group on OLIG2-S147 goes hand in hand with MN or OL generation, respectively. In our of OLIG2 . These datsoever . It is vS147A has an enhanced ability to bind NGN2, coupled with a diminished ability to form dimers with itself or OLIG1. Consistent with this, we found that OLIG2S147A inhibits NGN2-mediated transcriptional activation of the HB9 promoter more efficiently than does OLIG2WT, in cotransfection assays with an HB9:luciferase reporter (We found that OLIG2reporter . NGN2 isreporter . OLIG2 areporter . There ireporter . NGN2 exreporter . HoweverS147A:OLIG2+/− mice (data not shown) or in OLIG2S147A-electroporated chick , homodimers of S147-phosphorylated OLIG2 act to repress OL lineage genes in pMN and create a permissive environment for MN development—in which NGN2 plays an important role in concert with homeodomain transcription factors ISL1/2 and LHX3 . Subsequed chick L, which Sox10-U2 promoter in the OL lineage , OLP formation in the ventral spinal cord is disrupted . It willOur study focused on reversible phosphorylation of OLIG2-S147, an evolutionarily conserved site that is predicted to be a PKA target. We provided evidence that OLIG2-S147 can be phosphorylated by PKA in cultured Cos-7 cells and that dnPKA inhibits phosphorylation. Despite this, we cannot conclude that PKA is the only—or even the primary—protein serine kinase to target S147 in vivo. For example, the consensus target sequence for PKA (R[R/K]X[S/T]) can overlap with that of PKC ([R/K]X[S/T]U[R/K]) . Indeed, PKA and PKC are thought to phosphorylate common acceptor sites on the bHLH proteins HAND1 and HAND2 .S147AOlig2 mice failed to develop MNs suggests that PKA might normally play a positive role in MN development. This seems counter to the popular idea that PKA negatively regulates the SHH pathway (Drosophila Cubitus interruptus (Ci). SHH activates GLI3 (and GLI2) by inducing its proteolytic conversion from a full-length transcriptional activator into a truncated N-terminal repressor  coreceptor, promotes SMO accumulation on the primary cilium and triggers HH pathway activation , a cell-permeable analog of cAMP, and found that the number of NG2-positive OLPs was significantly decreased compared to untreated cultures . This isOur data raise the obvious question: What is the key event that signals S147 dephosphorylation and triggers the MN-OLP switch in pMN? Notch signaling is known to play an important role in glial cell development in the CNS . ConstitOlig1, Olig2, Sox10, Ngn2, Mash1, and Nkx2.2 were amplified by RT-PCR from RNA extracted from mouse embryonic spinal cord tissue and subcloned into Invitrogen's pCDNA3.1-V5 and/or pCDNA4-myc vectors, or Promega's pBIND and/or pACT vectors (for two-hybrid assays). Catalytic PKA and dnPKA vectors were kindly provided by Dr. Judy Varner . S147A mutant vectors were generated using Stratagene's QuikChange Site-Directed Mutagenesis Kit following the manufacturer's instructions. The HB9- luciferase reporter plasmid HB9:luc -KIA (residues 137–150 of OLIG2). Rabbits were immunized four times with peptide, and antiserum was collected 25 days after the last injection. The antiserum was first absorbed on a column containing nonphosphorylated peptide and then affinity purified on a phosphopeptide column. The purified antibody was used in WB at a dilution of 1:250. Primary antibodies used for immunofluorescence labeling were: goat anti-OLIG2 ; rabbit anti-OLIG2 ; chick anti-GFP ; rabbit anti-V5 ; rabbit anti-Pax6 ; mouse anti-Nkx2.2 ; mouse anti-HB9 ; guinea pig anti-SOX10 ; and rat anti-PDGFRa . Secondary antibodies for WB were bought from Thermo Pierce and used at 1:20,000. Alexa Fluor secondary antibodies were from Invitrogen (used at 1:750 dilution). The in situ hybridization probes for mouse Sox10 and Pdgfra were described previously ; rabbit anti-OLIG2 ; mouse anti-Myc ; rabbit anti-Myc ; and mouse anti-V5 . The specific anti-OLIG2 eviously . The chiOlig2 KO line was obtained from Charles Stiles  and David Rowitch   was provided by David Anderson   and WTOlig2 (WT:Olig2−/−Olig2) offspring for analysis. For certain experiments  . The Olihnology) . To crea E. coli . The 5′-+/− mice  to obtaits e.g., , we crosg2 locus , to obta2. Plasmid transfection was performed using Lipofectamine 2000 reagent (Invitrogen). Total DNA concentrations were normalized with empty vector DNA where required. The P19 EC-derived cell line was purchased from LGC-ATCC and maintained in Alpha minimal essential medium with ribonucleosides and deoxyribonucleosides, supplemented with 5% fetal bovine serum (Invitrogen) at 37°C with 5% CO2. For differentiation assays, 5 × 104 P19 cells were plated on 35 mm diameter plates in medium with 1% serum. After 5–6 days, the aggregated cells were treated with 1 μM RA and 100 nM SHHAg1.2 . Cells were fixed for immunolabeling after two more days in culture.Cos-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) at 37°C with 5% (v/v) COCultured cells were lysed by homogenization in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and one tablet of protease inhibitor mix per 50 ml buffer. Lysis buffer for spinal cord tissue was purchased from Sigma (CelLytic MT); sometimes phosphatase inhibitors were included. After lysis, cell debris was removed by centrifugation at 30,000 × g, then 500 μl cell lysate was treated with 50 U of DNase1 and precleared with 30 μl of protein G beads  for 3 hr at 4°C on a rotating wheel. The supernatant was decanted, incubated with the antibody of interest at 4°C for 1 hr, mixed with 30 μl of protein G beads, and incubated overnight at 4°C. The beads were washed twice in lysis buffer, twice in high-salt buffer  and once in low-salt buffer . The beads were resuspended in 30 μl of SDS gel sample buffer, boiled for 5 min, and subjected to SDS-PAGE followed by WB. Band intensity was quantified using ImageJ software. For 2D PAGE the beads were treated with Invitrogen ZOOM Protein Solubilizer, and protein samples were separated on the Invitrogen ZOOM 2D gel system following the manufacturer's instructions. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes. Protein bands were visualized by chemiluminescence .33P]orthophosphate (Amersham) were added to phosphate-free medium, and cells incubated for 4 hr at 37°C. The medium was then removed, and the cells washed twice with ice-cold PBS and immediately lysed on ice (see above). Proteins were immunoprecipitated, separated by SDS-PAGE, and visualized by autoradiography.Thirty-six hours after transfection, Cos-7 cells were washed twice with phosphate-free DMEM (Invitrogen). Thirty mega-becquerels [Renilla Luciferase activity. Three independent transfections were conducted in parallel for each condition, and each experiment was repeated three times.Two-hybrid assays for protein-protein interactions were performed using Dual Luciferase Assay System (Promega). The amounts of transfected DNAs were normalized with empty pCDNA vector. The measured firefly luciferase activity was normalized against Fertilized chicken eggs were supplied by Henry Stuart Inc. and incubated at 38°C in a humidified atmosphere. The embryos were staged according to HH and electroporated at HH12–14 . Expresshttp://www.ucl.ac.uk/∼ucbzwdr/Richardson.htm.Mouse and chick embryos were dissected in cold PBS and fixed in 4% (w/v) paraformaldehyde (PFA) in PBS for 1 hr at 20°C or overnight at 4°C (for SOX10 immunolabeling). The tissues were cryoprotected with 20% (w/v) sucrose in PBS, embedded in OCT, and frozen for cryosectioning. Tissue sections (15 μm) were permeabilized and preblocked in 0.1% (v/v) Triton X-100, 2% (v/v) calf serum in PBS for 1 hr at 20°C, then incubated in primary antibodies diluted in 2% calf serum in PBS overnight at 4°C followed by secondary antibodies at 20°C for 1 hr. Sections were counterstained with Hoescht 33258  to visualize cell nuclei. For RNA in situ hybridization, see


Nucella lapillus by reciprocal transplantation of snails between two shores differing in wave action and rearing snails of the same provenance in a common garden. Results were compared with those reported for similar experiments conducted elsewhere. Microsatellite variation indicated limited gene flow between the populations. Intrinsic growth rate was greater in exposed-site than sheltered-site snails, but the reverse was true of absolute growth rate, suggesting heritable compensation for reduced foraging opportunity at the exposed site. Shell morphology of reciprocal transplants partially converged through plasticity toward that of native snails. Shell morphology of F2s in the common garden partially retained characteristics of the P-generation, suggesting genetic control. A maternal effect was revealed by greater resemblance of F1s than F2s to the P-generation. The observed synergistic effects of plastic, maternal and genetic control of shell-shape may be expected to maximise fitness when environmental characteristics become unpredictable through dispersal.Assessment of plastic and heritable components of phenotypic variation is crucial for understanding the evolution of adaptive character traits in heterogeneous environments. We assessed the above in relation to adaptive shell morphology of the rocky intertidal snail  The relative contributions of plasticity and inheritance to phenotypic expression are crucial quantities for understanding the evolution of adaptive character traits in spatially and temporally variable habitats Nucella lapillus (L.) have been studied in relation to contrasting selection regimes associated with high and low wave exposure N. lapillus is a predatory snail with limited dispersal ability owing to direct development and a restricted crawling range N. lapillus. The shell of N. lapillus is more globular at sites exposed to wave action and more elongated at sheltered sites Littorina saxatilisN. lapillus.Aquatic snails have become popular models for investigating environmental and heritable components of phenotypic expression because they are easily reared in the laboratory, readily transplanted between habitats in the field, and have measurable phenotypic characters whose variation is closely correlated with physical and biological environmental factors critical to survival Nucella lapillus from sites differing in exposure to wave action. Snails from the more exposed site had a relatively larger foot, affording greater adhesion, than those from the more sheltered. Progeny of exposed- and sheltered-site snails developed a similar relative foot size when reared under calm-water conditions in the laboratory, indicating plastic rather than heritable phenotypic variation. Reciprocal transplantation of progeny between sites, however, revealed strong asymmetry in the response to environment. Progeny from the sheltered site transplanted to the exposed came to resemble the native snails by developing a relatively much larger foot  than controls replanted on the sheltered site. Progeny transplanted from the exposed to the sheltered site, on the other hand, developed a pedal area only slightly smaller than that of controls. Trussell Littorina obtusata, again finding asymmetric plasticity with sheltered-site snails developing a relatively larger foot in response to exposure to wave action, but not vice versa.Etter Etter N. lapillus, other studies have linked phenotypic variation (shell shape) to genotype In contrast to Etter's N. lapillus, we performed reciprocal-transplant and common-garden experiments, complemented by population genetic analysis, on an exposed- and a sheltered-site population free of karyotype polymorphism. While substantially corroborating previous studies of N. lapillus, our data also reveal new insights on the plastic and heritable components of phenotypic variation of this species.To further assess the symmetry of plasticity and heritable variation in adaptive morphology of N. lapillus , they were labelled with a waterproof pen. Labels were covered with superglue (Loctite™) to protect against abrasion and the marked juveniles were released as summarized in Each of the above treatments was given a unique colour-code. On release, individuals were gently irrigated with seawater to encourage them to emerge from their shells and grip the substratum. In early November 2008, marked animals were recaptured (two visits to each site), photographed as described below, re-marked and returned to the field. In September 2009 the experiment was completed by returning marked individuals to the laboratory where they were photographed.Nucella lapillus juveniles (≤12 mm shell length) were collected from each shore . Each experiment incorporated a duplicated common garden lacking the effects of wave exposure typical of the exposed field site and of crab predation typical of the sheltered field site. Quantitative comparison of traits shown by successive generations would potentially distinguish plastic, maternal and genetic components of variation.Experiments were run for F1 generation) were apportioned between two tanks: 25 of sheltered site ancestry and 90 of exposed site ancestry per tank . Seawater was supplied via two constant-head cisterns at a rate of 3 ml s−1 with permanent aeration supplied from air-diffusion stones. Ambient temperature fluctuated seasonally between 8–16°C. To avoid position effects, tanks were transposed each month. Subjects were photographed, as below, after 6 months and again after 12 months from the beginning of the experiment. Since eggs were laid in both tanks, opportunity was taken to continue the experiment through the F2 generation, retrospectively assigning parentage by genetic analysis (below).Adults from the exposed and the sheltered site were collected in February 2008 and allowed to spawn separately in aquaria as for the reciprocal transplant experiment (above). Having grown large enough for marking (≤12 mm shell length), hatched juveniles  and 16SNucRV (5′-CTCAGTCGGCCCAACTAAAA-3′) . PCR amplifications were carried out in 25 µl reactions containing 1 µl of cDNA, 0.3 pmol of each primer, 1× PCR Buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP (Promega) and 0.5 U Taq DNA polymerase (Promega) on an Biorad DNA engineTetrad2 Thermal cycler. An initial denaturation step of 5 min at 95°C was followed by 35 cycles at 95°C for 1 min, 53°C for 1 min, and 72°C for 1 min followed by a final extension at 72°C for 10 min. PCR results for both genes were sequenced using the Macrogen™ (www.macrogen.com) sequencing facility and subsequently aligned and compared using the software Bioedit Chromosome counting was complemented by comparison between populations of mitochondrial (16S) and nuclear (mMDH) genes, which vary in association with karyotypic and phenotypic polymorphism, in turn correlated with environmental variables such as wave exposure N. lapillus from Cable Bay (N = 96) and Llanfairfechan (N = 96) (0) and expected heterozygosity (HE) under Hardy-Weinberg assumptions, estimates of STF, ISF, and their significance per population over all loci were calculated according to Weir and Cockerham Microsatellites were used to assess gene flow between the two populations, separated by approximately 45 km or 90 km of coastline depending on dispersal route. Adult (N = 96)  were col1, n = 112) and offspring  were genotyped as described above, using seven microsatellite markers . Parental analysis was performed using CERVUS Potential parents of known gender . Shell growth at 3 months after initial release was ranked ES>EE>SS>SE , scores were standardized as residuals from linear regression. All paired comparisons  were sta1 parents comprised 36 males and 54 females of exposed-site ancestry and 10 males and 14 females of sheltered-site ancestry. Of the F2 progeny, 112 snails survived for 12 mo and genotyping unequivocally assigned 85 of these to known parentage, 5 having sheltered-site ancestry, 52 exposed-site ancestry and 28 mixed ancestry. The P generation was represented by controls from the reciprocal transplant experiment.F1 generations but not in the F2 generation except by planned comparison  and exposed-site snails (n = 51) differed on RW1 but not on RW2 or RW3, although difference on the latter was marginally non-significant . For the F2 generation, RWs 1–3 accounted for 30%, 20% and 11% of variance respectively. RW1 was correlated with centroid size and therefore corrected scores were used for subsequent analysis. Uncorrected scores were used for RW2 and RW3. Difference in mean score between sheltered-site (n = 5) and exposed-site snails (n = 27) was marginally non-significant on RW1 and RW2, and non-significant on RW3 .To gain resolution within generations, GM analyses were run separately for the full FE) per locus ranged from 0.332 to 0.892 and the observed heterozygosity (HO) from 0.313 to 0.917 , revealing significant structuring between the two sampling sites . There was therefore no evidence of karyotype polymorphism. All microsatellite loci were polymorphic for both populations. The number of alleles per population per locus ranged from 2 to 19, with a total number of 108 alleles in the global sample. The expected heterozygosity .Nucella lapillus is reported to have higher size-specific somatic growth rate but similar size-specific shell-growth rate at exposed sites than at sheltered Littorina obtusata, in which snails from exposed sites grew faster than those from sheltered under laboratory conditions of low flow velocity L. saxatilis where snails transplanted from high to low shore grew faster than low-shore residents Nucella spp. observed at exposed sites  persisted into the Fite loci  coincideite loci , S5 suggite loci  is probaN. lapillus aggregate in protective microhabitats in order to mate and spawn and although lacking specific homing behaviour they tend to use a restricted number of spawning sites distributed within a radius of up to about10 m that also encompasses their foraging range  and recaptured in November 2008; Jul-09: percentage of snails released at the beginning of the experiment and recaptured at the end of the experiment in July 2009; Jul-09b = percentage snails re-released in November 2008 and recaptured at the end of the experiment. E: exposed; S:sheltered; f: juveniles collected from the field; l: laboratory-hatched juveniles; fSS ; fES ; fEE ; fSE ; lSS ; lES ; lEE ; lSE .(TIF)Click here for additional data file.Figure S2Reciprocal transplant experiment: extreme deformation grids – Rw1/Rw2 (top), Rw2/Rw3 (bottom).(TIF)Click here for additional data file.Figure S3Location of study sites along the North Wales coastline. CB = Cable Bay , Thomas Exposure Index (TEI) = 14; CAE = Caethle , TEI = 15; FB = Friars Bay , TEI = 3; LL = Llanfairfechan , TEI = 2; MB = Menai Bridge , TEI = 0; RP = Ravens Point , TEI = 14; RWB = Redwharf Bay , TEI = 8.(TIF)Click here for additional data file.Figure S4Experiment 1: ontogenetic changes in shell morphology. Mean aperture external width adjusted to shell length (ANCOVA). For each size class, the first group represents snails reared in the laboratory and the second group snails collected from the ancestral field-population. Sample size (N) and covariate value (CV) for adjusting mean aperture width were as follows. Group 1: N exposed 1 = 10, N exposed 2 = 15, N sheltered 1 = 11, N sheltered 2 = 12; CV = 2.264 mm. Group 2: N exposed 1 = 11, N exposed 2 = 12, N sheltered 1 = 15, N sheltered 2 = 15; CV = 2.726 mm. Group 3: N exposed 1 = 13, N exposed 2 = 14, N sheltered 1 = 12, N sheltered 2 = 6; CV = 7.915 mm. Group 4: N exposed 1 = 15, N exposed 2 = 15, N sheltered 1 = 15, N sheltered 2 = 15; CV = 7.670 mm. Group 5: N exposed 1 = 17, N exposed 2 = 17, N sheltered 1 = 15, N sheltered 2 = 18; CV = 12.331 mm. Group 6: N exposed 1 = 15, N exposed 2 = 15, N sheltered 1 = 15, N sheltered 2 = 15; CV = 12.753 mm. Group 7: N exposed 1 = 35, N exposed 2 = 30, N sheltered 1 = 28, N sheltered 2 = 22; CV = 18.066 mm. Group 8: N exposed 1 = 39, N exposed 2 = 30, N sheltered 1 = 30, N sheltered 2 = 30; CV = 16.872 mm.(TIF)Click here for additional data file.Figure S5Comparison of relative aperture width of adult laboratory-reared and field-collected N. lapillus from shores differing in exposure to wave action. Adjusted means (see text) are shown with standard errors. Shores are ranked in increasing order of wave exposure (see sure see : Menai B(TIF)Click here for additional data file.Data S1Supplementary data. Experiment 1: ontogenetic changes in shell morphology; Experiment 2: wave-exposure gradient.(DOC)Click here for additional data file.


Not only does MK-STYXactive induce stress granules, but also it has the capacity to dephosphorylate G3BP-1. Taken together, these results provide evidence that the pseudophosphatase MK-STYX plays a key role in the cellular response to stress.The pseudophosphatase MK-STYX (mitogen-activated protein kinase phosphoserine/threonine/tyrosine-binding protein) has been implicated in the stress response pathway. The expression of MK-STYX inhibits the assembly of stress granules, which are cytoplasmic storage sites for mRNA that form as a protective mechanism against stressors such as heat shock, UV irradiation and hypoxia. Furthermore, MK-STYX interacts with a key component of stress granules: G3BP-1 (Ras-GTPase activating protein SH3 domain binding protein-1). Because G3BP-1 dephosphorylation at Ser149 induces stress granule assembly, we initially hypothesized that the inhibition of stress granules by MK-STYX was G3BP-1 phosphorylation-dependent. However, in the present study, using MK-STYX constructs and G3BP-1 phosphomimetic or nonphosphorylatable mutants, we show that MK-STYX inhibits stress granule formation independently of G3BP-1 phosphorylation at Ser149. The introduction of point mutations at the ‘active site’ of MK-STYX that convert serine and phenylalanine to histidine and cysteine, respectively, is sufficient to generate an active enzyme. In separate experiments, we show that this active mutant, MK-STYX Protein phosphorylation signalling cascades regulate many diverse cellular responses, including the stress response. Kinases and phosphatases control the activity of target proteins by coordinating a fine balance between phosphorylation and dephosphorylation. Disruption of this balance leads to diseases such as cancer, diabetes, chronic inflammation and Alzheimer's. In a newly recognized level of complexity, phosphorylation cascades are further modulated by a variety of pseudokinases and pseudophosphatases Caenorhabditis elegans pseudophosphatases EGG-4 and EGG-5 inhibit the activity of the dual-specificity tyrosine-regulated kinase minibrain kinase homologue-2 (MBK-2) by binding to its activation loop, preventing substrate phosphorylation Many pseudophosphatases have been identified 11HC(X5)R, the corresponding MK-STYX sequence (IFSTQGISRS) lacks both the critical histidine and cysteine residues HCTQGISRS, create an active phosphatase In the present study, we focus on the pseudophosphatase MK-STYX, a member of the mitogen-activated protein kinase (MAPK) phosphatase class In earlier studies concerning the cellular functions of MK-STYX, we discovered that MK-STYX binds directly to a protein that regulates both the Ras signalling pathway and stress granule assembly The present study focuses on understanding how MK-STYX inhibits stress granule formation. Initially, we hypothesized that MK-STYX inhibited stress granule formation by locking G3BP-1 in its phosphorylated state. However, in the present study, we show that, in heat-shocked HeLa cells, the expression of MK-STYX significantly inhibits stress granule formation in the presence of either phosphomimetic (S149E) or nonphosphorylatable (S149A) G3BP-1 mutants. These data indicate that MK-STYX inhibits stress granule formation independently of the phosphorylation state of G3BP-1 at Ser149, and thus suggest that MK-STYX acts at another point in the signalling pathway. Nevertheless, an interaction between MK-STYX and G3BP-1 is supported both by our immunoprecipitation studies active) . Because we had previously shown that MK-STYX does not suppress the expression of G3BP-1 Stress granule assembly can be induced by a variety of stressors, including the overexpression of G3BP-1 and the dephosphorylation of G3BP-1 at Ser149 active) . As antiDephosphorylation of G3BP-1 at Ser149 has been shown to induce stress granule assembly To determine how the pseudophosphatase MK-STYX might alter these levels, we co-expressed the S149A-G3BP mutant with MK-STYX. Based on our initial model, we predicted that MK-STYX would inhibit stress granule formation by shielding the phosphate group at Ser149 of G3BP-1 from phosphatase activity; hence, the expression of MK-STYX was not expected to alter stress granule formation. However, when we measured stress granule formation in cells co-expressing MK-STYX and S149A-G3BP, MK-STYX was found to suppress stress granule assembly A,B. Thisactive) would enhance stress granule formation in cells co-expressing either the nonphosphorylatable or phosphomimetic G3BP-1 mutants. In the presence of MK-STYXactive, not only did cells expressing the nonphosphorylatable S149A-G3BP mutant form stress granules ]. In the presence of MK-STYXactive, phosphoG3BP-1 at site 149 was not detectable  18active. Furthermore, the active mutant induces the assembly of intermediate-sized granules in cells expressing phosphomimetic S149E-G3BP-1. In the absence of MK-STYXactive, cells expressing S149E-G3BP-1 do not exhibit elevated levels of stress granule formation. This result suggests that the active mutant causes a conformational change, independent of dephosphorylation, that induces stress granule assembly. The N-terminus of G3BP-1 is characterized by the presence of a nuclear transport factor 2 (NTF2)-like domain, and a segment rich in acidic acids that has a key role in protein–protein interactions Our results demonstrate that the phosphorylation of G3BP-1 has different effects on MK-STYX and MK-STYXbona fide modulator in the stress response signalling pathway.In conclusion, the data obtained in the present study enhance our understanding of MK-STYX and its effects on stress granule dynamics, offering insights into G3BP-1-induced stress granule formation. Our data provide evidence that MK-STYX prevents stress granule accumulation independently of G3BP-1 phosphorylation at site 149. It will be of interest to investigate the dynamics between MK-STYX and G3BP-1 that result in the inhibition of stress granules. For example, is the role of MK-STYX in stress granule dynamics via polysome accumulation or stress granule disassembly? The precise mechanism of the pseudophosphatase MK-STYX still remains to be defined. However, our results dispel the notion that pseudophosphatases serve exclusively as ‘protectors’ that block protein tyrosine phosphatases. Instead, they support a model in which pseudophosphatases have very distinctive roles in regulating phosphorylation cascades. Taken together, our findings point toward a pivotal role of the pseudophosphastase MK-STYX as a active-FLAG were generated as described previously by pMT2-FLAG-MK-STYX-FLAG and pMT2-FLAG-MK-STYX2 in MEM  supplemented with 10% fetal bovine serum. Transfections were performed using Lipofectamine 2000 (Invitrogen). When heat shock experiments were required, cells were maintained at 41 °C for 1 h followed by immediate fixation or lysing for either immunoprecipitation or immunoblotting.HeLa cells were maintained at 37 °C in 5% CO5 cells were plated onto coverslips in six-well dishes . Twenty-four hours post-plating, cells at 40–60% confluence were transfected with 2 μg of G3BP-1, S149A-G3BP-GFP, S149E-G3BP-GFP, pMT2, or wild-type or mutant FLAG-tagged MK-STYX expression plasmid DNA and 4 μL of Lipofectamine 2000 per well, in accordance with the manufacturer's instructions. The medium was replaced 5 h after transfection. Twenty-four hours post-transfection, cells were washed with PBS and fixed with 3.7% formaldehyde. The coverslips were mounted to a slide using GelMount containing 4′,6-diamidino-2′-phenylinodole dihydrochloride  (0.5 mg·mL−1).For immunofluorescence assays, HeLa cells were grown to 80–90% confluence and 2 × 10active. To visualize stress granule assembly with cells expressing MK-STYX or MK-STYXactive, endogenous G3BP-1 was used as the marker and visualized with anti-G3BP (dilution 1 : 100) and Cy3 (indocarbocyanine)-conjugated goat anti-mouse  sera. To visualize the localization of MK-STYX or MK-STYXactive, anti-FLAG-fluorescein isothiocyanate (FITC)  sera was used.For experiments examining the effect of MK-STYX and the active mutant on stress-induced stress granule assembly, cells were transfected with S149A-G3BP-GFP or S149E-G3BP-GFP and either pMT2, wild-type or mutant FLAG-tagged MK-STYX expression plasmid DNA. The medium was replaced 5 h after transfection and, at 23 h post-transfection, cells were stressed at 41 °C for 1 h, and then processed as above. For localization studies, cells were transfected with pMT2, MK-STYX or MK-STYXiplab software  were used for image acquisition and primary image processing. imagej , adobe photoshop and adobe illustrator  were used for secondary image processing.Counting and image collection were performed on an Olympus BX60 microscope with U-MNU filter cube for DAPI and Omega Optical XF100-2 for GFP and an Olympus × 40 UPlanFL × 40/0.75 objective . A Cooke SensiCam camera and Approximately 100 cells were scored per treatment. Samples were scored blind with respect to treatment and were scored independently by two different individuals. Cells were scored into two categories: no stress granules and stress granules.l-glutamine. Subsequently, cells were incubated for 3 h in medium containing 1 mCi [γ-32P]ATP, and tagged or endogenous G3BP-1 proteins were immunoprecipitated from cell lysates with anti-G3BP sera (Becton-Dickinson Biosciences). Samples were eluted with 25 mm imidazole (pH 7.2), and used for the protein phosphatase activity assay.Forty-eight hours after post-transfection with G3BP-1-GFP, HeLa cells were incubated for 30 min in DMEM minus phosphate (Invitrogen), supplemented with 10% dialyzed fetal bovine serum and 1 : 100 active-FLAG). Forty-eight hours post-transfection, cells were lysed and MK-STYX proteins were immunoprecipitated using anti-FLAG sera (Sigma). Phosphatase activity in the immunoprecipitates was assayed by incubation with 32P-labelled G3BP-1 or G3BP-GFP as the substrate at 30 °C in 25 mm imidazole (pH 7.2), 0.1 mg·mL−1 bovine serum albumin and 1 mm dithiothreitol. G3BP-1 was labelled by [γ-32P]ATP and immunoprecipitated with anti-G3BP (Becton-Dickinson Biosciences). The reaction was terminated by the addition of a 300 μL charcoal mix  followed by centrifugation and counting of 250 μL supernatant in scintillant. The activity was determined with a LS 6500 Multi-purpose Scintillation Counter . The presence of immunoprecipitated MK-STYX and the active mutant was confirmed by 10% SDS/PAGE and autoradiography.HeLa cells were transfected either with expression vectors for native MK-STYX (pMT2-FLAG-MK-STYX-FLAG) or the catalytically active mutant MK-STYX  and protease inhibitor cocktail tablets ]. Lysates were centrifuged at 14 000 g for 10 min, and the supernatant protein concentration was determined by NanoDrop quantification . For immunoprecipitation, the lysates were precleared and incubated with anti-FLAG (Sigma) or anti-G3BP (Becton-Dickinson Biosciences) for 1 h at 4 °C, followed by incubation with Protein G beads . Samples were washed three times in lysis buffer and boiled in Laemmli sample buffer. Lysates were resolved by 10% SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane  for immunoblot analysis with anti-FLAG (Sigma) and anti-G3BP sera, followed by chemiluminescent detection. 32P-labelled immunoprecipitates were eluted with imidazole and stored for phosphatase analysis. For phospho-G3BP studies, lysates were resolved by 10% SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane with the iBlotter (Invitrogen) for immunoblot analysis with anti-phoshoG3BP (pSer149) (Sigma), anti-FLAG (Sigma) and β-tubulin  sera. Blots were probed first with pSer149 (dilution 1 : 1000), and then β-tubulin for the loading control. Aliquots of the same sample lysates were resolved simultaneously and probed for anti-FLAG.Forty-eight hours post-transfection, HeLa cells were harvested in lysis buffer [50 m


The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC)—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role) were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40 Nevertheless, these cells are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation , 11. Thephox), the first identified and the best studied member of the NOX family. During activation, the cytosolic oxidase subunits p47phox, p67phox, p40phox, and Rac2 translocate to the plasma membrane and associate with the membrane-bound cytochrome b558 complex. Cytochrome b558 is formed by two subunits—gp91phox  and by p22phox, and this heterodimer transfers electrons from NADPH to molecular oxygen. Flavin adenine dinucleotide (FAD) and two heme groups serve as the redox pathway which enables the transfer of electrons across the membrane [Formation of ROS is initiated by the activation of phagocyte NADPH oxidase  facilitate the recognition and clearance of apoptotic neutrophils by macrophages, resulting in safe removal of these cells from the site of inflammation. Since the delayed apoptosis and impaired clearance of neutrophils aggravate and prolong tissue injury, pharmacological intervention focused on neutrophil apoptosis is studied as an original approach for the design of new anti-inflammatory strategies , 19, 20.α, βI, βII, δ, and ζ, which participate in NADPH oxidase activation as well as in proapoptotic and antiapoptotic signalling [Modulation of protein kinase C activity represents a prospective method to regulate neutrophil functions. Immunochemical studies have shown that human neutrophils express five PKC isoforms, gnalling –25.α, PKCβII, and PKCδ (the most abundant PKC isoforms in neutrophils) was assessed.The present paper investigated the impact of piceatannol on the viability and oxidative burst of human neutrophils. We analysed separately the effects of this stilbenoid on the concentration of ROS produced by neutrophils extra- and intracellularly. Protein kinase C activity was examined as an assumed target of piceatannol action and the phosphorylation of PKCβ-phorbol-12β-myristate-13α-acetate), Ca2+-ionophore A23187, superoxide dismutase, dextran (average MW 464 000 kDa), zymosan (zymosan A from Saccharomyces cerevisiae), luciferase from Photinus pyralis, and D-luciferin sodium salt were from Sigma-Aldrich Chemie ; HRP (horseradish peroxidase) and catalase were obtained from Merck  and lymphoprep (density 1.077 g/mL) was purchased from Nycomed Pharma AS . Propidium iodide and rh Annexin V-FITC (produced in E. coli and conjugated with fluorescein isothiocyanate—FITC) was received from eBioscience  and PKC kinase activity kit was from Enzo Life Sciences AG . Phosphospecific antibodies versus PKC isoforms and versus p40phox were obtained from Cell Signaling Technology . Secondary anti-rabbit antibody and Lumigen Detection Reagent were supplied by GE Healthcare Life Sciences . Piceatannol was purchased from Acros Organics . Luminol, isoluminol, PMA  who had not received any medication for at least 7 days. The samples were mixed with 3.8% trisodium citrate, in the ratio of 9 mL of blood to 1 mL citrate. Erythrocytes were allowed to sediment in 1% dextran solution  and the suspension of leukocytes and platelets in plasma (buffy coat) was used for flow cytometric analyses or for neutrophil isolation. For neutrophil isolation, the buffy coat was centrifuged, cells were resuspeded in phosphate-buffered saline, and neutrophils were separated on Lymphoprep . The contaminating erythrocytes were removed with hypotonic cold haemolysis and neutrophils were washed with phosphate-buffered saline. Neutrophil count was assessed by Coulter Counter  and adjusted to a final concentration of 10μmol/L) was stimulated with phorbol myristate acetate , opsonized zymosan (0.5 mg/mL), or Ca2+-ionophore A23187 (1 μmol/L). Chemiluminescence of isolated human neutrophils (5 × 105/sample) was initiated by PMA (0.05 μmol/L). Oxidants released extracellularly were determined in the system containing isoluminol (5 μmol/L) and HRP (8 U/mL); HRP was added to the system, ensuring sufficient extracellular peroxidase concentration. Intracellular chemiluminescence was enhanced with luminol (5 μmol/L) in the presence of the extracellular scavengers superoxide dismutase (100 U/mL) and catalase (2 000 U/mL). Concentration of oxidants was evaluated on the basis of integral values of chemiluminescence over 1 800 s (isolated neutrophils and A23187 stimulated whole blood) and over 3 600 s (whole blood chemiluminescence initiated with PMA or zymosan).Oxidative burst of neutrophils was recorded on the basis of enhanced chemiluminescence , 27, in μmol/L), HRP (2 U/mL), luminol (10 μmol/L), and hydrogen peroxide (100 μmol/L) in 50 μL aliquots. Chemiluminescence was determined for 10 min at 37°C [The antioxidative activity of piceatannol was measured in cell-free system containing piceatannol (0.01–100  at 37°C . 5 cells) were incubated with piceatannol (1–100 μmol/L) for 30 min at 37°C and stimulated with PMA (0.15 μmol/L) for 3 min. Stimulation was stopped by the addition of a tenfold volume of ice-cold phosphate buffer. After centrifugation, neutrophils were broken by lysing solution and by sonication, centrifuged, and the supernatant was used for the determination of protein kinase C activity using an enzyme-linked immunosorbent assay kit.Isolated neutrophils (5 × 106 cells) were incubated at 37°C with piceatannol , stimulated with PMA  and lysed by sonication in a lysing solution containing protease, and phosphatase inhibitors. Debris from the lysed cells was pelleted by centrifugation and the supernatant was taken for blotting assay. Protein concentration was measured using Bradford Dye Reagent detection kit . Proteins (20 μg per lane) were separated by SDS polyacrylamide gel electrophoresis and transferred to Immobilon-P Transfer Membrane. From each membrane, the area between 60 and 100 kDa was detected with primary anti-phospho-PKCα/βII (Thr638/641) antibody (1 : 5 000) and with the secondary antibody conjugated to HRP (1 : 5 000). Phosphorylated PKC isoforms α and βII were visualised with Lumigen Detection Reagent kit, scanned, and quantified densitometrically using ImageJ programme. The membrane was then stripped, using stripping buffer , and reprobed with anti-phospho-PKCδ (Thr505) antibody (1 : 1 000). The membrane area between 30–60 kDa was detected with regard to the presence of the internal standard β-actin  and the phosphorylated subunit of NADPH oxidase p40phox (anti-phospho- p40phox (Thr154) antibody, 1 : 5 000).Western blot analysis was performed as previously described , 30. Iso4 cells) was incubated with piceatannol (1–100 μmol/L) for 15 min at 37°C. Then, the mixture of luciferin (1.6 μg) and luciferase (45 000 U) was added and chemiluminescence was recorded for 60 seconds. Chemiluminescence of ATP standards (1–500 nmol/L) was measured in each experiment and concentrations of ATP in samples were calculated from the calibration curve. Total ATP content was assessed immediately after sonication of neutrophils for 10 seconds.Damaging effect of piceatannol on the integrity of plasma membranes was evaluated on the basis of ATP liberation, measured by the luciferin-luciferase chemiluminescence method . Suspensμmol/L) for 10 min at 37°C. The cells were double-stained with annexin-V conjugated with FITC (in dark at 4°C for 10 min) and with propidium iodide and analysed by cytometer Cytomics FC 500 . From the granulocyte area, 5 000 cells were gated and the percentage of apoptotic (annexin positive and propidium iodide negative), dead (double positive), and viable cells (double negative) was determined as described previously [Human plasma buffy coat see  was incueviously , 33. t-test. P values below 0.05 were considered to be statistically significant and were indicated in the figures by *P ≤ 0.05 and **P ≤ 0.01.All values were given as the mean ± SEM and the statistical significance of differences between means was established by Student's μmol/L (PMA), 2.71 ± 0.41 μmol/L (A23187) and 9.43 ± 0.53 μmol/L (zymosan).Piceatannol reduced the oxidative burst of human neutrophils measured in whole blood . It inhiμmol/L and 12.59 ± 0.96 μmol/L; in cell-free system, the EC50 of piceatannol was 0.63 ± 0.01 μmol/L. The phosphorylation of p40phox  was increased more than three times after PMA stimulation. This increase was not modified by the treatment of neutrophils with piceatannol  was also decreased after piceatannol treatment  and 1.87 μmol/L (extracellular). It means that the radicals formed within neutrophils (fulfilling a regulatory role) were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular) membranes [δ  was affoxidants . Yet theThe more pronounced extracellular activity results from the structure of piceatannol. Compared to resveratrol, the additional hydroxyl group makes the molecule of piceatannol more hydrophilic and less able to pervade biological membranes . Thus, pα and PKCδ, as these PKC isoforms participate in antiapoptotic signalling in neutrophils [μmol/L piceatannol, that is, in a concentration several times higher than assumed piceatannol plasma levels obtained by dietary intake. Yet it should be taken into account that, in comparison to in vivo conditions, in these samples neutrophil count was substantially higher and the exposure of the cells to piceatannol lasted only 30 minutes. Similarly, the accelerated apoptosis of leukemic cells was detected after 24–48 h incubation with 10–60 μmol/L piceatannol [The observed reductions in chemiluminescence and in the activity of protein kinase C were not associated with neutrophil damage because in the presence of piceatannol no increase in extracellular ATP concentration was recorded. As confirmed by flow cytometry, this stilbene enhanced spontaneous apoptosis of neutrophils. This was indicated by an increased number of annexin-positive cells, that is, cells displaying a more pronounced externalisation of phosphatidylserine. The expression of phosphatidylserine on the external side of plasma membrane facilitates the recognition of apoptotic neutrophils by macrophages and their safe removal from the site of inflammation . The abitrophils , 53. Inceatannol , 55.Piceatannol decreased the concentration of ROS produced by neutrophils and accelerated spontaneous apoptosis of these cells. The observed effects classified piceatannol as a potentially useful complementary medicine in states associated with persistent activation of neutrophils, oxidative damage of tissues, and persistent inflammation. However, the bioavailability and toxicity of this promising natural substance is a decisive question requiring further studies.


Saccharomyces cerevisiae, is the simplest and most well-known representative of eukaryotic cells and thus a convenient model organism for evaluating toxic effects in human cells and tissues. Yeast cell sensors are easy to maintain with short generation times, which makes the analytical method of assessing antifungal toxicity cheap and less-time consuming. In this work, the toxicity of test compounds was assessed in bioassays based on bioluminescence inhibition and on traditional growth inhibition on agar plates. The model organism in both tests was a modified S. cerevisiae sensor strain that produces light when provided with D-luciferin in an insect luciferase reporter gene activity assay. The bioluminescence assay showed toxic effects for yeast cell sensor of 5,6-benzo-flavone, rapamycin, nystatin and cycloheximide at concentrations of nM to μM. In addition, arsenic compounds, cadmium chloride, copper sulfate and lead acetate were shown to be potent non-specific inhibitors of the reporter organism described here. The results from a yeast agar diffusion assay correlated with the bioluminescence assay results.Baker's yeast,  Ethanol was from Altia Plc , antibiotic assay disks of diameter of 6 mm were from Schleicher & Schuell . Yeast nitrogen base w/o amino acids and agar-agar (Difco) were purchased from Voigt Global Distribution  and D-Luciferin was supplied by Biothema AB .Zinc(II)chloride  nystatin and B-NF into DMSO. Other chemicals were dissolved into sterile deionized water. Sample sets were prepared by diluting stock solutions in a ratio of 1:10 with the respective solvent. Rapamycin solutions were stored at -20°C and CdCl3.3S. cerevisiae transformed with a modified firefly  luciferase gene (luc) inserted in the plasmid as a reporter for genetic response, BMA64-1A+pRS316luc [S. cerevisiae. Firefly luciferase catalyses the following reaction: Luc + D-luciferin +ATP → oxyluciferin + AMP + CO2 +PPi + light. The resulting luminescence (yellowish light) can be measured very sensitively and in our assay simply by adding D-luciferin substrate after an exposure of few hours or even in real-time. We use the D-luciferin substrate at pH of 5.0, because in the modified firefly luciferase the last three amino acids of the enzyme have been truncated. The natural peroxisomal targeting signal (Ser-Lys-Leu) [C-terminus of the enzyme results in cytoplasmic expression, which leads to high levels of light emission.The yeast strain used in this study was RS316luc . The insLys-Leu)  lacking S. cerevisiae was cultivated overnight at 30°C and 300rpm shaking in liquid synthetic dextrose medium (SD) that contains yeast nitrogen base w/o amino acids (6.7 g/L) and D-glucose 40 mL/L of a 40% (w/v) supplemented with adenine (5 g/L) and the essential amino acids l-histidine (2 g/L), l-leucine (10 g/L) and l-tryptophan (2 g/L). Yeast nitrogen base w/o amino acids and D-glucose were autoclaved (121°/15 min) and adenine and amino acids were filter sterilized (pore size 0.2 μM). pH of the SD medium was checked time to time and pH was 4.8±0.2. For the bioluminescence assay, the yeast cells were grown to an optical density (OD600) of about 3.0 and after that the culture was diluted with the same medium to OD600 of 0.8 and right after were used in the assay. For agar diffusion assay, the same medium described above was solidified with agar-agar.For both bioluminescence and conventional agar diffusion assay, 3.43.4.1Ten or 1 μL aliquots of the sample sets and reference samples (blank solvent) were pipetted in triplicate into a white 96-well plate  with hydrogen peroxide  as a positive control. Ninety or 100 μL aliquots of the diluted yeast culture were then added into the wells containing samples. In the wells, the final amount of water or acetate buffer was 10 % and DMSO 1%.50 of all tested chemicals was calculated. Because the bioluminescence response curves of nystatin were different from the curves of other tested chemicals, an exposure time of 10 hours was used to see if the same phenomenon could be seen under a prolonged incubation time.Plates were then incubated at 30°C and 300 rpm for 2.5 h or 5 h. After the incubation, 100 μL aliquots of 1 mM D-luciferin (in 0.1 M Na-citrate buffer pH 5) were pipetted into the wells and the bioluminescence was immediately measured with a Chameleon Multilabel Detection Platform . Furthermore, for real-time monitoring, 5,6-benzoflavone and rapamycin were measured continuosly  and ligh3.4.2To compare and validate the results from the bioluminescence assay, a conventional agar diffusion assay (ADA) was performed. Aliquots of test sample (five dilutions) and reference solutions were pipetted onto filter papers (Ø 6 mm) which were then spotted on the surface of the SD –agar plates containing the logarithmic overnight grown yeast sensor strain. After overnight incubation at 30 °C, the growth inhibition zones were measured. The lowest chemical concentration at which an inhibition zone could be observed was defined as a value of minimal inhibitory concentration (MIC).4.S. cerevisiae transformed with a modified firefly  luciferase gene (luc) inserted in the plasmid as a reporter is an alternative method in screening non-specific toxicity of several chemicals in real-time. The toxicity of the test compounds was assessed in the bioassays based on bioluminescence inhibition. The traditional growth inhibition on an agar plate was performed to compare and validate the results from the bioluminescence assay.The goal of this study was to show that our toxicity assay based on a representative of eukaryotic cells S. cerevisiae is easy to cultivate in a pour medium with short generation time, the bioluminescence assay could be a fast and inexpensive tool suitable for toxicity assessments.It naturally needs to be emphasized that when toxicity testing is concerned, all systems have their advantages and disadvantages. Even though the results from the agar diffusion assay (ADA) correlated with the results from the bioluminescence assay, the ADA has many disadvantages. Making plates for each concentrations and each chemical is time-consuming, and the results can be read only after 1-3 days, whereas in the bioluminescence assay different concentrations of different chemicals can be performed in a multi-well plate and the luciferase activity can be measured from intact living yeast cells even in real-time just by adding D-luciferin. Furthermore, since the ADA reveals only a level of the toxicity, the bioluminescence assay can give some ideas about the character of the toxicity like during 5,6-benzoflavone, rapamycin and nystatin monitoring. Thus, because of the advantages mentioned above and due to the fact that


Ricin is a highly toxic type II ribosome-inactivating protein that has potential as a biochemical weapon and as the toxic component of immunotoxins. The unfolded protein response (UPR) is a survival response that helps cells to recover from endoplasmic reticulum (ER) stress. Failure to recover from ER stress leads to apoptosis. In yeast, ricin-A-chain (RTA), the enzymatic component of ricin, inhibits UPR. Our goals were to determine if RTA inhibits UPR in two epithelial cell lines and if this affects RTA cytotoxicity. RTA alone did not induce UPR. However, RTA inhibited both phosphorylation of inositol-requiring enzyme 1 (IRE1) and splicing of X-box binding protein1 mRNA by the UPR-inducing agent tunicamycin (Tm). The ability of dithiothreitol (DTT) to activate eukaryotic translation initiation factor 2 alpha (eIF2α), a component of the PERK pathway, was also inhibited by RTA. Treatment with RTA in combination with Tm or DTT inhibited protein synthesis more than either agent did alone in one cell line, while caspase cleavage was enhanced by the treatment combination in both cell lines. These data indicate that RTA is more cytotoxic when UPR is inhibited. This ability to inhibit UPR may enhance the potential of RTA as a therapeutic immunotoxin in solid tumors. Ricinus communis. Due to its ease of isolation and extreme toxicity it has been classified by the Centers for Disease Control and Prevention as a category B select agent  methionine  were added to each sample during the last 30 min of toxin treatment. Cells were washed twice with cold PBS and scraped into cold 5% trichloroacetic acid (TCA). Samples were collected on glass microfiber filters (Whatman) using a vacuum manifold, washed three times with ice-cold 5% TCA and washed two times with cold 95% ethanol. ScintiVerse cocktail (Fisher) was added to each sample and radioactivity was determined by scintillation counting (Beckman Coulter).Confluent cells were serum-starved for two h, washed two times with methionine-free media (Invitrogen) and incubated in methionine-free media for 45 min prior to treatment with toxin. Approximately 8 μCi [Post-hoc test or by student’s paired t-test using GraphPad Prism 5 Software.Data were analyzed by one-way ANOVA with Bonferroni’s Multiple Comparison i.e., spliced plus unspliced) XBP1 mRNA expression was not different between cells treated with Tm alone and those treated with Tm plus 0.1 μg/mL RTA while total XBP1 mRNA levels were 50% lower in Tm-treated cells exposed to the higher concentration of RTA relative to cells treated with Tm alone. These data indicate that the decrease in XBP1 splicing was not a direct reflection of decreases in unspliced XBP1 mRNA levels.XBP1 splicing subsequent to IRE1 phosphorylation is a primary component of the UPR response. Therefore we first examined whether RTA would affect this arm of UPR in the non-transformed mammary epithelial cell line MAC-T. We have previously shown that these cells are sensitive to RTA in terms of depurination, protein synthesis inhibition, and apoptosis within 4 h of treatment . As showTo determine if the holoenzyme acts similarly to RTA, experiments were repeated with ricin holotoxin, RTA and RTB. None of these alone stimulated XBP1 splicing. Ricin and RTA produced similar inhibition of Tm-induced XBP1 splicing, while RTB had no effect on XBP1 splicing induced by Tm C. These UPR is also induced by thapsigargin (Tg), which disrupts calcium homeostasis in the ER, as well as DTT, which reduces protein disulfide bonds. Therefore, we determined if RTA could inhibit the ability of either of these chemicals to induce XBP1 splicing in MAC-T cells. As shown in In order to determine if the inhibition of Tm-induced XBP1 mRNA splicing by RTA was also observed in transformed cells, the experiments were repeated in HeLa cells, a human cervical carcinoma cell line, which is also responsive to RTA as shown in our previous work . SimilarSince RTA inhibited Tm-induced XBP1 mRNA splicing, we looked upstream of XBP1 at IRE1 phosphorylation. As shown in PERK, another arm of UPR, shares a similar activation mechanism during induction of ER stress. To determine if the PERK pathway was affected by RTA, western blotting of phospho-eIF2α was performed. eIF2α is a direct substrate of phospho-PERK, the activated form of PERK. As expected, phosphorylation of eIF2α in HeLa cells was induced 2- to 4-fold compared to untreated controls by exposure to Tm, Tg or DTT A. RTA alProtein synthesis inhibition is often used as a marker of ricin-induced cytotoxicity. To determine if inhibition of UPR by RTA affected this parameter, de novo protein synthesis was measured after 4 h of RTA treatment . MAC-T cActivation of the UPR pathway represents a survival response that allows the cell to recover homeostasis after an ER overload. Since RTA inhibited the IRE1 pathway induced by Tm and the PERK pathway induced by DTT, we were interested in determining if RTA would activate caspases to a greater degree in the absence of the UPR-survival pathway. As shown in A wide variety of stressors including nutrient deprivation, disruption of calcium homeostasis and hypoxia activate ER stress and elicit the UPR ,28,29. RRTA inhibits Tm-induced HAC1 mRNA splicing in yeast . TherefoWhen expressed in yeast, both pre-RTA, which contains the ER localization sequence, and mature RTA, which lacks this sequence and thus fails to enter the ER, inhibit Tm-induced UPR . Based oWhen MAC-T cells were treated with Tm or DTT together with RTA, protein synthesis was inhibited to a greater degree that it was with RTA alone, indicating that the two agents are more cytotoxic in combination. Since Tm and DTT each inhibited protein synthesis on their own, this could be an additive effect. Interestingly, in HeLa cells where Tm and DTT had minimal effects on protein synthesis, the addition of either UPR-inducing agent did not alter RTA-induced protein synthesis inhibition. Since UPR is a survival response that pushes the cell toward apoptosis if homeostasis is not restored, we asked the question of whether or not inhibiting the UPR could alter RTA-induced caspase activation as a marker for apoptosis. Treatment of HeLa cells with RTA in combination with Tm or DTT evoked greater caspase cleavage than that observed in response to RTA, Tm, or DTT alone while the ability of RTA to inhibit protein synthesis when UPR was inhibited was not altered. These data indicate that inhibiting the UPR allows RTA to increase cell death through a mechanism that is independent of protein synthesis inhibition. This supports previous findings in yeast using RTA mutants which show that protein synthesis inhibition and cell death can be uncoupled . RTA hasThe present work shows that RTA can inhibit ER stress, leading to an enhancement of its cytotoxicity. ER stress has been shown to be an important component of several disease conditions, including cancer . The abi


Ricinus communis L.  is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis.The allergenicity of in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction.The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment. Ricinus communis L.) contains approximately 50% oil, which has special characteristics such as a high viscosity, a high stability under heat and pressure, a low freezing point, and the ability to form waxy substances upon chemical treatment. As 0energy demands increase and fossil fuels are limited, the development of alternative renewable fuels becomes imperative. Interest in biodiesel has been increasing owing to its environmental benefits and renewability Castor bean (N-ethyl-5-phenylisoxazolium-3′-sulfonate) In the present study we identify critical amino acids for IgE binding and investigate cross-reactivity with allergens typically used for allergy diagnosis. Initially, we utilized the glutamic acid-specific Woodward's Reagent K, WRK,  seeds were obtained from the Instituto Agronômico de Campinas, São Paulo/Brazil. The 2S albumin fractions were isolated and characterized by SDS-PAGE and immunoblotting experiments as described previously Castor bean . Immunizations with the 2S albumin pool (Ric c 1 plus Ric c 3) and serum separation were conducted as previously described IgG and IgE from human and rat sera were separated by affinity chromatography using protein G-Sepharose beads in batch mode with 1.5 mL polypropylene tubes (Sigma protocols). Thirty micro litres of total serum of immunized R/A Tor rat was mixed with the 100 µL of resin and with 50 µL of equilibration buffer. This mixture was incubated for 16 hours under stirring at 4°C. The mixture was centrifuged and the supernatant denoted FE (fraction likely enriched in IgE) was removed and stored at 4°C. After removal of the supernatant, the resin was washed ten times with 100 µL of equilibration buffer to remove the remaining uncoupled material. The immunoglobulin coupled to protein G-Sepharose was eluted using 100 µL of a 0.5 M acetic acid. The acidic solution was immediately neutralized by the addition of 1 M Tris-HCl pH 9.0. After washing and centrifugation, the eluted fraction, denoted FG (fraction likely enriched in IgG), was isolated. The presence of IgG was investigated in both FE and FG by dot blotting. Mast cell degranulation assays in the presence of FG or FE were also performed to identify the immunoglobulin involved in mast cell activation.R. communis by dot blotting. After the first evaluation, patient serum with high intensity recognition of castor bean allergens was used in subsequent assays.Human serum analysis: sera from five patients who had total IgE values of 106 to 1527 KU/L were evaluated for their ability to recognize 2S albumin from 2O2.For dot blot assays, 2S albumin or synthetic peptide (10 µg in 10 µL/dot) was spotted onto a nitrocellulose membrane and allowed to dry. The nitrocellulose membrane was incubated with total serum (1∶50) or affinity supernatant or eluted fractions, FE and FG, from human or rat serum. Secondary anti-rat IgG or anti-human biotin IgE (0.5 mg/mL) (both diluted 1∶2000) was then added to the membrane for 1 hour. Two hours later, IgG was detected using a rabbit anti-rat IgG- HRP conjugate (1∶2500). For IgE detection, the membrane was subsequently incubated with streptavidin-biotinylated HRP complex for 1 h. The colour of all probes was developed with a substrate mixture: 5 mg of DAB in 4.9 mL of water, 300 µL of 0.1 M imidazole, 100 µL of Tris-HCl 2 M buffer (pH 7.5) and 5 µL of 30% H2 and a peritoneal wash was performed by injection of 20 mL of DMEM (Dulbecco's Modified Eagle Medium) containing 12 U/mL of heparin. The abdomen was gently massaged for approximately 90 s. The peritoneal cavity was carefully opened and the fluid containing peritoneal cells was aspirated with a Pasteur pipette. Thereafter, the cells were transferred to Petri plates and incubated for 30 min at 37°C. Two-thirds of the supernatant was aspirated and discarded. The mast cell-rich supernatant (1.8×105 mast cells/mL) was separated into 100 µL aliquots and kept at room temperature.Wistar rats were obtained from the animal facility of the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF). All experimental procedures were approved by the animal research ethics board of the UENF (Proc. CEUA-UENF/112). Rats (weighing ∼250 g) were euthanized with CORat peritoneal mast cells (100 µL) were incubated with pre-immune serum (control) and activated for 60 min at 37°C using 2S albumin polyclonal anti-rat IgE . After sensitization with 2S alb AR IgE, cells were washed twice with DMEM. Each experiment was carried out in the presence or absence of the synthetic peptides and a 2S albumin pool (100 ng). After incubation with antibodies and potential allergens , histamine contents were determined (see below) and the cells (in 10 µL) were stained for 15 min with 10 µL of a solution containing 0.1% toluidine blue, 10% formaldehyde and 1% acetic acid, pH 2.8, allowing the visualization of degranulated mast cells. Granulated and degranulated mast cells were counted under a light microscope using the 40 X objective in a Neubauer chamber.R. communis and allergens used for allergy diagnosis, mast cells previously sensitized by 2S alb AR IgE were incubated with airborne and food allergens from FDA Allergenic (FDA-PRICKIT- Lot 04AK00001 and FDA-FOODKIT lot 04AK00004).In order to investigate cross-reactivity between 2S albumin from The airborne allergens that were tested were from dust, airborne fungi, wool, dust mites, grass, flowers, tobacco, cat epithelium, dog epithelium, horse epithelium, bovine epithelium, epithelia mix, feathers, cotton, horsehair, grass, chamomile, cockroaches, pyrethrum, silk, and cattails. The food allergens were from poultry, shrimp, beef, pork, egg, egg yolk, milk, fish, rice, gluten, corn, wheat, soybean, pepper, pineapple, cocoa, cashew, peanuts, orange, lemon, tomato and strawberry. Protein content measurements of the allergen solutions from FDA allergenic were performed using the Bradford method using egg albumin as a standard. Stock solutions containing 10 µg/mL were prepared and each test used 10 µL of these allergen solutions. Mast cell activation was evaluated by taking the percentage of granulated and degranulated cells as described previously and by the quantification of histamine released.After incubation with antibodies and allergens, the cell suspensions (100 µL) were centrifuged at 170 xg for 10 min. An aliquot of the supernatant (20 µL) was removed for histamine detection. The released histamine was submitted to ion exchange chromatography and quantified using a previously described protocol The peptides previously identified as IgE-binding epitopes in Ric c 1 or Ric c 3 allergens by Felix et al. WRK is a specific reagent for modifying dicarboxylic amino acids −1 using 0.1% trifluoroacetic acid (TFA) as solvent A and 80% of acetonitrile containing 0.1% TFA as solvent B.) using a Shimadzu apparatus. The elution profile was monitored by on-line measurement of the absorbance at 220 nm. Fractions containing the major peaks were dried in a speed Vac system. The UV spectrum was obtained using a photo-diode detector.To verify the modifications on the proteins after chemical treatments, the samples, synthetic peptides and 2S albumin were subjected to reverse-phase liquid chromatography on a C18 column . Amino acids were dissolved in DEMEN. After free amino acid treatment, cells were incubated with 100 ng of each synthetic peptide or allergenic fractions (Ric c 1 or Ric c 3). Mast cell degranulation was evaluated as described, and the percentages of degranulation and histamine release were compared with those triggered by synthetic peptides or by allergenic isoforms in the absence of free amino acid treatment.Mast cells sensitized with IgE were treated with 100 ng of aspartic acid, glutamic acid or a mixture containing 100 ng of each other amino acids present in the synthetic peptide  followed by streptavidin-peroxidase conjugate (1∶1000 diluted) (Pharmingen). The peroxidase reaction was developed and the optical density (OD) was measured at 492 nm. Each value represents the mean of two assays. OD Values <0.1 were considered negative.I/ODT X 100); ODI represents the absorbance after incubation of glutamic acid-treated human serum; and ODT represents the absorbance of untreated human sera. Mast cell granulation or degranulation was counted by optical microscopy and histamine release was quantified as described above.The ability of glutamic acid to inhibit the binding of allergic patients' IgE to Ric c 1 and Ric c 3 was assessed by ELISA inhibition and mast cell activation assays. Briefly, serum from an atopic patient (427 KU/L- 1∶5 diluted) and Wistar rats were preincubated for 1 h with glutamic acid (end concentration 10 µg/mL). The ELISA or mast cell degranulation assays were performed as previously described. The percentage of IgE binding inhibition achieved by the pre-incubation treatment was calculated as follows: percentage of IgE binding  = 100– were separated by affinity chromatography. Two fractions denoted FE (supernatant fraction) and FG (eluted fraction) were obtained and the presence of IgG was investigated in both fractions by dot blotting. The synthetic peptides present in Ric c 1 and Ric c 3 structures, which were recognized by the pool of sera and induced mast cell degranulation as described by Felix et al. N-ethyl-5-phenylisoxazolium-3′-sulfonate group (data not shown) by each glutamic residue. WRK-modified peptides were employed in mast cell degranulation assays and the results are shown in We found that the peptides identified as IgE epitopes had at least two glutamic acid residues . The rationale of our experiment is illustrated in The process of isolation of mast cells from rat peritoneal cavities induces ∼30% degranulation and 2% histamine release; thus, these values were set as a baseline for the negative controls. Castor bean 2S albumin induced 70% mast cell degranulation and more than 40% histamine release when incubated in the presence of IgE .When mast cells were incubated with untreated serum or with serum treated with neutral or basic amino acids, degranulation triggered by allergens could be observed   . The dicllergens . We noteFigure S1(A) Hypersensitivity reactions: a) Allergenic proteins are processed by antigen-presenting cells (APC) by the MHC class II pathway with subsequent increases in IgE production; b) IgE sensitizes mast cells by binding to FcεRI; c) A second exposure to the allergen activates these cells to degranulate and release vasoactive amines. (B) Blockage of IgE: a) Sensitized mast cells are incubated with glutamic or aspartic acid; b) These dicarboxylic amino acids bind to IgE-sensitized mast cells resulting in protection from mast cell degranulation.(TIF)Click here for additional data file.


AMPK has been recognized as an important upstream signaling intermediate intimately involved in the regulation of the mTOR pathway [722/Ser792 which it is essential for inhibition of the raptor-containing mTOR complex I (mTORCI). Moreover, AMPK is activated by the adenosine analogue, 5-aminoimidazole-4-carboxamide (AICA)-riboside (AICAR) and metformin. These drugs have been used to inhibit the growth and survival of breast cancer and glioblastomas cells [AMPK is a physiological cellular energy sensor that is activated by phosphorylation at Thr473 of Akt/PKB, which is essential for full Akt/PKB activation. We have evidence in TSC-deficient proximal tubular cells accumulates significant amounts of ROS compared to wild type cells (data not shown). Accumulation of ROS is associated with activation/phosphorylation of Akt at Ser473, inactivation/phosphorylation of tuberin at Thr1462 and activation mTOR/rictor complex II [1135 by p70S6K, which negatively regulates mTORC2 protein complex as part of a negative feedback mechanism controlling Akt activity [mTOR is a large protein kinase with two different complexes. One complex contains mTOR, GβL and raptor, which is a target of rapamycin . The othTargeting mTOR is emerging as an important approach in cancer therapeutics . Early c1387 and suppressed the mTOR activity. Moreover, rapamycin treatment led to a robust phosphorylation and activation of AMPK and ACC to a degree similar to that observed with AICAR stimulation in TSC2+/− treated with HG further supporting the importance of rapamycin as an activator of AMPK. In addition, treatment of TSC-deficient mice with rapamycin showed inhibition of mTORC1 through increasing AMPK phosphorylation at Thr172 and it is downstream, ACC at Ser79 and accompanied by increased OGG1 activity [722/Ser792 and inhibit mTOR that lead to activate the DNA repair enzyme OGG1 in cancer cells.Upregulation of OGG1 was detected in several cancer cells treated with AMPK activator, AICAR . Activat


In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3.The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis.Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation.Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner. While anesthetic drugs primarily act on neuronal cells in the central nervous system αIIbβ3 is the most abundant receptor in platelets and plays a critical role in platelet aggregation and clot stability through the interaction with its Arg-Gly-Asp (RGD)-motif –containing ligands fibrinogen, von Willebrand factor and fibronectin 2 at 37°C. 293T cells  were cultured in DMEM with HEPES modification, 10% FBS at 37°C in 5% CO2.Chinese hamster ovary (CHO) -K1 cells stably transfected with αIIb-wild type (WT)/β3-WT or αIIb-WT/β3-N305T were previously described and kindly given by Dr. Springer 2, 137.5 mmol/L NaCl, 12 mmol/L NaHCO3, 2.6 mmol/L KCl, pH 7.4) as described The activation of αIIbβ3 was probed using PAC-1, an IgM antibody that binds only to the activated αIIbβ3 2/CaCl2 (inactivating condition) or HBS Containing 1 mM MnCl2/0.4 mM CaCl2 (activating condition) in the presence of various concentrations of isoflurane, sevoflurane or propofol. Isoflurane and sevoflurane were administered to cells in the closed chamber using a Fluotec vaporizer, and their concentrations were measured using infrared spectroscopy. Goat anti-mouse IgM-FITC  was used as a secondary antibody. Cells were analyzed with a FACScan. In addition, the cell surface expression of αIIbβ3 was probed with AP3 antibody . PAC-1 binding % was calculated as [(MFI of sample at various concentrations of anesthetics)-(MFI of isotype control sample)]/(MFI of sample without anesthetics)-(MFI of isotype control sample)]×100%.CHO-K1 cells transfected with αIIbβ3 were detached in HEPES-buffered saline (HBS)/10 mM EDTA and washed three times with HBS. Cells were incubated with 10 µg/ml PAC-1 (BD Biosciences) in HBS containing 1 mM MgClThe purification of full-length ectodomain and headpiece αIIbβ3 was previously described 2/CaCl2 or 1 mM MnCl2/0.4 mM CaCl2 for 1 hour. Isoflurane and sevoflurane were administered to wells in a closed chamber using a Fluotec vaporizer and their concentrations were measured using infrared spectroscopy. Attached αIIbβ3 was captured with anti-mouse IgM- HRP . Color was developed with substrate . Optical density (OD) at 405 nm was read using an ELISA plate reader . PAC-1 binding % was defined as [(OD of sample at various concentrations of anesthetics) – (OD of background)]/[(OD of sample without anesthetics) - (OD of background)]×100%.Capturing antibody AP3 was coated on 96 wells overnight. Wells were blocked with 2% BSA and then incubated with recombinant αIIbβ3 (full length or headpiece). Following washing, wells were incubated with PAC-1 in the presence of various concentrations of isoflurane, sevoflurane or propofol containing 1 mM MgClSequest software to determine the adduct attachment sites. Mass spectrometry work was performed at the University of Pennsylvania Proteomics Core Facility.Photolabeling experiments were performed using azi-isoflurane, isoflurane with a diaryzinyl moiety. The details of experiment have been previously described Alanine scanning mutagenesis was performed using Quikchange XL kit . DNA sequence was confirmed. Transfection was performed using Lipofectamine 2000  per company protocol.Data were analyzed using an analysis of variance (ANOVA) with Tukey post hoc pairwise comparisons or student's t-test as indicated in corresponding figure legends. Statistical significance was defined as P<0.05. Statistical analysis was performed using PRISM 5 software .From clinical observational studies on hemostasis as summarized in 2+/Ca2+) or an activating condition (1 mM Mn2+/0.4 mM Ca2+). In 1 mM Mg2+/Ca2+, PAC-1 does not binds to αIIbβ3 WT  sequence that is analogous to the RGD sequence in the complementarity determining region 3 of the heavy chain. This region is speculated to interact with the activated MIDAS We demonstrated that isoflurane and sevoflurane attenuated the activation of αIIbβ3 in cell-based assays using CHO transfectants. Anesthetics are well appreciated as promiscuous molecules , Our cell-free and cell-based assays strongly suggested that isoflurane and sevoflurane directly interacted with αIIbβ3 and attenuated its activation. Previously we reported that a novel photoactivatable compound, azi-isoflurane 2+/0.4 mM Ca2+), indicating the importance of this residue. This was consistent with the previously reported results of the mutants of the other SyMBS forming residues To confirm the functional role of the azi-isoflurane adducted residues (Asp-158 and Lys-159), we made β3-D158A and –K159A mutants to alter the chemical texture of this site. As shown in In this study, we demonstrated that isoflurane and sevoflurane, but not propofol, attenuated the activation of integrin αIIbβ3. Furthermore, the photolabeling experiment using azi-isoflurane suggested that isoflurane bound to the residues around the SyMBS of the I domain of the β subunit. That these two findings were linked was suggested by the mutagenesis experiments, which indicated the importance of this site for expression and activation.2+ ion of the I domain via one of the oxygen atoms in the compound's Asp carboxyl or an equivalent carboxyl With the appreciation of its profound effect on platelet aggregation, αIIbβ3 has been an attractive therapeutic target to prevent platelet aggregation in specific disease states. For example, peptides containing the RGD sequence competitively prevent αIIbβ3 from binding to its ligands 2+ and allosterically activates integrins for ligand binding by stabilizing the MIDAS site 2+ and MIDAS Mg2+ at the same time Surprisingly, the adducted residues of photolabeling experiments were located around the SyMBS, which was not at the binding pocket of the aforementioned αIIbβ3 small molecule antagonists and Fab. The SyMBS coordinates CaInterestingly, we found the adducted residues only on the I domain of the β3 subunit in αIIbβ3 with two different preparations. In addition to the αIIb subunit, the β3 subunit couples with the αV subunit to form integrin αVβ3. The number of αVβ3 copies on platelets is small compared with that of αIIbβ3 Clinical significance of functional alternation in αIIbβ3 is apparent from a familial bleeding disease, Glanzmann thrombasthenia. Bleeding in this disorder derives from the failure of platelet aggregation due to reduced or absent αIIbβ3 In conclusion, we have demonstrated that the inhalational anesthetics isoflurane and sevoflurane, not but the intravenous anesthetic propofol, impairs the activation of integrin αIIbβ3 via a direct novel allosteric mechanism.