Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites . We also analyzed lamin C (A-type) and its mutant S37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R64H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S45E mutant was insoluble, in contrast to lamin C S37E. Lamin Dm T435E  and R64H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S45E and T435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T435E was cytoplasmic and showed higher mobility in FRAP assay.Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used  Lamins are the major components of the nuclear lamina, a dense, filamentous meshwork which provides structural support for the nuclear envelope (NE), although a fraction of lamins are present in the nuclear interior as well. Lamins serve as an organizing center for essential cellular processes such as transcription, DNA replication, cell differentiation, nuclear migration and others The level and pattern of phosphorylation of lamins vary throughout the cell cycle 404 by Akt kinase in response to insulin 392) and B1 (S393) by cdk5 kinase can directly cause neuronal cell death observed in neurodegenerative diseases, including Alzheimer's disease Drosophila melanogaster.While the function of mitotic lamin phosphorylation is well characterized, very little is known about the possible role of specific lamin modification during interphase. One of them is the control of subcellular distribution of lamins by regulation of their uptake into the nucleus 1, Dm2 and Dmmit1 and Dm2 occur as an insoluble polymer that is mostly underlying the nuclear envelope during interphase 2 isoform arises from Dm1 by phosphorylation on the N-terminal site at or around S25in vivo. During mitosis this interaction is lost 1 and Dm2 change during mitosis into a soluble form (lamin Dmmit) In vitro, lamin Dm polymers can be solubilized by cdc2, PKC (protein kinase C) and PKA kinase (protein kinase A) as well Fruit fly has two lamin genes coding for lamin Dm and lamin C, which are equivalents of vertebrate B- and A-type lamins, respectively. There are at least three posttranslationally modified lamin Dm isoforms: Dmin vivo phosphorylation on soluble lamin Dm was demonstrated using mass spectrometry. The identified phosphopeptide suggested location of one of the phosphorylation sites on S45 residue within the sequence of typical, lamin N-terminal cdc2 kinase motif 19, S25, S595 and T435 (candidate cdc2/PKC/PKA site) as the late embryo in vivo phosphosites in vivo more phosphorylation sites may be involved and that they may be distributed along the entire protein Early, syncytial embryo specific in vivo site by its conversion into glutamic acid . Thus, we were able to see the effect of single site modification with respect to major lamin properties .It is commonly known that one of the main functions of the phosphorylation of lamins is to facilitate nuclear envelope breakdown, which probably occurs due to effects on the ability of lamins to polymerize. But we do not know whether, and if so which, other functions and interactions of lamins, such as chromatin binding or lamin location during mitosis, are affected by mitotic phosphorylation. Moreover, we do not know anything about the role of the interphase phosphorylation of lamins and if there is any difference in the function of phosphorylation between B and A type lamins. Besides, for better understanding of lamin functions it is important to know what is the exact effect of phosphorylation of a particular site. We know that lamins may be phosphorylated on the same site by different kinases and typically up to three phosphorylation sites are modified at the same molecule (lamin Dm) so different combinations of phosphorylated sites may result in different phenotypic effects. It is essential to know the effect of single sites before we start to elucidate the function of simultaneous phosphorylation on many sites, as it is in vivo. That is why we started the project of generation of “stably phosphorylated” (pseudophosphorylated) lamin Dm mutants. The idea was to use a simple model system to mimic single phosphorylation of a particular known Drosophila lamins may be used as a good model for studying the effect of phosphorylation on both types of lamins. Because to date there are no reports about experimentally confirmed phosphosites on the fruit fly lamin C, we used Clustal W for detection of potential, evolutionary conservative sites located in this protein. The results showed that S45 in lamin Dm and S37 in lamin C lie within the very conservative, classical cdc2 phosphorylation motif present in all lamins. The tail domain of Drosophila lamins contains two typical regions of phosphorylation sites: one located immediately next to the coiled-coil domain and near the NLS sequence and the second at the end of the tail domain. The site containing T435 represents a potential cdc2 site but this is not a typical, conservative cdc2 site present in vertebrate lamins (435 lies near or within a less conserved region responsible for interaction of lamin Dm with histones H2A and H2B in vitro (TRAT sequence) 405 (S405PGR). Thus Drosophila lamins lack a typical, conservative C-terminal cdc2 motif characteristic of vertebrate lamins but possess a classical N-terminal cdc2 site.e lamins . Instead37E mutant representing modification of the N-terminal cdc2 site as a control for A-type lamin. For lamin Dm study we used wild type protein, lamin Dm S25E, lamin Dm S45E, lamin Dm T435E and lamin Dm S595E. We also used lamin Dm R64H as an additional, control protein that is very well characterized in vitro, and unable to polymerize longitudinally Hence for the current study we decided to choose the following lamin proteins: wild-type lamin C and lamin C S435E was more resistant to thermal denaturation than wild type protein, while lamin Dm S595E was the least resistant. The calculated contents of secondary structures of analyzed proteins are shown in In order to assess the secondary structure of bacterially expressed lamins, we analyzed their circular dichroism (CD) spectra . All wilin vivo phosphorylation of lamins is to affect lamin polymerization. We decided to confirm which particular lamin Dm phosphosites are responsible for modulation of polymerization using single pseudophosphorylation mutants. We examined the solubility of lamins in a sedimentation assay using western blotting for lamin detection in the pellet fraction  and in the supernatant fraction   increased solubility of the protein. This indicates that phosphorylation of the C-terminal alone, but not the N-terminal conserved cdc2 site on lamin Dm may be sufficient for solubilization of lamin Dm in vitro.The only well-documented function of specific ed form) . As an ain vivo should be the loss of interaction between lamins, chromatin and nuclear envelope components. To test this assumption for Drosophila lamins, we used isolated Xenopus sperm chromatin as a target for lamin binding in vitro. Only lamin C S37E mutant failed to bind to chromatin in vitro  as well as when chromatin was decondensed using interphase extracts from unfertilized Xenopus eggs. All lamin Dm proteins were able to associate with Xenopus pronuclei  fusion proteins. As a control, we used human wild type lamin A, B and C also as EGFP fusion proteins  of these proteins. This suggests that lamin Dm S45E and T435E mutants have impaired ability to be incorporated into the cell nucleus and the nuclear lamina compared to lamin Dm and other mutants. Since all lamin Dm mutants bind to chromatin in vitro and lamin Dm T435E does not polymerize while lamin Dm S45E polymerizes in vitro, this suggests that for lamin Dm location in the nuclear lamina in vivo, at least one property other than polymerization ability and chromatin binding ability is important as well. This also indicates that phosphorylation of S45  alone is not sufficient for solubilization of lamin Dm in vivo, at least in S2 cells. Similarly, the phosphorylation of T435, which affects polymerization in vitro, has only a moderate effect on location of this mutant in vivo.Overexpression of lamin Dm, lamin Dm Slamin Dm . Lamin DXenopus sperm chromatin and HeLa chromatin in our in vitro studies, we overexpressed the same lamin Dm mutants in HeLa and Xenopus XTC cells. As a control, we used human wild type lamin A, B and C. In order to test the generality of the lamin Dm interactions and due to the use of 25E and S595E demonstrated predominant nuclear location while S45E and T435E mutants were located both in the nucleus and cytoplasm. Over 20% of cells showed, beside nuclear, also cytoplasmic location of these mutants  modification had a greater effect on location of lamin in HeLa cells than in Drosophila cells. This may indicate the additional role played by interacting protein partners of lamins in their nuclear lamina retention. In Drosophila, abnormal location is less frequent, probably due to the interactions, while in human cells lamin Dm may not interact or interact with lower affinity. Observed differences can also be connected with different physiological temperatures of human and insect cells lines, which can affect the protein properties . It should be pointed out that transfection of wild type human lamin A, B1, C as well as lamin Dm and mutants into HeLa cells similarly decreased the mitotic index of cells (below 0.5%) and induced apoptosis. The highest level of apoptosis (about 35%) was shown by lamin Dm T435E, the lowest by lamin A (about 10%). Preliminary analyses of cell cycle progression in connection with cytoplasmic/nuclear location of lamins do not show any specific correlation. There is, however, a correlation between the level of overexpression of exogenous protein and apoptosis. An approximately 50% decrease is observed in the number of cells highly overexpressing exogenous lamin between 24 h and 48 h. This effect is due to the increased rate of apoptosis in such cells and is characteristic for all lamins, both human and fly.Wild type lamin Dm-GFP and mutants S mutants . All lamDetailed analyses of distribution of lamin Dm and endogenous lamins in HeLa cells demonstrated that most lamin Dm does not colocalize with endogenous lamins  at the nuclear envelope. Lamin Dm proteins localize at the nuclear envelope as small “domains” or form bigger fragments up to “budding” fragments of nuclear envelope (not shown).In order to test the hypothesis that the effect of lamin Dm pseudophosphorylation can be moderated or hidden by the effect of farnesylation of the C-terminal CAIM motif and nuclear or nuclear lamina retention may be the effect of nuclear envelope targeting by the farnesyl moiety, we prepared the same set of mutants (as EGFP fusion proteins) but with additional mutation in the C-terminus which prevents farnesylation.435E mutant, which was predominantly cytoplasmic after 24 and 48 h post-transfection  analysis. Experiments on transfected S2 cells and transgenic flies  expressing wild type lamin Dm-GFP demonstrated that lamin Dm in transgenic flies and all mutants in S2 cells have the same mobility. The mobility was similar to mobility of human lamin B-GFP transfected into HeLa cells. It is noteworthy that our previous experiments indicated that lamin Dm mutants transfected into S2 cells were predominantly nuclear and that farnesylation of lamin Dm facilitates its targeting to the nuclear envelope. This may affect mobility independently of polymerization ability of a particular lamin Dm mutant in vivo.In order to gain insight into mobility of transfected fusion proteins and polymerization ability of lamins 435E fusion protein showed higher mobility than other tested mutants or wild-type lamins and only about two times lower than nuclear EGFP alone.Therefore we decided to measure the mobility of farnesylation incompetent lamin Dm mutants, using the FRAP method. We expressed lamins as fusion proteins with EGFP in HeLa cells. In FRAP experiments, fluorescent molecules were irreversibly photobleached in a defined area by a single, high-powered laser pulse. Next, the recovery of the fluorescent signal in the bleached regions was measured and expressed as the recovery. in vivo several sites may be phosphorylated on a single molecule in vivo lamin Dm can be phosphorylated on S25 and S59519 and T597in vitro and no phosphorylation is possible, wild type lamins seem to be an adequate control. Besides, for sterical reasons, threonine should have been substituted by an amino acid with an identical side chain, which does not exist. Moreover, substitution of serine or threonine for alanine may affect the structure or interaction of such a mutant. A recent example might be the identical effect of mutations T435D and T435A in a 30 kDa lamin Dm fragment interacting in vitro with histones In order to keep the experimental system as simple as possible we decided to analyze only single phosphorylation sites, although in vivo phosphorylation sites on lamin C have been published, in our study we used only lamin C S37E mutant, representing the putative N-terminal cdc2 site. Based on the structure and the sequence of lamins, S37 seems to be the equivalent of the identified in vivo S22 in mammalian lamin A, S28 from goldfish lamin A 45 in lamin Dm. The lamin C region containing the C-terminal cdc2 site did not show such a high level of similarity to other lamins, as the N-terminal region shows  sites flanking the rod domain ase site . The potIn vitro lamin Dm can be either depolymerized or its polymerization inhibited by cdc2 kinase, PKC or PKA 64C, R64H, I396V) showed decreased polymerization ability D. melanogaster lamin polymerization in vitro.in vivo phosphorylation sites were detected for mitotic lamin B The lack of the effect of modification of the N-terminal cdc2 kinase site at lamin Dm on the polymerization is supported by the similar data obtained for vertebrate B-type lamins where C-terminal in vitro37 pseudophosphorylation is sufficient for inhibition of polymerization of lamin C in vitro. Our discovery that phosphorylation of lamin C on the N terminal cdc 2 site is sufficient  for increasing solubility in vitro  also support our thesis. The FCS data indicated that increased mobility of lamin Dm mutant T435E in vivo is the result of decreased polymerization since in vitro diffusion times for lamin Dm T435E and Dm R64H were similar  and ten times lower than for lamin Dm . Similar differences in diffusion were obtained in FRAP experiments.The presented FRAP data also indicated that pseudophosphorylation of T in vivo . Prelimiin vivo phosphorylation on lamin Dm in fly embryos suggested S45  and T432 or T435  as phosphosites during embryogenesis with molar stoichiometry of about 0.75 and 0.2 mol phosphate/mol protein respectively 45 was identified in the soluble lamin Dm fraction in early (0–5 h old) embryos. The C-terminal cdc2 site (T432/435) was identified in the late embryos in the lamin Dm fraction, which was insoluble. These results seem, at the first look, to be in contradiction to what should be expected about the function of B-type lamin phosphorylation at the cdc2 site (e.g. S405 is a predominant “mitotic” phosphorylation site on human soluble lamin B1 in vivo) 435), but not the N-terminal site (S45) is sufficient for lamin Dm solubilization. There may be several plausible explanations for such differences. Typically, lamin Dm in somatic cells is phosphorylated on two to three sites Drosophila early embryos on S45 may serve either as a storage form of maternally provided lamin Dm or as a necessary component of semi-closed mitosis in syncytial Drosophila embryos (with other unidentified phosphosites) or both. Moreover, early embryo soluble lamin Dm named Dmmit was found to be copurified with a 90 kDa protein 404 interacting with nuclear 14-3-3 protein in vitro experiment in which interphase insoluble lamin Dm can be made soluble by addition of anti-lamin Dm IgGs 45 is probably not the only early embryo  site and definitively not the only site in somatic cells because mutant S45E has the same properties as wild type lamin Dm. Instead, T435 residue seems to be the best available candidate, since mutant T435E is soluble.The last report on the 37E perfectly fits the ideal model of the effect of mitotic phosphorylation. This single site mutant was soluble, was not able to bind to chromatin or the nuclear envelope, and was unable to associate with the reforming cell nucleus in in vitro nuclear assembly. This indicates a much stronger effect of the N-terminal cdc2 site phosphorylation than in vertebrate A-type lamins, where both cdc2 sites have to be modified to observe the full effect Among all tested lamins, only lamin C S45 mutant and T435 mutant show only partial mislocalization in S2 cells is that farnesylation, by providing the mechanism for nuclear lamina retention as well as the possibility for ER lateral transport into the nucleus, “hides” the effect of phosphorylation on lamin location  in response to phorbol ester treatment significantly reduced the import of lamin B2 into the nucleus Transient transfection studies on HeLa cells with farnesylation incompetent lamin mutants demonstrated that all lamin Dm proteins, except T nucleus , 6. The in vivo directly or indirectly. Lamin Dm interacts directly with DNA in vivo during interphase but not during mitosis Drosophila lamin C does not interact in vivo with DNA at all. In vitro invertebrate and vertebrate lamins bind scaffold associated or matrix attachment regions of DNA (SAR/MAR DNA) in vitro to chromatin or nucleosomes D. melanogaster lamin Dm, which binds to chromatin through histones H2A and H2B in vitro to chromatin through histones H2A and H2B 432D, T435D but also T432A and T435A inhibited binding. The authors suggested the conservative sequence T432RAT is essential for binding. Deletion of the NLS sequence also decreased binding to chromosomes similarly to human lamin A binding to DNA Drosophila lamins, only lamin C mutant S37E did not bind to any chromatin. All other proteins did bind to chromatin from Xenopus (Drosophila embryos (not shown), which suggested that other regions of lamins may also be involved in interactions with chromatin. This binding ability is not simply dependent on polymerization since lamin Dm R64H and T435E did bind to chromatin. Lamins interact with chromatin in at least two modes, through nucleic acids and through histones, and each way of binding requires different lamin fragments for interaction. Thus phosphorylation of the C-terminal cdc2 site on lamin Dm, while possibly affecting interactions through histones, may not affect interactions with nucleic acids or other protein(s) associated with nucleic acids.Lamins bind chromatin components  Xenopus , S4, HeLXenopus (ARTI) (432RATTPS) is present in the N-terminal fragment of lamin C (RV13RASSTSTP). Phosphorylation of S37 on lamin C may affect a hairpin-like structure of the head domain of lamin C and “hide” this potential histone binding motif. The best example of such a sterical change may be the report that in vitro phosphorylation of S25 on lamin Dm inhibits binding of monoclonal antibody ADL84 to its epitope RPPS25AGP. But also single point mutations in lamin Dm protein distant to this epitope or on the rod domain  abolish antibody binding 37 phosphorylation on lamin C may also induce conformational change in the head domain of lamin C, which in turn prevents binding to chromatin through histones.The lamin Dm sequence flanking from the C-end the C-terminal cdc2 site is the site which is necessary for binding to the chromatin through histones s (ARTI) . SurprisExperimental data indicate that in vertebrates, lamins A/C reassociate with the decondensing chromatin and reforming nuclear envelope later than B-type lamins Xenopus laevis Lamin B1 (AAC31543); Lamin B2 (AAC31544); Lamin-L (Lamin-B3) (P10999); Lamin-A (P11048). Gallus gallus (chicken) lamin B2 (NP 990616); Homo sapiens (human): Lamin B1 (AAA36162).Lamin Dm (AAF52262); Lamin C (NP_523742); Lamin-1 (CeLam-1) (Ce-lamin) (Q21443); Xenopus laevis LAP2 (xLAP2) protein was a kind gift of Prof. Kathrin Wilson  64H in expression vector pET20b(+) was a kind gift of Prof. Yosef Gruenbaum  The expression vector pET23a xLAP2 1–165 for the N-terminal 165 aa fragment of Drosophila melanogaster lamin C coding sequence from previously cloned pET17xblamC vector gaggcgggtccgcagc-3′5′-ccggccgcca; ctctggcggccgg-3′5-gctgcggacccgc; LamDmS45E: gagcccacccggcactcg-3′5′-ctccagccccctc; ctcgagggggctggag-3′5′-cgagtgccgggtggg; LamDmT435E: gagccatcgcgtcgcac3′5′ctccacgcgagcc; ctcggctcgcgtggag3′5′gtgcgacgcgatgg; LamDmS595E: gaggtgaccgccgtggacgg3′5′ctgagtcgtcgtcgc; ctcgcgacgacgactcag3′5′ccgtccacggcggtcac; LamCS37-E: gagcccacgcgcaccagc3′5′cctcacccacc; ctcggtgggtgagg3′5′gctggtgcgcgtggg. Sequences of the mutants were confirmed by DNA sequencing.The full length E. coli BL21DE3(pLysS) (Stratagene) strain was transfected with derived expression vectors. Protein expression was induced with 1 mM IPTG for 6 h. Bacteria were harvested by centrifugation, washed with PBS and suspended in PBS buffer containing 0.1% Tween 20. Bacterial cells were lysed by sonication, extracted on ice for 10 min followed by centrifugation at 12000 g for 15 min. Resultant supernatant was collected for analysis. The obtained pellet was extracted successively with PBS buffer containing 1% Triton X-100, 3 M urea and 6 M urea. His-tagged lamin fusion proteins were purified on Ni-NTA-agarose columns (Qiagen).For protein overexpression in bacteria, the Proteins were incubated in PBS buffer at room temperature for 60 min at the concentration of 100 µg/ml. After incubation, samples were centrifuged at 12 000 g for 20 minutes. Pellet fractions were solubilized for SDS PAGE. Supernatant fractions were TCA precipitated  for 10 min on ice and proteins collected by centrifugation . Resultant pellets were washed twice with ice cold acetone and solubilized for SDS PAGE. Pellets and supernatants from the sedimentation assays were analyzed in immunoblotting. Immunoblots were digitalized and analyzed in gel documentation equipment with a CCD camera and using Bio-Profil V99 software (Vilber Lourmat). Quantitative data were obtained from 3 independent experiments and were expressed as the ratio of the amount of a particular protein in the pellet or supernatant fraction compared to the total amount of protein in both fractions.Egg collection and extract preparation were according to Salpingidou et al. Xenopus was used in the condensed form and in the decondensed form prepared by incubation of sperm chromatin with poly L-glutamic acid in Pfaller buffer 5 sperm was incubated in Pfaller buffer with 0.5 µg/µl poly L-glutamic acid for 20 min at room temperature in a total volume of 40 µl. For sperm chromatin binding assays the bacterially expressed proteins (0.25 µg–1 µg) were added to the condensed or decondensed chromatin (about 105) in extraction buffer in a total volume of 50 µl. The reaction proceeded for 15 min at room temperature. Chromatins were recovered on glass coverslips and processed for immunofluorescence analyses according to Drummond et al. Isolated and demembranated sperm chromatin (DSP) from Xenopus laevis. The Local Ethical Committee License for experiments with Xenopus is permanent and was issued on 02.07.2003 . The Polish National Ethical Committee (KKE) issued a license to the Faculty of Biotechnology, University of Wrocław on behalf of the Laboratory of Nuclear Proteins for animal maintenance and performing experiments on animals . The Laboratory of Nuclear Proteins has certification from the Polish Ministry of Science and Higher Education for cultivation of animals and performing research .The Laboratory of Nuclear Proteins has all necessary documents, licenses and the Local Ethical Committee license for work with Xenopus sperm pronuclei were reconstituted according to Salpingidou et al. Xenopus XTC cells were grown on 12 mm cover slips (in a 50 mm culture dish plate) up to 80% confluency as described previously Drosophila lamin Dm (ADL 67) and lamin C (ALC 28). The HeLa cell line was obtained from Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław. The XTC cell line was obtained from the Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, UK.HeLa cells or Xenopus XTC cells were trypsinized, centrifuged at 118× g for 5 min in 50 ml Falcon tubes and resuspended in NIB buffer nucleus isolation buffer  1 ml/106 cells. After 15 minutes on ice, cells were homogenized and centrifuged through 30% sucrose in NIB in Eppendorf tubes . Cell nuclei were suspended in NIB buffer. Isolation of cell nuclei from Drosophila embryos and S2 cells was described previously Human cells or 5 of cell nuclei or equal amounts of chromatin in 20 µl of PBS and incubated for 20 min at room temperature. Fixation was in 100 µl of 1% EGS in PBS for 30 min at 37°C. Samples were layered over 30% glycerol in PBS-Tween (0.05%) and centrifuged onto 22 mm coverslips , fixed with formaldehyde and processed for immunofluorescence.Between 1 and 0.25 µg of each protein was added to the 10Donkey anti-rabbit-FITC conjugated and donkey anti-mouse-TRITC conjugated (Jackson ImmunoResearch) were used at a dilution of 1∶50. ADL67 mouse monoclonal anti-lamin Dm was used at a dilution of 1∶2. LC 28 mouse monoclonal antibodies against lamin C were used at a dilution of 1∶3. R389 rabbit serum against lamin C was used at a dilution of 1∶50 for IF. Affinity purified antibodies for bacterially expressed human lamin C were used at 1∶20 dilution. Affinity purified IgGs from rabbit serum against lamin Dm were used at a dilution of 1∶50 2 dmol−1 using the following equation: [θ] = [θobs/(10×lxc)]/n, where θobs is the observed ellipticity in degrees, l is the optical path length of the cell in cm, c is the protein molar concentration, and n is the number of residues in the protein. Measurements were performed in phosphate buffer .CD (circular dichroism) spectra were collected with a J-175 spectropolarimeter (Jasco) using 2 nm bandwidth and response time of 1 s. Sampling was every 1 nm. Each curve represents the medium value for 3 scans. All experiments were carried out using cuvettes of 0.1 cm path length. Ellipticity was expressed as mean residue ellipticity [θ] in deg cmDrosophila Schneider 2 cells, Invitrogen) were transfected by electroporation with CLB-Transfection Device (Lonza), program CELL5, using approx. 107 cells and 10 µg of plasmid DNA. Transfection efficiency was about 30–50% in the case of HeLa cells and up to 10% in S2 cells. Based on calculations performed on transfected HeLa, S2 and HEK293 cells (90% efficiency of transfection) followed by western blot and IF analyses, it is possible to estimate that most of the transfected HeLa cells expressed exogenous lamins at a comparable amount to endogenous lamins. It was similar in S2 cells.HeLa cells  were transfected by plasmids encoding lamins by using Metafectene reagent  according to the manufacturer's instructions. S2 cells  were previously adapted to grow at room temperature without carbon dioxide. XTC cells and S2 cells normally grow at room temperature without carbon dioxide. Cells were cultured on glass coverslips for 24 h and 48 h after transfection and analyzed under a 40× (water immersion) objective in culture medium under a Zeiss LSM 510 Meta microscope according to the manufacturer's instructions. For each mutant FRAP analyses were performed on lamin proteins located in the nuclear interior, nuclear envelope and cytoplasm (if available). Mobility analyses were performed many times for each compartment. For calculations, at least three measurements, of the best quality from each compartment were selected. We used FRAP analysis software (Zeiss LSM510 software - ZEN 2008) for experiment setup and data collection  and calculation of values for the mobile fraction (Mf measured in %) and time required for 50% fluorescence plateau recovery of the bleach zone (t1/2 measured in seconds), using normalized fluorescence intensity. The diffusion coefficient [D measured in µmFigure S1Overexpression and purification of lamin Dm, lamin C and mutant proteins. 12% SDS-polyacrylamide gel electrophoresis of fractions from the purification of wild type lamin Dm and Lamin Dm S45E (Panel A and B respectively) together with lamin C and mutant lamin C S37E (Panel C and D respectively) and all purified lamins (Panel E). The lamin proteins were overexpressed in E. coli BL21DE3(pLysS) bacterial cells. Bacteria were lysed and proteins were solubilized from inclusion bodies in buffer containing 6 M urea and purified by metal affinity chromatography on Ni-NTA-agarose columns. Please note that relative electrophoretic mobility of particular protein may vary from gel to gel and lane to lane due to the different proteins concentration, buffer composition, position etc. Panel E illustrates the quality of purified lamin proteins. M – Prestained Protein Molecular Weight Marker (Fermentas), BSA  – 2.5 µg, S4 – proteins extracted in buffer containing 6 M urea, U – unbound fractions, E – elution fractions.(TIF)Click here for additional data file.Figure S2Circular dichroism (CD) spectra of the purified wild type lamin C and lamin Dm, and their mutants. Curves representing different temperatures are marked by different colours. Initial curves on both panels have two peaks, one at 208 nm and the second at 220 nm, demonstrating typical shape for native proteins containing a large proportion of α-helical structure. The denaturation was found to be largely reversible (R - renatured CD spectra).(TIF)Click here for additional data file.Figure S3All lamin Dm proteins bind to chromatin and nuclear envelope in in vitro nuclear assembly system from Xenopus.In vitro Xenopus sperm pronuclei assembly reaction was used to assess the ability of lamin Dm mutants to bind to assembling chromatin and nuclear envelope structures. Assembly reaction was carried out for 40 min in the presence of bacterially expressed proteins.(TIF)Click here for additional data file.Figure S437E protein does not bind to Mutant lamin C Sin vitro assembled Xenopus sperm pronuclei.In vitro Xenopus pronuclei assembly reaction was used to assess the ability of bacterially expressed lamin C and lamin C S37E mutant to bind to assembling chromatin and nuclear envelope structures. The control experiment was without addition of any exogenous protein. Assembly reaction was carried out for 30 min in the presence or absence of bacterially expressed proteins.(TIF)Click here for additional data file.Figure S5Localization of fusion EGFP: lamin A, lamin B and lamin C 48 hours post-transfection into HeLa cells visualized under confocal microscope in order to visualise differences in phenotype depending on fusion protein expression level. Cells were stained for DNA with DAPI and for lamin C with rabbit affinity purified antibodies. Staining with secondary antibodies was with goat anti-mouse secondary antibodies conjugated with TRITC and goat anti-rabbit secondary antibodies conjugated with Cy-5 respectively. Particular lamin fusion proteins were visualized by EGFP fluorescence. Single confocal Z-sections are shown through the center of nuclei. Only the most typical phenotypes are shown.(TIF)Click here for additional data file.Table S1The content of distinct secondary structure types present in analyzed lamins and residue molar ellipticity. The content of distinct secondary structures in lamin proteins was calculated using CDFIT software. Molar ellipticity values were measured at 20°C in PBS with 0.6 M urea.(DOC)Click here for additional data file.


The role of the high affinity IgE receptor, FcεRI, in IgE-mediated immune responses of the gastrointestinal (GI) mucosa is poorly understood. Currently, a detailed characterization of FcεRI expression throughout the human gut is lacking. The aim of this study was to define the expression pattern of FcεRI in the GI tract.We compared FcεRI expression in children with gastritis/esophagitis (n = 10), celiac disease (n = 10), inflammatory bowel disease (IBD) (n = 9), and normal mucosa (n = 5). The α–subunit of FcεRI (FcεRIα), detected by immunohistochemistry, was found on cells infiltrating the mucosa of the esophagus, the stomach, and the duodenum, but was rarely detected in more distal sections of the GI tract. Accordingly, quantitative RT-PCR analysis on esophagus, stomach, duodenum, colon, and rectum biopsies revealed that FcεRIα and -β expression levels decreased towards the distal intestine. mRNA transcripts of the common Fc-receptor-γ chain were present in the entire GI mucosa. Double-immunofluorescence staining of esophageal specimens confirmed that FcεRIα was expressed on intraepithelial mast cells and Langerhans cells. The mRNA expression levels of the α, β, and γ subunits of FcεRI did not correlate with total serum IgE but were associated with mucosal inflammation.Our data define the upper GI tract as the main site for IgE-mediated immune activation via FcεRI. Tissue mRNA levels of FcεRIα are regulated by inflammatory conditions rather than serum IgE, indicating that FcεRI might also play a role in pathologies other than allergy. The gastrointestinal (GI) mucosa is a large interface area for pathogens and environmental antigens and, therefore, is under constant surveillance of the immune system. Immunoglobulin (Ig) receptors are gatekeepers of host defense at mucosal surfaces; they shuttle Ig-antigen complexes across the healthy epithelium and activate protective immune responses. Misguided immune responses, however, can lead to inflammation of the gut or other allergic reactions towards harmless allergens. IgE and its cellular receptors are key players in allergic reactions and parasite defense. Humans express three IgE-receptors, the high affinity IgE-receptor, FcεRI, and two low-affinity IgE receptors, FcεRII (or CD23), and ε-binding protein εBP  2) on the surface of mast cells and basophils, and in a trimeric form (FcεRIαγ2) on eosinophils, macrophages, and dendritic cells (DCs) FcεRI is a multimeric receptor of the immunoglobulin receptor superfamily and binds the Fc-part of IgE with its immunoglobulin domain-containing α-chain. Allergen-mediated crosslinking of IgE-FcεRI complexes on the surface of blood and tissue cells then triggers the allergic cascade via the receptor’s signaling subunits, FcεRIβ and FcεRIγ In the GI tract, FcεRI-expressing DCs of the Langerhans cell type have been described in the oral mucosa A detailed analysis of mucosal FcεRI expression throughout the GI tract is currently not available. The aim of the present study, therefore, was to characterize the expression pattern of FcεRI throughout the GI tract and to investigate the impact of serum IgE levels and mucosal inflammation on FcεRI expression levels.We investigated mucosal specimens from a total of 34 pediatric patients . Patients had a diagnosis of gastritis/esophagitis (n = 10), celiac disease (n = 10), or inflammatory bowel disease (IBD) (n = 9). Biopsies of 5 patients did not show any mucosal pathology and served as normal controls. Total serum IgE was measured at the time of endoscopy. Fifteen patients had elevated serum IgE levels . In 19 patients, IgE levels were within the normal range. Patients’ characteristics are summarized in We conducted immunohistochemistry staining of snap-frozen mucosal biopsies from all sections of the GI tract accessible by endoscopy. FcεRIα was frequently found on cells infiltrating the mucosa of the upper GI tract , in 5/34 gastric samples, 8/33 duodenum samples, 1/12 terminal ileum samples, 2/13 colon samples, and 1/13 rectum samples . FcεRIβ IgE-binding to FcεRIα stabilizes the receptor complex at the cell surface Our experiments permit us to conclude that our findings regarding the correlation of serum IgE levels and FcεRI expression in peripheral blood We have previously shown that FcεRI-bearing immune cells infiltrate the esophageal mucosa of children with reflux esophagitis and EoE, two inflammatory conditions of the esophagus We show here that the high affinity IgE receptor, FcεRI, is highly expressed on cells infiltrating the mucosa of the esophagus, the stomach, and the duodenum, and shows a low level of expression in more distal parts of the GI tract. Accordingly, tissue mRNA of all three FcεRI subunits was detected in the intestinal mucosa. FcεRIα-, β-, and γ-mRNA levels did not correlate with total serum IgE, but were modulated by gastrointestinal inflammation.FcεRIα mRNA was most abundant in the esophageal mucosa and could be detected in every patient. FcεRIβ mRNA levels, on the other hand, were rather low in the esophagus and peaked in the gastric mucosa. This could reflect the predominance of cells expressing the trimeric receptor isoform, such as dendritic cells and eosinophils, in the esophagus. In the stomach, cells bearing the tetrameric FcεRIαβγ2, such as mast cells and basophils, could be the main FcεRI-expressing population. This interpretation is supported by previous studies showing that the gastric mucosa is particularly rich in mast cells The binding of IgE, the natural ligand to FcεRI, increases FcεRI expression on peripheral blood cells and Langerhans cells Total IgE values in mixed-age pediatric populations are not directly comparable due to differing age-related normal ranges. To overcome this problem, absolute IgE levels are commonly converted into age-normalized z-values as published by Kjellman et al. We observed that mucosal inflammation was associated with FcεRIα mRNA levels, based on our finding that patients with a histopathological diagnosis of esophagitis displayed the highest FcεRIα mRNA expression. The highest copy numbers of esophageal FcεRIα mRNA were found in two celiac disease patients. According to routine pathology records, these two patients also had intraepithelial inflammatory infiltrates in the esophagus. Therefore, high FcεRIα mRNA expression could be a surrogate for invading FcεRIα-positive inflammatory cells. Further supporting this argument, previous studies identified intraepithelial Langerhans cells and mast cells in the inflamed esophageal mucosa of children with EoE and reflux esophagitis as the main cellular sources of FcεRI The role of the low affinity IgE receptor, CD23, in transepithelial transfer of IgE-allergen complexes is well established In summary, we show here that FcεRI is robustly expressed in the upper GI mucosa of pediatric patients at steady state. Inflammatory conditions, independent of the underlying pathology, lead to increased FcεRI tissue mRNA expression. In conclusion, our data define the upper GI tract as the main site for IgE-mediated immune activation via FcεRI. Once the immune homeostasis is disturbed by an inflammatory condition such as IBD, the lower GI tract becomes susceptible to IgE-mediated immune reactions as well. The fact that we see tissue FcεRIα mRNA regulation by inflammatory conditions independent of IgE-mediated disorders indicates that this receptor might also play a role in pathologies other than allergies.The study protocol was approved by the Ethic’s Committee of the Medical University of Vienna. Patients or their legal guardians provided written informed consent.Children between 2 and 18 years of age who underwent elective diagnostic endoscopy of the upper and/or lower GI tract at the Department of Paediatrics and Adolescent Medicine, Medical University of Vienna were randomly invited to participate. Subjects with increased risk of bleeding or perforation and patients scheduled for endoscopic interventions or undergoing non-elective endoscopy were excluded. Endoscopic biopsies from the esophagus, the stomach, the duodenal bulb, the terminal ileum, the ascending colon, and the rectum were either fixed in OCT Tissue Tek  and stored at −80°C until further processing for RT-PCR or snap-frozen for immunohistochemistry analysis. Tissue specimens were obtained from sites free of macroscopic mucosal pathology.FcεRIα in frozen sections of gastrointestinal specimens was detected with mAb 15.1 Double immunofluorescence staining for FcεRIα and the mast cell marker c-kit or the Langerhans cell marker CD1a were performed on frozen esophagus specimens. Sections were acetone-fixed, incubated with 1 mg/ml sodium borohdyride , and blocked with 5% v/v normal donkey serum . Sections were then exposed to mouse anti-FcεRIα  and goat anti-CD1a  or rabbit anti-c-kit  overnight. CY5-conjugated donkey anti-mouse-, Dylight 549 donkey anti-goat-, and Dy549 donkey anti-rabbit-antibodies  were used as secondary antibodies. Secondary antibody controls were performed in parallel with each experiment. After they were washed, the sections were mounted with Prolong Gold anti-fade mounting media containing DAPI . FcεRIα/CD1a double-staining was investigated on a Zeiss AxioImager M1 wide-field fluorescent microscope with a 20x/0.8 N.A. plan-apochromat objective lens and the AxioVision software. FcεRIα/c-kit double staining was investigated on a Zeiss LSM510 Meta confocal system and Zeiss LSM510 image acquisition software. The images were taken with a 20x/0.8 plan-apochromat objective and a 40x/1.3 oil plan-apochromat objective.Total mRNA was isolated from mucosal specimens using the GenElute™ Total RNA MiniPrep Kit  and subjected to reverse transcription with the iScript™ cDNA synthesis kit  following the manufacturer’s instructions. Quantification of mRNA was performed using the iQ SYBR Green Supermix (Bio-Rad) on a CFX96 Real time System (Bio-Rad). The thermal profile of the PCR reaction was: initial denaturation at 95°C for 3 minutes, followed by 30 cycles of denaturation at 95°C for 10 seconds and annealing/elongation at 63°C for 30 seconds, and a final melting curve. The following primers were used to amplify cDNA: FcεRI α: forward: 5′GAA ATG TGA CCT GCT GCT GA3′, reverse: 5′TGT GGC AGC TGG ACT ATG AG3′; beta-Actin: forward: 5′CTC TTC CAG CCT TCC TTC CT3′, reverse: 5′AGC ACT GTG TTG GCG TAC AG3′. Primer sets for FcεRIβ and -γ subunits and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were purchased from Qiagen . Relative expression of FcεRIα,-β and -γ was calculated according to the Pfaffl method Serum samples for IgE measurement were collected at the time of endoscopy.nd year of life, 150 kU/l for the 3rd year of life, 190 kU/l for the 4th and 5th years of life, 150 kU/l for the 6th year of life, 120 kU/l for the 7th to the 16th year of life, and 100 kU/l for patients older than 16 years.Total IgE was determined using the Phadia ImmunoCAP® solid phase immunoassay . Total serum IgE levels are given in kU/l. For some analyses, patients were subgrouped into elevated- and normal-IgE-patients. Expected normal ranges for this assay are 40 kU/l for age < one year, 100 kU/l for the 2Statistical analyses were carried out with SPSS software . Differences in tissue mRNA levels between groups were tested for statistical significance with two-sided Kruskal-Wallis-H- test and two-sided Mann-Whitney U-test with Bonferroni-Holm correction. Correlations of data with serum IgE were determined using Spearman rank correlation test. For certain analyses, total serum IgE values were transformed into z-units to adjust for age dependent normal values as published by Kjellman et al.


The ArfA/RF2 and ArfB systems rescue stalled ribosomes directly by hydrolyzing the immature peptidyl-tRNA remaining on the ribosome. Here, the biochemical aspects of these systems, as clarified by recent studies, are reviewed.During protein synthesis in cells, translating ribosomes may encounter abnormal situations that lead to retention of immature peptidyl-tRNA on the ribosome due to failure of suitable termination processes. Bacterial cells handle such situations by employing three systems that rescue the stalled translation machinery. The transfer messenger RNA/small protein B (tmRNA/SmpB) system, also called the  During cellular protein synthesis, ribosomes interact with a variety of macromolecules to enable the precise translation of genetic information into functional polypeptides. One of the most frequent ribosomal interactions is the process of sense codon decoding, in which a codon of mRNA interacts with an anticodon of tRNA in the A site of the ribosome. In bacteria, elongation factor Tu (EF-Tu) plays a crucial role in this process by delivering aminoacyl-tRNA to the ribosomal A site as part of a ternary complex (aminoacyl-tRNA/EF-Tu/GTP). When the anticodon of aminoacyl-tRNA in the ternary complex matches the mRNA codon in the ribosomal A site, GTP hydrolysis is stimulated and EF-Tu changes its conformation, leading to dissociation of the EF-Tu/GDP binary complex, accommodation of aminoacyl-tRNA at the A site of the ribosome, and transfer of a nascent polypeptide to the aminoacyl-tRNA.Although its molecular mechanism is similar to that of sense codon decoding, termination codon decoding does not require both tRNA and EF-Tu. In this process, a termination codon of mRNA interacts with class-1 release factors (RFs) including RF1 and RF2, at the A site of the ribosome. Like the anticodon of tRNA in sense codon decoding, a specific region of class-1 RFs, known as a peptide anticodon , the alternative ribosome rescue factor A (ArfA)/RF2 system, and the alternative ribosome rescue factor B (ArfB) system are involved in the rescue of stalled ribosomes. This review focuses on the biochemical aspects of these three processes, as elucidated by recent studies.In bacteria, the transfer messenger RNA/small protein B (tmRNA/SmpB) system  of the tmRNA, which contains an authentic translation termination codon. The tmRNA-encoded peptide sequence attached to the C-terminus of newly synthesized proteins, known as an SsrA tag, acts as a degradation signal for proteolysis. Through this process, stalled ribosomes are recycled for new translation reactions and the SsrA-tagged proteins are degraded immediately, which may contribute to the maintenance of both the translation process and the quality of cellular proteins .The Escherichia coli tmRNA consists of 363 nucleotides and contains a tRNA-like domain (TLD), an ORF region encoding an SsrA tag, and four pseudoknot structures . One of the characteristic features of E. coli tmRNA is the TLD, in which the 5′ and 3′ terminal regions of tmRNA combine to form a tRNA-like structure. Like standard tRNA, tmRNA has an acceptor stem with a 3′-CCA end, as well as a TψC stem and loop structure that includes conserved bases. These features of tmRNA not only facilitate its recognition and processing by RNase P  and encodes a peptide with the sequence ANDENYALAA . Together with the alanine residue attached by tmRNA itself, the AANDENYALAA peptide forms an SsrA degradation signal tag. The YALAA portion of the tag is important for degradation by ClpXP or ClpAP protease  in E. coli and Salmonella typhimurium. Deletion of the smpB gene in E. coli produces a phenotype identical to that of ssrA-defective cells and SmpB binds specifically to tmRNA with high affinity  region (NMR (nuclear magnetic resonance) analyses of SmpB from ) region . The cor) region  that is ) region .trans-translation process. Consistent with its oligonucleotide-binding fold configuration, the core domain of SmpB plays a role in the interaction with tmRNA. In the crystal structure of the complex composed of the entire TLD of tmRNA and SmpB from T. thermophilus . Binding of SmpB to the TLD of tmRNA contributes to the formation of an L-shaped structure by compensating for the lack of a D stem in tmRNA and thereby assisting D and TψC arm interaction, and by stabilizing the coaxial structure of the TψC and acceptor stems . SmpB enhances alanylation of tmRNA or the TLD by alanyl-tRNA synthetase . This arrangement locates the C-tail region of SmpB close to the decoding region of the 30S ribosomal subunit during entry of tmRNA into the ribosomal A site .During the eriments , cryo-eleriments , X-ray ceriments , and sineriments . These sIn this scheme, codon–anticodon interactions play a critical role in both the first and second stages of the sense codon decoding process. In the first stage, codon–anticodon interaction in the A site induces structural rearrangements of the 30S ribosomal subunit; formation of the first and second base pairs expose the A1492 and A1493 residues from helix 44 of 16S rRNA and the G530 residue of 16S rRNA also undergoes a conformational change that enables it to interact with the second and third base pairs . MoreoveIn contrast to the sense codon decoding process, codon–anticodon interactions do not occur during tmRNA/SmpB entry because tmRNA does not possess an anticodon and the ribosome does not contain mRNA codon in its A site. Despite this situation, the tmRNA/SmpB complex binds to and facilitates the hydrolysis of EF-Tu  to enablT. thermophilus ribosome, a partial fragment of tmRNA, SmpB, and EF-Tu, which was trapped in the GDP form after GTP hydrolysis using the antibiotic kirromycin . Considering that key residues responsible for controlling the peptidyl-transfer rate are dispersed in the entire region of the C-tail  was originally identified as a protein factor essential for the viability of n system . Using gE. coli arfA gene encodes a small protein of 72 amino acids. A conserved hairpin structure within the arfA mRNA .Although ArfA can bind to the 50S subunit of the ribosome and peptidyl-tRNA in the ribosomal P site can be hydrolyzed in cell-free protein synthesis reaction mixtures containing S30 cell extract , ArfA alin vitro. However, this mutation does not inhibit binding of RF2 to the ribosome  plays a role in stalled ribosome rescue was reported independently by two research groups . Handa etrans-translation and ArfA/RF2 systems, the molecular mechanism of ArfB-dependent ribosome rescue is relatively simple. ArfB consists of a C-terminal unstructured region and a globular N-terminal domain containing a GGQ amino acid motif that resembles domain 3 of class-1 RFs. Analysis of the crystal structure of E. coli ArfB bound to the T. thermophilus ribosome with the initiator tRNAfMet .In addition to mutation of the N-terminal domain, truncation of the C-terminal unstructured region of ArfB also suppresses peptidyl-tRNA hydrolysis on the ribosome . Analysin system , the C-tn system . Severale rescue . In summA common feature of the three ribosome rescue pathways described here is that they may be structurally designed to selectively target stalled ribosomes lacking a codon in the A site. The C-tail regions of SmpB and ArfB are both accommodated in the mRNA path downstream of the ribosomal A site , suggestin vitro studies showed that the presence of an mRNA downstream of the A site inhibits the three ribosome rescue pathways. An in vitro study of the trans-translation system showed that the rate of alanyl-transfer to the nascent polypeptide is inversely proportional to the length of the mRNA downstream of the A site (in vitro (Accordingly, e A site . Furthere A site . The ribe A site . The facin vitro . These sin vitro .in vitro studies described above suggest that the ArfB system may interfere with the normal translation system because it can cover a wider range of ribosomal situations than the trans-translation or ArfA/RF2 systems. There is currently no explanation for the broader specificity of the ArfB system. Notably, endogenous ArfB cannot restore the viability of E. coli cells depleted of both ArfA and tmRNA, whereas overexpression of exogenous ArfB does restore this defect (E. coli (about 0.5 and 15 protein molecules/cell, respectively; E. coli cells. However, the situation may be different in other bacterial cells because only a subset of β- and γ-proteobacteria contains ArfA homologs (trans-translation system suggests that these two systems have complementary roles in ribosome rescue (E. coli cells at least. Therefore, it is reasonable to speculate that the trans-translation and ArfA/RF2 systems are structurally designed to target stalled ribosomes more precisely.The s defect . This lahomologs . The tige rescue , perhapsIn vivo studies showed that the trans-translation system targets ribosomes that are stalled at certain regions of mRNA due to amino acid starvation (in vitro studies because such pausing reportedly induces cleavage of the A site mRNA codon (arvation  or the parvation  or speciarvation , in whicNA codon . The mecNA codon . AdditioThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Newer chemotherapeutic agents target extracellular signaling, including the mitogen-activated protein kinase kinase (MEK) pathway. We present a case of a 54-year-old female who developed bilateral multifocal central serous-like chorioretinopathy shortly after starting MEK inhibition for metastatic cutaneous melanoma. There was a complete resolution of findings after drug stoppage. After resuming a lower dose of the MEK inhibitor, the findings recurred again but resolved after drug stoppage. Other etiologies were unlikely given the clinical course. The presumed mechanism involves toxicity to the retinal pigment epithelium, with breakdown of the blood-retinal barrier. Recognition of this side effect is important with this new class of chemotherapy. Recently developed chemotherapeutic agents target the mitogen-activated protein kinase kinase (MEK) pathway . CentralA 54-year-old Caucasian female presented for an eye examination prior to initiation of dabrafenib  and trametinib  chemotherapy for cutaneous melanoma with axillary and cervical lymph node metastases. Past medical history was positive for hypertension and past ocular history was negative. Current medications included diltiazem, meloxicam, losartan, and hydrochlorothiazide. Visual acuity (VA) was 20/20 OU with normal exam findings.Three weeks after beginning therapy, she presented with decreased vision of 20/60 OD and 20/50 OS. Fundus examination showed bilateral multifocal neurosensory retinal detachments that were hyperautofluorescent on fundus autofluorescence Figures . SubretiFour months later, her vision gradually deteriorated to 20/30 OU with recurrence of SRF Figures . DabrafeDysregulation of extracellular signaling is an increasingly recognized factor in the development of human cancers. Three kinase enzymes are part of this pathway: mitogen-activated protein kinase (MAPK), MEK, and extracellular signal-regulated kinase (ERK) . InhibitSeveral cases of MEK inhibitor-induced retinopathy have been reported. In a prospective, randomized, phase I/II study of a B-raf/MEK inhibitor, 2% of patients in the higher dose group developed chorioretinopathy . Velez-MMechanisms underlying retinopathy in the setting of MEK inhibition are unknown, but we hypothesize that MEK inhibition results in acute retinal pigment epithelial (RPE) toxicity and dysfunction with breakdown of the blood-retinal barrier. In support of this, the MEK pathway appears to be important in maintaining the blood-retinal barrier and protecting RPE cells against oxidative and light-induced damage. Animal models demonstrate that MEK inhibition induces oxidative stress and inflammation with subsequent endothelial and blood-retinal barrier damage and RPE hyperpermeability , 6, provThe combination of B-raf and MEK inhibition in patients with cutaneous melanoma significantly improves progression-free survival . In addi


The presence of autoantibodies in systemic lupus erythematosus, particularly those of the IgG subclass, have long been associated with disease onset and activity. Here we explored the prevalence of autoreactive IgE in SLE and its relevance to disease in French (n = 79) and United States (US) (n = 117) cohorts with a mean age of 41.5±12.7 and 43.6±15.3 years and disease duration of 13.5±8.5 and 16.6±11.9 years, respectively. Our findings show that approximately 65% of all SLE subjects studied produced IgE antibodies to the seven autoantigens tested. This positivity was increased to almost 83% when only those subjects with active disease were considered. SLE subjects who were positive for anti-dsDNA, -Sm, and -SSB/La -specific IgE showed a highly significant association in the levels of these antibodies with disease activity similar to that of the corresponding IgG's. A strong association of IgE autoantibodies with active nephritis was also found in the combined cohort analysis. A test of the predictive value of autoreactive IgE’s and IgGs for disease activity (SLE Disease Activity Index (SLEDAI) ≥4) revealed that the best predictors were dsDNA-specific IgE and IgG, and that the age of an SLE subject influenced this predictive model. The finding argue that the overall levels of IgE autoantibodies, independently or in combination with IgG autoantibodies, may serve as indicators of active disease. Systemic Lupus Erythematosus (SLE) is a chronic systemic autoimmune disease characterized by the loss of immune tolerance to self-antigens, dysregulated autoantibody production, and multiple clinical manifestations IgE is the rarest of immunoglobulins in the blood with serum levels in a healthy human at approximately 150 ng/ml whereas that of IgG is 10 mg/ml. Normally, IgE functions to protect against various parasitic infections Recently, we reported that the presence of autoreactive (dsDNA) IgE is linked to activation of basophils, which function to amplify autoantibody production in SLE through support of plasma cell survival To further advance the understanding of IgE in SLE we measured the levels of IgE’s with specificity to the most common SLE antigens, dsDNA, Sm, SSA (Ro), and SSB (La) in a sample of 196 SLE subjects in France or the US with a mean age of 41.5±12.7 and 43.6±15.3 years and disease duration of 13.5±8.5 and 16.6±11.9 years, respectively . The hea. The demographics and characteristics of all SLE patients and healthy controls are summarized in This collaborative study was approved by the Institutional Review Board of NIAMS  and by the Comité Régional de Protection des Personnes . A written consent was obtained from all healthy donors and SLE patients. In the US, samples were obtained from SLE patients enrolled in a long-term natural history study of SLE. In France SLE samples were obtained from in- and out- patients enrolled in a long term prospective study of SLE, and clinical data were used after approval by the Comission Nationale de l’Informatique et des Libertés (CNIL). All patients fulfilled the American College of Rheumatology classification criteria for SLE Whole blood from SLE and healthy donors was collected in 4 ml SST (serum-separating tube) tube for serum. Serum samples were aliquoted and kept at −80°C.The level of antibodies against the four common nuclear antigens  was measured by an ELISA. Horseradish peroxidase-conjugated antibodies to human IgE  or IgG  were used for colorimetric detection was with tetramethylbenzidine substrate  and optical density (OD) was measured at 450 nm. Positive values were considered as greater than two standard deviations over the mean absorption units obtained from samples of healthy individuals. dsDNA-specific IgG levels were quantified using standard laboratory procedures by the NIH Clinical Center and by the INSERM U699 Total levels of IgE were determined by ELISA  following the manufacturers’ instructions.To determine the level of IgG and IgE autoantibodies to CLIP4, MPG and APEX nuclease 1, we used purified recombinant human proteins, CLIP4 , MPG , and APEX nuclease 1  in ELISA. Detection of reactive autoantibodies was as described above.A Protoarray Human Protein Microarray v5.0 (Invitrogen Life Technologies) with over 9000 purified human proteins was used for novel antigen discovery. The purified recombinant proteins spotted on nitrocellulose-coated glass slides were reacted with the serum of SLE subjects or healthy controls and subsequently incubated with biotinylated goat anti-human IgE antibody (5 µg/ml) (Vector Laboratories) to detect IgE antibody binding. Then either an AlexaFluor 647-conjugated streptavidin  or an AlexaFluor 647-conjugated anti-V5 antibody  was added for detection and normalization, respectively. Arrays were scanned using fluorescent microarray GenePix 4000B Scanner and GenePix 6.0 software was used for data acquisition. Data analysis was done using Invitrogens proprietary Protoarray Prospector software. For each protein spotted in every array, Z-Score, Z-factor, CI-P value  was measured. M-Statistic is used to identify those proteins which show a significant differential signal between two populations. The “cutoff” value used corresponded to the value of 200 RFU above the M-Statistic signal threshold established for a specific protein.t test or a non-parametric Mann-Whitney U-test (when necessary) was used to compare SLE and control samples. A Chi2 assessment of the non-parametric Fisher exact test was used to determine the association of autoreactive IgE with active nephritis as well as the association of autoreactive IgE with active disease. One-way ANOVA with Bonferroni correction test or Kruskal-Wallis with Dunns correction test was applied when more than two groups were compared. Mean and SEM is shown in the scatter plot graphs. The analysis was performed using Graphpad Prism 5.0. Grubbs’ outlier test was used to assess the outliers. In some instances some outliers detected in the healthy control group were excluded from the analysis because of the identification of allergic disease or a urine infection and no clinical indications of these conditions in the studied SLE subjects. For forest plot analysis, a fixed effects model was applied and Hedges g statistics was used to calculate the standardized mean difference (SMD).Student Stepwise Logistic Regression offered by IBM SPSS Statistics 20 software was used to determine a predictive model of disease activity . Probability to enter a predictor in the model p<0.1 and to remove it p>0.05. Model 1 included as predictors: positive levels of dsDNA IgE, Sm IgE, SSA/Ro IgE, SSB/La IgE, dsDNA IgG, Sm IgG, SSA/Ro IgG and SSB/La IgG; as confounders age, gender and ethnicity . Model 2 included as predictors: positive levels of autoreactive IgEs, autoreactive IgGs (positive for at least one of the autoantibodies against the four common antigens); as confounders age, gender and ethnicity. Factors not affecting the models were eliminated from consideration. ROC Curve with AUC (area under the curve) analysis and Youden’s J Index was calculated by IBM SPSS in order to determine the optimal predictive model selection.The characteristics and demographics of both French and US SLE subjects studied herein are described in 2 = 0%) in the levels of IgE autoantibodies among the cohorts and no significant difference was observed in the effect of the autoreactive IgE on the two cohorts relative to healthy controls as shown by the overall standardized mean difference (SMD) for each individual autoreactive IgE. This argues that the two cohorts showed similar differences in levels of autoreactive IgE relative to healthy controls and that the distribution of the detected autoreactive IgE is similar in these two cohorts. No meaningful differences in the levels of IgE autoantibodies were detected when either gender or ethnicity were considered and no correlation was found between the total levels of IgE and the levels of IgE autoantibodies in these cohorts (data not shown).It was not known if IgE autoantibodies are generated to all of the antigens most frequently detected in the serologic profile of SLE. Thus, we assayed for the presence of dsDNA, Sm, SSA/Ro and SSB/La in both US and French SLE cohorts. IgE autoantibodies to all four self-antigens were present with anti-dsDNA and anti-Sm IgE antibodies having the most significant association with SLE relative to healthy controls . AnalysiThe relationship between disease activity and the presence of any one of the four auto-antigen specific IgE’s was explored. Disease activity was based on a SLEDAI score of ≥4 as active disease, >0 to <4 for mild disease, and 0 for inactive. As shown in To further verify the association of autoreactive IgE’s with disease activity, we analyzed its relationship to hypocomplementemia, a commonly used serological marker of increased disease activity or disease relapses While more than 50% of SLE subjects had autoreactive IgE to one or more of the four common SLE auto-antigens, the prevalence of these IgE’s in active SLE subjects (SLEDAI ≥4) was 73.7% and 74.1% for the French and US subjects, respectively, with dsDNA specific-IgE being the most prevalent   is known to be associated with SLE disease activity, we set out to compare the relationship of autoreactive IgG and IgE with disease activity. Analysis of SLE subjects, with active disease, having positivity for autoreactive IgE or IgG to the four common SLE autoantigens showed a similar significance (p<0.0001) in the association of anti-dsDNA IgE or IgG with active disease . HoweverAnalysis of the predictive value of autoreactive IgE’s or IgG’s for active disease in SLE subjects demonstrated that anti-dsDNA IgE and anti-dsDNA IgG were significant in predicting SLE subjects with active disease . Thus, tRoughly, 40–50% of SLE subjects tested did not have IgE autoantibodies to the four common SLE autoantigens . To explOf the 146 SLE subjects analyzed from the US and French cohort who were positive for autoreactive IgE’s to all seven autoantigens tested, 94 were positive for increased levels of autoreactive IgE whereas 107 were found to have increased levels of autoreactive IgG’s. Of this latter group, 76 subjects (71.1%) were positive for both  where detection of IgE in the kidney has been difficult. One must also consider that the concentration of IgE in both the circulation and tissues is extremely low and that this immunoglobulin does not fix complement (a major participant in tissue damage in the lupus kidney), thus the pathological consequence of any IgE deposition in the kidney is unclear and the contribution of IgE to tissue damage would most likely occur through induction of proinflammatory responses. While the relationship between autoreactive IgE and autoantibody production in human disease is difficult to unravel, our current analysis on the combined cohorts tested for the common SLE autoantigens uncovered evidence of a significant correlation between increased levels of autoreactive IgE and increased levels of autoreactive IgG  (A) or in SLE subjects with hypocomplementenemia  is shown. The percent of SLE subjects with positive dsDNA IgE levels was the reference point. Percent of subjects with Sm IgE had no dsDNA IgE, percent of subjects with SSA/Ro IgE had no dsDNA or Sm IgE, and the percent of subjects with SSB/La IgE had no detectable levels of dsDNA, Sm or SSA/Ro IgE. Figure S3, Association of novel autoreactive IgE levels  with nephritis (A) and complement levels (B). Low levels of complement were defined as C3<80 mg/dl or C4<15 mg/dl. Kruskal-Wallis with Dunn’s multiple comparisons test was used to compare the different groups. AU, Arbitrary Units. Mean ± SEM is shown. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Figure S4, Increased levels of dsDNA and Sm –specific IgE’s are associated with increased levels of their respective IgG counterparts. A. Analysis of the relationship between IgE and IgG in SLE subjects positive for all seven autoantigens  tested in this study. Stacked bar (left panel) and analysis  of the combined number of French and US SLE subjects. Positivity was considered as a value over the mean +2SD of healthy controls for at least one of the aforementioned autoantigens. B. Spearman correlation between autoreactive IgE and IgG levels for dsDNA and Sm in the group of subjects with double positivity shown on a logarithmic scale. Table S1, Correlation of positive levels of autoreactive IgE with active nephritis. Analysis of the combined and individual US and French cohorts.(DOCX)Click here for additional data file.


Inhibiting human neutrophil elastase (HNE) is a promising strategy for treating inflammatory lung diseases, such as H1N1 and SARS virus infections. The use of sivelestat, the only clinically registered synthesized HNE inhibitor, is largely limited by its risk of organ toxicity because it irreversibly inhibits HNE. Therefore, potent reversible HNE inhibitors are promising alternatives to sivelestat.in vitro HNE inhibition assay was employed to screen a series of triterpenes. Six pentacyclic triterpenes, but not tetracyclic triterpenes, significantly inhibited HNE. Of these pentacyclic triterpenes, ursolic acid exhibited the highest inhibitory potency (IC50 = 5.51 µM). The HNE inhibitory activity of ursolic acid was further verified using a mouse model of acute smoke-induced lung inflammation. The results of nuclear magnetic resonance and HNE inhibition kinetic analysis showed that the pentacyclic triterpenes competitively and reversibly inhibited HNE. Molecular docking experiments indicated that the molecular scaffold, 28-COOH, and a double bond at an appropriate location in the pentacyclic triterpenes are important for their inhibitory activity.An Our results provide insights into the effects of pentacyclic triterpenes on lung inflammatory actions through reversible inhibition of HNE activity. Neutrophils are major mediators of inflammation. They are involved in the pathogenesis of various lung inflammatory diseases, including acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchiectasis, pulmonary emphysema, and chronic obstructive pulmonary disease (COPD) Human neutrophil elastase  is a 30 kDa serine protease stored in the azurophilic granules of neutrophils. Intracellular HNE breaks down foreign proteins , whereas the extracellular HNE released by neutrophils and mostly bound to the neutrophil plasma membrane, assists neutrophil migration to inflammation sites by degrading various host proteins, such as extracellular matrix proteins 1-antitrypsin (α1-AT), secretory leukocyte proteinase inhibitor, α2- macroglobulin, and elafin Under normal physiologic conditions, HNE is controlled by its endogenous inhibitors, including αSivelestat sodium hydrate (ONO-5046) is the only clinically registered chemically synthesized selective HNE inhibitor. It attenuates pulmonary disorders and improves pulmonary function. ONO-5046 is clinically used to treat pneumonia and ALI caused by viral infections in vitroin vivo remain unclear.Natural compounds are a potential source of HNE inhibitors. Some natural compounds reportedly inhibit HNE activity in vitro and in vivo. The results indicated that the compounds with a pentacyclic triterpene scaffold significantly inhibit HNE through reversible competitive binding mode, whereas tetracyclic triterpenes did not exhibit notable inhibitory activity. The molecular scaffold, the carboxyl function at position 28, and a double bond at the appropriate location in the pentacyclic triterpenes are crucial to their inhibitory activity.In the present study, we used molecular docking to analyze the binding capabilities of HNE with a series of compounds isolated from herbs that inhibit lung inflammation. We subsequently confirmed their inhibitory activity Pentacyclic triterpenes, such as ursolic acid and oleanolic acid, are widely distributed in apples and other fruits; these compounds are used in nutraceuticals for treating various diseases HNE (EC 3.4.21.37) from human leukocytes was purchased from Innovative Research Company . ONO-5046, HNE substrate , soybean trypsin inhibitor, and DMSO were obtained from Sigma  and used without further purification. The Mouse NE ELISA Kit  and Mouse TNF-α ELISA MAX™ Deluxe Set (Lot Number: B138014) were purchased from Cusabio  and Biolegend , respectively. All other reagents were of analytical grade.1 to 6 . The geometries of all compounds were optimized by applying Gasteiger–Marsili atom charges The crystal structure (PDB ID: 1B0F) of HNE in complex with the inhibitor SEI was downloaded from the RCSB protein data bank The advanced docking program Autodock 3.0.3 was used to perform automated molecular docking to handle the interaction mode of the triterpenes with HNE The direct inhibition assay of HNE activity was performed as previously described max′) and the apparent Michaelis–Menten constant (Kapp) were determined by fitting the initial velocity at each HNE substrate concentration The enzyme inhibition kinetic analysis was performed by maintaining the ursolic acid concentration  and using six different HNE substrate concentrations (0.2 mM to 2 mM) in a manner analogous to the assay described above. The UV absorption at 405 nm was monitored for 5 min using an automatic microplate reader. The apparent maximum velocity  and were carried out in accordance with internationally accepted guidelines. Female BALB/c mice (18 g to 20 g) were obtained from the SLAC Laboratory . All mice were kept under a 12-hour light–dark cycle, temperature of 22°C to 23°C, and humidity of 50% to 60%.in vivoThe mouse model of acute smoke-induced airway inflammation was established to assay the activity of triterpenes 1), astragaloside IV (7), and ONO-5046, which was used as the positive control in the vehicle (20% DMSO in double distilled water), were intraperitoneally injected into the mice 45 min before smoke exposure. The control mice, which were exposed to fresh air and smoke without drug treatment, were administered the vehicle 45 min before exposure to fresh air or smoke. The mice were sacrificed 16 h after the final smoke exposure through an overdose of urethane, and their BALF was analyzed. Total and differential BALF cell counts were performed, and the NE and TNF-α in the BALF samples were analyzed using a Mouse NE ELISA Kit  and Mouse TNF-α ELISA MAX™ Deluxe Set , respectively.Ursolic acid  containing 80% d6-DMSO. The concentrations of the tested compounds and HNE were 1.0 mM and 5.0 µM, respectively. All measurements were performed on a Bruker Advance 400 MHz NMR spectrometer at 298 K. The suppression of residual water signals was achieved through the Watergate pulse program with gradients.Stock solutions of pentacyclic triterpenes and HNE were dissolved in d1H NMR spectra were recorded using a BBO broadband probe. A total of 64 scans were collected into 4 K data points at a spectral width of 4000 Hz. The spin-lattice relaxation rates were measured using the (180°-τ−90°-t)n sequence. The τ values used for the selective and nonselective experiments were as follows: 0.00001, 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.2, 1.5, 2, 3, and 5.0 s, and the delay time (t) was 0.5 s. The 180° selective inversion of the proton spin population was obtained via a 20 ms selective Gaus1_180i.1000 shape pulse at 50 dB; these measurements correspond to an excitation width of approximately 45 Hz Table S1in File S1) that protect lung function according to traditional Chinese medicine Table S2 (in File S1).A total of 323 compounds from 11 herbs  in a dose-dependent manner (1) showed the most potent inhibitory activity among the six pentacyclic triterpenes, with a minimal inhibition (12.2%) at 1 µM and a maximal inhibition (>80%) at >10 µM. Consistently, the IC50 values of the six natural pentacyclic triterpenes were approximately 5.51 µM (ursolic acid), 8.05 µM (oleanolic acid), 36.90 µM (glycyrrhetinic acid), 27.58 µM (glycyrrhizin), 6.83 µM (betulinic acid), and 8.20 µM (lupeol). Considering ursolic acid exhibited the highest inhibitory activity against HNE, it was further studied in a subsequent in vivo bioassay.The inhibitory effects of the compounds selected through molecular docking and our other available compounds were further investigated using an HNE t manner Table 1.50 of 16.59 µM  minimally affected HNE activity at concentrations up to 100 µM (Table S3 in File S1). This result indicates that the pentacyclic triterpene scaffold is important for HNE inhibition. In the following assay, astragaloside IV (7), a tetracyclic triterpene, was used as the negative control.In contrast to the inhibitory potencies of pentacyclic triterpenes, the six tested tetracyclic triterpenes (compounds app increased with increasing ursolic acid concentration from 0 µM to 10 µM (P<0.05). The trend of Vmax′ was basically considered to maintain invariable as a function of ursolic acid concentration (P>0.05). These results suggest that ursolic acid competitively inhibits HNE with respect to substrate concentration.The inhibitory kinetics of ursolic acid was determined in the presence of six HNE substrate concentrations Fig. 4. Fig. S1 in File S1). In the BALF complement, the TNF-α from activated macrophages and the NE released from the activated neutrophils were augmented after exposure to smoke (1) significantly reduced the number of total leukocytes and neutrophils. At the same dose, it also inhibited the increase in BALF NE by 54% and the increase in TNF-α by 38%. We further assayed the effects of ursolic acid (1) on airway inflammation in mice at 10, 30, and 100 mg/kg. The results showed that ursolic acid reduces the levels of BALF NE and TNF-α in a dose-dependent manner, with significant inhibition at 30 mg/kg (P<0.05) (and7) did not inhibit airway inflammation  Fig. 6B.seR) and nonselective (nsR) spin-lattice relaxation rates of the ligand with and without the enzyme. The change in the formation of ligand–enzyme adducts affected nsR and seR at different extents, and the slower rotational tumbling primarily affected seRnsR is affected to a lesser extent and that the nsR/seR ratio decreased during the binding process. The change in the nsR/seR ratio from the ligand with and without the enzyme occurs only when the ligand binds to the enzyme in a fast reversible exchange manner nsR/seR ratio indicates the reversible interaction between the ligand and the enzyme.NMR evaluation was based on the comparison of the selective  1 to 6 and the tetracyclic triterpenes 7 to 12 showed that the molecular scaffold, the 28-COOH, and a double bond in the appropriate location in their skeleton increased their inhibitory activity. The placement of the ligand into the HNE catalytic pocket explains the need for its hydrophobicity. The six pentacyclic triterpenes selected interacted with the extended substrate-binding domain of HNE at subsites S3, covered S2, S4, and S5, and pointed to the oxyanion hole (Gly193 and Ser195) in the S1 pocket, whereas the six tetracyclic triterpenes occupied subsite S2, subsite S4, or none of the subsites. This result suggests that the skeleton shape and the molecular size of the ligand are important in HNE inhibitory activity. Important hydrogen bonds that anchor the pentacyclic triterpenes into the HNE binding site were also identified. 1) pointed toward the backbone NHs of Ser195 and Gly193 (the oxyanion hole), and that the distances were suitable for the optimal hydrogen bonding of Ser195 and Gly193 7) had no hydrogen bonds with the appropriate key residues (6), reduced the inhibitory potency of its methylation analogue betulinic acid (5). This finding indicates that the 28-COOH in the pentacyclic ring system of the triterpenes contribute to HNE binding. The difference in HNE inhibitory activity between oleanolic acid (2) and glycyrrhetinic acid (3) also suggests the importance of the 28-COOH. The pentacyclic triterpenes, including ursolic acid  and were therefore fully protected against d-galactosamine with LPS-induced hepatitis (1) and astragaloside IV (7), reportedly have anti-inflammatory and antioxidant functions through multiple mechanisms, such as by inhibiting NF-κB, iNOS, COX-2, and other proinflammatory mediators in vivo. However, we did not observe the inhibitory effects of astragaloside IV on NE and TNF-α. These results imply that the anti-inflammatory effects of the pentacyclic triterpenes are closely related to direct inhibition of HNE activity. This inhibition reduced the TNF-α level, which prevented neutrophil activation and their subsequent chemotaxis to inflammatory sites with the excessive HNE release.Recent studies have demonstrated that HNE stimulates diverse proinflammatory signaling pathways through multiple mechanisms. Therefore, HNE is an important regulator of inflammatory processes and is an interesting target for new therapeutic approaches against inflammatory disorders, particularly those involving the lungs In conclusion, pentacyclic triterpenes, but not tetracyclic triterpenes, protect against inflammation-induced lung injury by reversibly and competitively inhibiting HNE activity. Furthermore, we deduced that the scaffold, the carboxyl group at position 28, and a double bond at the appropriate location in the pentacyclic ring system of the triterpenes contributed to HNE binding. To the best of our knowledge, this study is the first to demonstrate the structure–activity relationship between pentacyclic and tetracyclic triterpenes, thereby providing insights into the molecular mechanism underlying their pharmacologic effects on lung inflammatory actions. Therefore, compared with synthetic HNE inhibitors, the less toxic pentacyclic triterpenes, which are present in many fruits, have better therapeutic value in preventing HNE-induced inflammation.File S1Combined file of supporting figures and tables.(DOC)Click here for additional data file.


Anabolic resistance is the inability to increase protein synthesis in response to an increase in amino acids following a meal. One potential mediator of anabolic resistance is endoplasmic reticulum (ER) stress. The purpose of the present study was to test whether ER stress impairs the response to growth factors and leucine in muscle cells.Muscle cells were incubated overnight with tunicamycin or thapsigargin to induce ER stress and the activation of the unfolded protein response, mTORC1 activity at baseline and following insulin and amino acids, as well as amino acid transport were determined.ER stress decreased basal phosphorylation of PKB and S6K1 in a dose-dependent manner. In spite of the decrease in basal PKB phosphorylation, insulin (10–50 nM) could still activate both PKB and S6K1. The leucine (2.5–5 mM)-induced phosphorylation of S6K1 on the other hand was repressed by low concentrations of both tunicamycin and thapsigargin. To determine the mechanism underlying this anabolic resistance, several inhibitors of mTORC1 activation were measured. Tunicamycin and thapsigargin did not change the phosphorylation or content of either AMPK or JNK, both increased TRB3 mRNA expression and thapsigargin increased REDD1 mRNA. Tunicamycin and thapsigargin both decreased the basal phosphorylation state of PRAS40. Neither tunicamycin nor thapsigargin prevented phosphorylation of PRAS40 by insulin. However, since PKB is not activated by amino acids, PRAS40 phosphorylation remained low following the addition of leucine. Blocking PKB using a specific inhibitor had the same effect on both PRAS40 and leucine-induced phosphorylation of S6K1.ER stress induces anabolic resistance in muscle cells through a PKB/PRAS40-induced blockade of mTORC1. Amino acids activate mTORC1  by promoting the exchange of GDP for GTP in the RagA/B GTPase proteins mTORC1 is the central molecular player in anabolic resistance. mTORC1 is composed of mTOR (a Ser/Thr protein kinase), raptor (regulatory associated protein of mTOR) and mLST8/GβL (G protein beta subunit-like protein) and is sensitive to the macrolide rapamycin One potential mediator of anabolic resistance is endoplasmic reticulum (ER) stress In the present study, we sought to determine whether ER stress could induce anabolic resistance in muscle cells. Consistent with this hypothesis, low levels of ER stress were sufficient to prevent the activation of mTORC1 by leucine, assessed by a decrease in S6K1 phosphorylation on Thr389, whereas at the same level of ER stress insulin could still activate mTORC1 normally. The inability to activate mTORC1 was not due to a lack of leucine transport, but rather to the ER stress-induced decrease in basal PKB phosphorylation resulting in PRAS40 hypophosphorylation and allosteric inhibition of mTORC1.C2C12 murine skeletal muscle myoblasts  were seeded in 150 mm-diameter culture dishes and grown in Dulbeccos's Modified Eagle Medium  supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (5000 U/5000 µg/ml). Cells were then plated in 6-well plates until 90% confluent. At this time, the proliferation medium was replaced by a differentiation medium containing 2% horse serum and 1% penicillin/streptomycin (5000 U/5000 µg/ml). After 96 h of differentiation, tunicamycin  or thapsigargin (Tocris Bioscience) was added for 17 h in serum-free DMEM before stimulation of the mTORC1 pathway with insulin (10–50 nM) for 15 min or with leucine (2.5–5 mM) for 30 min. Tunicamycin and thapsigargin were used as they are well-documented chemical inducers of ER stress by blocking N-glycosylation and calcium entry into the ER, respectively. For PKB inhibition experiments, cells were serum-starved for 16 h to maintain the cells in the same conditions as those described for tunicamycin and thapsigargin experiments. PKB inhibitor (100 nM–1 µM), also known as Akt Inhibitor XIII or Akti2 , was then added 1 h before leucine (2.5–5 mM) stimulation for 30 min. At the end of the incubation period, cells were harvested and cell lysates were immediately frozen at −80°C for subsequent analyses or cells were immediately used for leucine uptake measurements. All experiments were performed at least in duplicate.Cells were rinsed once with PBS and harvested in a lysis buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM DTT  and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science). The homogenates were then centrifuged for 10 min at 10,000 g and the supernatants were immediately stored at −80°C. Protein concentration was determined using the DC protein assay kit (Bio-Rad Laboratories).Cell lysates (15 µg) were combined with Laemmli sample buffer and separated by SDS/PAGE. After electrophoretic separation at 40 mA for 1 h, the proteins were transferred to nitrocellulose membrane at 100 V for 1 h for western blot analysis. Membranes were then incubated in a 5% Blotto solution. Subsequently, membranes were incubated with the following antibodies overnight at 4°C: eEF2; S6K1; phospho-S6K1 Thr389 and Thr421/Ser424; phospho-PKB Ser473 and Thr308; phospho-PRAS40 Thr246; phospho-mTOR Ser2448; phospho-PDK1 Ser241; phospho-4E-BP1 Thr37/46; phospho-GSK3 Ser9/21; BiP; IRE1α; phospho-PERK Thr980; and phospho-eIF2α Ser51. All antibodies were from Cell Signaling except phospho-S6K1 Thr421/Ser424 and S6K1 that were acquired from Santa Cruz and phospho-PRAS40 acquired from Invitrogen.Membranes were washed in TBST and incubated for 1 h at room temperature in a secondary antibody conjugated to horseradish peroxidase. After an additional 3 washes, chemiluminescence detection was carried out using an Enhanced Chemiluminescent Western blotting kit . The films were then scanned on an ImageScanner using the Labscan software and quantified with the Image Master 1D Image Analysis Software . Equal loading was checked by probing membranes with an anti-eEF2 as previous experiments showed that the different treatments used in the present study did not affect eEF2 expression.−ΔΔCt method. The identity and purity of the amplified product was checked through analysis of the melting curve carried out at the end of amplification.Total RNA from the cells of one 35 mm-well (6-well plates) was extracted with 0.5 ml TriPure reagent according to the instructions provided by the manufacturer (Roche Diagnostics). RNA was quantified by spectrophotometry (260 nm) and its concentration adjusted to 1 µg/µl using RNase-free water. cDNA was prepared by reverse transcription of 1 µg total RNA using the reverse transcription system (Promega). SYBR®Green (Sigma Aldrich) was used for real-time PCR detection using an Eppendorf Light Cycler PCR machine. Real-time PCR primers were designed  for mous4 and 1 mM CaCl2, pH 7.4), pre-warmed at 37°C. Cells were incubated for 1 min with 0.25 µCi (or 0.5 µCi) [14C]leucine diluted in 500 µl HBS containing 10 µM (or 1 mM) leucine. The doses of cold leucine were chosen to be below the Km of the transporter and about ∼10 times higher  and thapsigargin (1000 nM). Tunicamycin decreased Thr308 phosphorylation in response to both low and high levels of insulin. The inhibitory action of thapsigargin was less potent, decreasing Thr308 phosphorylation at low but not high levels of insulin. Even though tunicamcyin and thapsigargin decreased PKB phosphorylation, the phosphorylation of PRAS40 on Thr246 was still enhanced by insulin, indicating that following insulin stimulation tunicamycin and thapsigargin did not repress mTORC1 via PRAS40. As with Thr389, the phosphorylation of S6K1 on Thr421/Ser424 mirrored that of PKB. When PKB phosphorylation was decreased, S6K1 phosphorylation followed suit Thr389; . NeitherSince activation of mTORC1 by amino acids generally occurs independent of PKB, it was not surprising that PKB phosphorylation was not changed by the addition of leucine . As descWe surmised that the low phosphorylation of PKB and PRAS40 resulted in the allosteric inhibition of mTORC1 in response to leucine. To test this hypothesis, PKB activity was blocked chemically In summary, whereas ER stress only partially prevents insulin stimulation of PKB and mTORC1, the lack of activation of PKB by amino acids results in a complete block of leucine-stimulated activation of targets downstream of mTORC1 likely through hypophosphorylation of the mTORC1 kinase inhibitor PRAS40.The main finding of the present study is that ER stress induces anabolic resistance in muscle cells through PKB/PRAS40-induced blockade of mTORC1. Anabolic resistance is defined as the inability to increase protein synthesis in response to an increase in amino acids following a meal It should be mentioned that when cells were stimulated with insulin, this was made in presence of basal levels of leucine (0.8 mM) usually found in cell culture medium, whereas when cells were stimulated with extra leucine (2.5 or 5 mM), there was no insulin or serum. As insulin is known to play a permissive role in the action of amino acids on the mTORC1 pathway, it is possible that the effect of additional leucine was relatively less optimal compared to insulin. This could have contributed to the fact that ER stress was more potent in reducing leucine- than insulin-induced stimulation of mTORC1.Having previously established a negative relation between ER stress and the mTORC1 pathway in vivo studies that have shown that anabolic resistance is not due to reduced amino acid availability Another possible mechanism of anabolic resistance is a decrease in amino acid transporter levels and/or amino acid uptake. Several transporters are known to participate directly or indirectly in leucine transport in muscle cells. The preferred transporter for leucine is the system L-type vice versaIn C2C12 muscle cells, ER stress seems to impair mTORC1 rather than In vitro, ER stress completely blocks any effect of amino acids on S6K1 and 4EBP phosphorylation, whereas the effect on insulin was less dramatic. This is in accordance with the fact that anabolic resistance in vivo is mainly due to decreased sensitivity to amino acids and impaired mTORC1 signaling, rather than an impaired response to insulin The present study suggests that ER stress is a potential mediator of the anabolic resistance observed in the muscle of aged