In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival.Mast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, Purified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA.IgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells.These data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect. Mast cells play a key role in many physiological and pathophysiological processes. They contribute to the maintenance of tissue homeostasis, wound repair ,2 and re2+ influx and the release of histamine, leukotriene C4 (LTC4) and CXCL8 [Mast cells can be activated by many diverse stimuli leading to mediator release but allergen-dependent activation occurs predominantly through the high affinity IgE receptor complex (FcεRI) following aggregation of allergen-specific IgE bound to FcεRI . IgE nd CXCL8 .Understanding the mechanisms of mast cell hyperplasia in diseased tissue structures is of interest because inhibiting this might offer new approaches to treatment. Increased mast cell recruitment by the asthmatic ASM for example appears to be one factor . HoweverIn this study, we have tested the hypothesis that IgE alone enhances HLMC survival through the production of the survival enhancing cytokines IL-6 and stem cell factor. We demonstrate for the first time that monomeric IgE in the absence of antigen enhances HLMC survival, and that this effect is mediated, at least in part, through the autocrine production of IL-6.Human lung mast cells undergo apoptosis with SCF and IL-6 withdrawal . We therIn order to determine the mechanism of the enhancement of mast cell survival with IgE we next investigated the autocrine production of the pro-survival cytokines SCF and IL-6. There was absolutely no effect on survival with the addition of neutralising anti-SCF (1 μg/ml) or isotype control antibodies Figure . Howevert even after 50 cycles  relative expression of IL-6 mRNA was 3.79 (1.01–577000) when compared to control.In order to confirm that IL-6 expression in HLMC was upregulated with the addition of IgE alone, comparative quantitative real time RT-PCR was carried out and the relative expression of IL-6 mRNA in IgE 3 μg/ml)-stimulated cells compared to unstimulated cells. The relative expression of IL-6 mRNA in the IgE-stimulated cells was increased 46997 ± 41171 fold compared to control Figure  . Thus using the 2-ΔΔCt calculation, the relative expression of IL-6 following IgE stimulation was upregulated by 29445 ± 21013  which uses a different algorithm. The relative expression using the REST algorithm was almost identical to the 2-ΔΔCt algorithm with an upregulation of 29445 ± 21013 , HLMC survival is enhanced following growth factor withdrawal. Although SCF is the major growth and survival factor for HLMC, and is released by them in an autocrine manner , IgE-depin vivo relevance of our in vitro findings regarding the ability of IgE to enhance mast cell survival. An important feature of mast cell activation in the presence of IgE alone is that while free IgE is present, intracellular signalling is sustained, whereas this ceases as soon as free IgE is removed [in vivo where IgE exposure is continuous. Bearing in mind the strong correlation between serum IgE and the presence of asthma [A study of the effects of anti-IgE therapy  on airway inflammation in asthma identified a downward trend in both epithelial and lamina propria mast cell numbers of approximately 20% compared to control although this did not reach statistical significance . However removed . The mecf asthma -32, it if asthma .4 and CXCL8 [4 was much greater in the presence of SCF, a vital growth and differentiation factor for human mast cells which also potentiates IgE-dependent mediator release [We have demonstrated recently that IgE in the absence of antigen directly activates cultured HLMC leading to the release of histamine, and the production of LTC release ,33. Howe release ,34. The  release . One fac release , and dem release ,16,36. A release .Matsuda and colleagues found that the sodium azide present in their myeloma IgE preparation enhanced survival in the absence of IgE. For this reason, we ensured that all of our experimental conditions contained an equal amount of sodium azide, and included an appropriate sodium azide control. We also found there to be a small survival enhancing effect with the addition of sodium azide to the cells, but this was far less marked than that reported by Matsuda et al . Howeverin vitro induce IgE to self aggregate at the receptor [2+ is a critical requirement for mediator release in mast cells activated by either IgE alone or by antigen, the channels carrying Ca2+ into the cell following monomeric IgE stimulation appear to be different [2+ influx requires PKCβII whilst antigen-induced Ca2+ influx does not [greater the percentage of IgE monomers, the greater the biological response. All of these observations point towards distinct signalling pathways in mast cells activated by IgE alone versus antigen-dependent FcεRI cross-linking.Mast cell signalling and responses to IgE alone raise the question as to whether the IgE preparations used are truly monomeric or just complexes of IgE which mimic receptor cross-linking with antigen, and whether the physiological concentrations of IgE used receptor ,38. In freceptor . Howeverifferent . In addiifferent ,17. Furtdoes not . Interesdoes not . FurtherIn summary, we demonstrate that IgE in the absence of antigen promotes HLMC survival in a dose-dependent manner, and that this survival is markedly attenuated with the addition of neutralising anti-IL-6 antibody. Since IL-6 mRNA transcription is dramatically upregulated with the addition of IgE, and there is an increased release of IL-6 into the supernatant of IgE stimulated cells, we can conclude that the prosurvival effect of IgE in HLMC is due, at least in part, to the autocrine production of IL-6. This study provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect.All human subjects gave written informed consent and the study was approved by the Leicestershire Research Ethics Committee, UK. Lung tissue was obtained by surgical resection for bronchial carcinoma and mast cells isolated as described previously . The fin2 for a minimum of 4 weeks prior to experiments. Half of the medium was changed every 7 days. HLMC purity remained unchanged during the culture period.Following isolation, HLMC were cultured in DMEM/Glutamax/HEPES containing 10% FBS, 1% MEM nonessential amino acids , 1% antibiotic/antimycotic solution (Sigma-Aldrich), 100 ng/mL recombinant human (rh)SCF, 50 ng/mL rhIL-6 and 10 ng/mL rhIL-10  at 37°C in a humidified incubator flushed with 5% CO4 HLMC were plated into each well of a 96 well cell culture plate in 50 μl of DMEM/10% FBS. 50 μl of 2× the final concentration of human myeloma IgE  or medium alone was added. IgE was centrifuged at 14000 g for 20 min to remove any large aggregates [Long term cultured HLMC were counted using an haemocytometer and cell viability assessed using exclusion of trypan blue stain. HLMC were washed to remove the cytokines present in the culture medium. 1 × 10gregates ,15. IgE 1), anti-human SCF (1 μg/ml)  or an isotype control (1 μg/ml) (mouse IgG1) antibodies  were added to cultures containing 3 μg/ml IgE.For the determination of pro-survival cytokine activity in the cultures, neutralising anti-human IL-6 (1 μg/ml)  according to the manufacturer's instructions.2 × 10m within 1°C of 60°C. Primer3 software was used for the design of the primers [Quantitative real time RT-PCR was performed using a single tube Full Velocity SYBR Green Kit . Primers were designed for IL-6 to span exon-exon junctions to eliminate DNA contamination issues. PCR products were designed to be between 200 and 300 base pairs in length in line with the optimum length for SYBR green use. The Full Velocity SYBR Green QRT-PCR kit uses a combined annealing/extension step of 60°C. Therefore, all primers were designed to have a T primers . Primer The QRT-PCR reaction was optimised for each gene and 200 nM final concentration of primers was assessed as the optimum for use over all conditions. Using this concentration of primers ensured no non-specific amplification occurred in the sample wells. Reverse transcription was carried out for 30 minutes at 50°C before 5 minutes at 95°C was used to inactivate the reverse transcriptase which otherwise interferes with the DNA polymerase. 50 cycles of 95°C for 20 seconds followed by 60°C for 30 seconds were carried out and fluorescence was collected at the end of each extension cycle using the Mx3000P QPCR machine (Stratagene). A SYBR green dissociation curve was carried out at the end of the reactions to ensure specificity and products were run on 1.5% agarose gels to determine product length. Products were gel purified  and sequenced using the Protein and Nucleic Acid Chemistry Laboratory, University of Leicester.t.For the comparative QRT-PCR, the internal normaliser gene used was β-actin and all mRNA expression data were normalised to β-actin and corrected using the reference dye (ROX). The β-actin primer sequences used were: forward primer – 5'-TTCAACTCCATCATGAAGTGTGACGTG – 3', reverse primer – 5'-CTAAGTCATAGTCCGCCTAGAAGCATT – 3'. The product length for β-actin was 310 bp. Only experiments where a distinct single peak was observed with a melting temperature different to that of the no template control were used. Expression data was expressed as the relative gene expression compared to the calibrator (unstimulated cells) as determined by the Mx3000P software's built in algorithm using an adaptive baseline to determine the CSupernatants removed from the survival assays were stored at -20°C prior to measurement of IL-6. Samples were measured for IL-6 from conditions following 7 days of cytokine withdrawal. IL-6 was measured using the Quantikine human IL-6 ELISA kit  according to the manufacturer's instructions.6 cells. Differences between paired data were evaluated using Student's paired two-tailed t tests. Comparative quantitative RT-PCR data is presented as the relative expression compared to control (untreated cells) after correction against the internal calibrator β-actin as determined by the Mx3000P software's built in algorithm using an adaptive baseline to determine the Ct. Due to the inherent variability of relative expression data according to the algorithm used, relative expression was also calculated using the 2-ΔΔCt method [t value was not always achieved so the final cycle (cycle 50) was assigned as the Ct which tends to underestimate the degree of upregulation. Traditional statistical analyses for quantitative PCR are not appropriate due to high variability of gene expression in ex vivo human cells. Thus comparative QRT-PCR statistics were obtained by a hypothesis test with randomised bootstrapping of values with 50,000 iterations using the Relative Expression Software Tool .Data is presented as the mean ± SEM unless otherwise stated. IL-6 release is presented as ng/10t method  and the GC designed and carried out the quantitative PCR, carried out survival assays, carried out IL-6 ELISA, performed statistical analyses and wrote the manuscript. SC carried out survival assays and blocking antibody experiments. PB conceived the study, and participated in its design and coordination and wrote and edited the manuscript. All authors read and approved the final manuscript.


Eur. J. Biochem. 269, 2002, 3329; Planta, 219, 2004, 298). Although this notion is based mainly on biochemical evidence and in vitro systems, the recent availability of Arabidopsis sigma knock-out lines for complementation by intact and mutant sigma cDNAs has opened up new strategies for the study of transcription regulatory mechanisms in vivo. Using Arabidopsis sigma factor 6 (AtSIG6) as a paradigm, we present data suggesting that: (i) this factor is a substrate for regulatory phosphorylation by cpCK2 both in vitro and in vivo; (ii) cpCK2 phosphorylation of SIG6 occurs at multiple sites, which can widely differ in their effect on the visual and/or molecular phenotype; (iii) in vivo usage of the perhaps most critical cpCK2 site defined by Ser174 requires (pre-)phosphorylation at the n + 3 serine residue Ser177, pointing to ‘pathfinder’ kinase activity capable of generating a functional cpCK2 substrate site.Plastids contain sigma factors, i.e. gene-regulatory proteins for promoter binding and transcription initiation. Despite the physical and functional similarity shared with their prokaryotic counterparts, the plant sigma factors have distinguishing features: most notably the existence of a variable extra sequence comprising their N-terminal portions. This distinct architecture is reflected by functional differences, including phosphorylation control by organellar protein kinase(s) closely related to nucleocytosolic, rather than bacterial-type, enzymes. In particular, cpCK2, a nuclear-coded plastid-targeted casein kinase 2, has been implicated as a key component in plant sigma factor phosphorylation and transcriptional regulation ( Plant cells are tripartite genetic systems consisting of three transcriptionally active compartments, i.e. the nucleus, the mitochrondria and the plastids. The latter contain two principally different forms of RNA polymerases for transcription of a full complement of organellar genes in normal (wild-type) plants. Nucleus-encoded polymerase (NEP) is a single-subunit enzyme closely related to those of T7/T3 phages and mitochondria. In contrast, plastid-encoded polymerase (PEP) is a multi-subunit bacterial-type enzyme with α-, β- and β′-equivalent core subunits that are encoded by plastid genes . It has An intriguing feature of PEP transcription is the involvement of typical ‘prokaryotic’, yet nucleus-encoded, sigma initiation factors  sigma knock-out and antisense plants as tools that allow us to establish causal relationships between sigma genes and phenotypic traits in vivo -specific transcription, with noticeable consequences for plant development and function. This is in part because of a certain level of functional overlap between factors, which helps maintain the overall transcription program, even in adverse situations . Anotherin vivo has not yet been reported.Available evidence , which win vivo compared with the bacterially expressed recombinant protein in vitro. Using site-directed mutagenesis and retransformation of an AtSig6 knock-out line, we present evidence suggesting phosphorylation control of SIG6 activity by cpCK2 and probably one other protein kinase.To help clarify this question, we focused our attention on one of the Arabidopsis sigma factors, AtSIG6, for which mutant lines with readily discernible phenotypes are available . To gain information whether AtSIG6 can be a potential substrate for CK2 phosphorylation, we therefore searched for putative sites in the derived protein sequence.on state . In addiConsensus phosphorylation site motifs for (nucleocytosolic) CK2 often conform to the sequence motifs S*/T*xxEx and S*/T*xxDx, respectively  Table 1. The plaetPhos 2.0  DNA binding experiments were carried out using bacterially expressed AtSIG6 in combination with promoter . The recE. coli core enzyme alone in the absence of AtSIG6 (lane 2) resulted in a small but significant portion of labelled material to a shifted position. The core enzyme preparation  did not contain detectable levels of bacterial sigma factor(s), and the shifted material was thus taken into account as the baseline in subsequent experiments.Following incubation of either unphosphorylated (not shown) or phosphorylated AtSIG6 alone (lane 3) in the presence of the labelled probe, but without core enzyme, the only detectable signal is at the position of the free probe (bottom band). The virtual absence of any additional band with retarded mobility indicates that the factor lacks DNA binding activity on its own. atpB promoter fragment as a competitor (lane 6), the intensity of shifted radioactive material was largely reduced. In contrast, a promoter-less fragment that was tested as a non-specific competitor did not negatively affect the binding signal  , gave shifted signals at the same position and strength as seen for the core alone (lane 2), suggesting that unphosphorylated AtSIG6 has little if any sigma activity in this assay. However, when the phosphorylated recombinant factor was used , this led to a considerable increase in intensity, along with an additional small shift in migration position of the DNA–protein complex. Carrying out the same reaction with an added twofold molar excess of unlabelled  lane 7) . Togethein vitro.To investigate this further, we next constructed and tested mutant versions of the AtSIG6 protein that contained altered residues at putative cpCK2 substrate sites . As is ein vitro system, at least for the atpB promoter.A Thr → Ala exchange at the ‘general’ position T249 results in a mutant protein that is still active in its phosphorylated state (lanes 16 and 17), although perhaps to a somewhat lesser extent than the wild-type factor (lanes 4 and 5). This would argue against a significant regulatory effect of phosphorylation at this site in the in vitro  and 2.0 kb (NEP-driven) (sig6-2 knock-out with SIG6 cDNA (sig6-com), the wild-type pattern is restored, i.e. both the 2.6- and 2.0-kb transcripts but not the 4.8-kb transcript are visible.In brief, with the When RNA samples from the SIG6 phosphorylation site mutants were analysed, the hybridization patterns obtained were as follows: (i) The 2.0-kb (NEP) transcript is detectable in all lines; (ii) the larger 2.6-kb (SIG6/PEP) transcript is present at wild-type levels in both the S176 and the S180 seedlings, but is highly reduced or virtually absent in the other retransformed mutant lines; (iii) the 4.8-kb transcript is only detected in the S174 lines.atpB gene expression in vivo. A noticeable, but less pronounced, effect is also obvious for Ser94 and Ser95, as well as for the ‘general’ site T249-Ala, whereas Ser176 and Ser180 do not seem to be required for activity.Based on these diagnostic transcript patterns, it seems that Ser174 is absolutely required for SIG6 activity in driving psbA probe. The single 1.2-kb transcript is present in about equal relative quantities in wild-type RNA from 7-day-old seedlings and 14-day-old plantlets. In the sig6-2 knock-out mutant the signal intensity is reduced only at the earlier stage, and its intensity is restored to wild-type levels following retransformation of the knock-out with SIG6 cDNA  would ‘open’ Ser177, which can then serve as the n + 3 position for Ser174. This explanation, which is solely based on our current knowledge of consensus CK2 substrate sites, is fully consistent with the data obtained in the g assays . It is aRNA data , except http://plantsp.genomics.purdue.edu/html/families.html) as a possible candidate. In recent localization analyses, a member of the RSK family, PK-like protein (At3g44610), was identified as a plastid kinase . It has a single T-DNA insertion in exon 5 of the AtSig6 gene  were grown on MS agar medium containing 0.4% (w/v) Gelrite and 1% (w/v) sucrose . Plates were maintained at 24°C under 8-h short-day conditions at a photofluence rate of 60 μmol m−2 sec−1. Cotyledons (day 7) and leaves (day 14) were collected, frozen and powdered in liquid nitrogen. Total RNA was prepared as decribed by http://www.stratagene.com) and the oligonucleotides listed in Rhizobium radiobacter (Agrobacterium tumefaciens) strain GV3101, and then transformed into the sig6-2 line by floral dip  as well as those from within the AtSig6 coding region (UKSIG6-RP and UKSIG6-LP) . The linearized plasmids were transcribed by SP6 or T7 RNA polymerase in the presence of DIG-11-UTP . Plant total RNA (1 μg) was gel-fractionated, transferred to positively charged nylon membrane (Roche) and hybridized with the DIG-labelled probe, followed by chemiluminescence detection (Gene-specific RNA probes (http://www.neb.com), and recombinant proteins were purified on amylose resin according to the pMAL manual. Purified proteins (15 μg) were phosphorylated in 50-μl reactions containing 20 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl2, 0.1 mm ATP und 15 μg cpCK2 at 30°C for 30 min, as described by in vitro .AtSIG6 cDNA or mutagenized derivatives thereof were amplified using the forward primer Sig6-TP , which pE. coli core RNA polymerase , 5 ng 32P-labelled double-stranded probe . After cutting out the fragment with BamHI/SalI (Promega), it was electrophoretically purified, eluted, and end-labelled using [γ-32P]ATP and polynucleotide kinase (NEB). The same fragment, but unlabelled, was used as a specific competitor. Bam0.5, a 500-bp DNA fragment from within the trnK intron . After drying, gels were analysed using an FLA-3000 phosphoimager .EMSA reactions  containied probe  and 3 μged probe . The proK intron , served


The antitumour agent 5-imidazole-4-carboxamide (DTIC) was found to inhibit competitively the low-Km cyclic AMP phosphodiesterase activity in an ammonium-sulphate-precipitable fraction of the 2,000g supernatant of rat liver. With substrate concentration at 0.25 microM, I50 was 790 microM for DTIC and 350 microM for theophylline. DTIC at 2 mM more than doubled the cAMP response to glucagon in hepatocytes and to adrenaline in MH1C1 hepatoma cells, indicating that it also exerts its inhibitory effect on the phosphodiesterase in intact cells. The possible contribution of the phosphodiesterase inhibition to the growth-inhibitory and cytotoxic effects of DTIC is discussed.


This hypothesis was confirmed by the ability of Ang II to induce tyrosine nitration of the MAP kinases ERK1/2 and of protein kinase B/Akt (Akt). Tyrosine nitration of ERK1/2 was required for their phosphorylation on Thr and Tyr and their subsequent activation, whereas it completely inhibited Akt phosphorylation on Ser473 and Thr308 as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3α phosphorylation in vitro. Inhibition of nitric oxide synthase and NAD(P)Hoxidase and scavenging of free radicals with myricetin restored insulin-stimulated Akt phosphorylation and GLUT4 translocation in the presence of Ang II. Similar restoration was obtained by inhibiting the ERK activating kinase MEK, indicating that these kinases regulate Akt activation. We found a conserved nitration site of ERK1/2 to be located in their kinase domain on Tyr156/139, close to their active site Asp166/149, in agreement with a permissive function of nitration for their activation. Taken together, our data show that Ang II inhibits insulin-mediated GLUT4 translocation in this skeletal muscle model through at least two pathways: first through the transient activation of ERK1/2 which inhibit IRS-1/2 and second through a direct inhibitory nitration of Akt. These observations indicate that not only oxidative but also nitrative stress play a key role in the pathogenesis of insulin resistance. They underline the role of protein nitration as a major mechanism in the regulation of Ang II and insulin signaling pathways and more particularly as a key regulator of protein kinase activity.Angiotensin II (Ang II) plays a major role in the pathogenesis of insulin resistance and diabetes by inhibiting insulin's metabolic and potentiating its trophic effects. Whereas the precise mechanisms involved remain ill-defined, they appear to be associated with and dependent upon increased oxidative stress. We found Ang II to block insulin-dependent GLUT4 translocation in L6 myotubes in an NO- and O With regard to this question, we have shown previously that ROS-dependent activation of ERK1/2 by Ang II −), which leads to their nitration on tyrosine residues 2−. with nitric oxide (NO) and is generated in many pathological conditions which involve oxidative stress, such as diabetes − regulates this kinase has however not been investigated so far although Akt has been reported to be S-nitrosated in diabetic mice and in response to NO donors − than required for S-nitrosation Although superoxide ions , indicating that both NO and O2−. generation are involved in this signalling cascade. These compounds had no significant effect per se on GLUT4 translocation under control or insulin-stimulated conditions but abolished the effect of Ang II alone (data not shown).In order to test whether this action of Ang II involves a ROS- or RNS-dependent pathway, the effect of compounds interfering with the generation of these reactive species on GLUT4 translocation was investigated. As shown in the same panel, the Ang II-mediated inhibition of insulin-dependent translocation was significantly (P<0.05) alleviated by the NADP(H) inhibitor AEBSF (0.5 mM), the selective iNOS inhibitor 1400 W (10 µM) and the O473 and Thr308 residues which is known to be required for its full activation by the upstream kinases PDK1 and mTORC2 and subsequent activation of AS160 which triggers GLUT4 translocation.As Akt is considered to be the upstream regulator of GLUT4 translocation we tested the effect of Ang II on its activation in response to insulin. This was achieved by determining its level of phosphorylation on Ser473 was more persistent than that of Thr308 (≥120 min. vs. 60 min.), suggesting partial inactivation of the enzyme after 1 hour. Incubation of the myotubes with 10 nM Ang II alone decreased Akt phosphorylation on Ser473 below control levels and inhibited insulin-mediated phosphorylation on both Ser473 and Thr308 by more than 80% . These data indicate that, as observed for GLUT4 translocation, NO and O2−. generation are required for the inhibition of Akt phosphorylation by Ang II. Likewise Ang II inhibited basal Akt phosphorylation below control levels (P<0.05) and the ROS and RNS modulators used restored basal phosphorylation (data not shown).Since NAD(P)H-oxidase and NOS inhibitors and a free radical scavenger significantly restored Ang II-induced inhibition of GLUT4 translocation to the plasma membrane A, we ex473 B. Ident2−. are known to react yielding the RNS peroxynitrite. The absolute requirement of NO and O2−. generation for the inhibition of Akt phosphorylation , is located in the α-helix E, close to β6 and the C-loop which contains the active site Asp166 with which it might interact.As the molecular mechanism through which tyrosine nitration regulates kinase activity is still unclear, we tried to map the nitration sites in both ERK 1 and Akt1 in order to see whether their location may give some clues. Both recombinant human enzymes were nitrated in vitro with SIN-1 and digested with trypsin and chymotrypsin. The tryptic fragments were immunopurified with an anti-nitrotyrosine antibody and analyzed by MALDI-TOF mass spectrometry. Whereas we were unable to analyze nitrated Akt peptides due to digestion and ionization problems, we identified two peptides displaying a mass difference of +45 by MS analysis, corresponding to the adjunction of a nitro group with ERK1. The peptides with monoisotopic masses of resp. 1552.91 Da and 2749.28 Da correspond to aminoacids 153 – 165 (GLKYIHSANVLHR) and 109–131 (ASTLEAMRDVYIVQDLMETDLYK). The first peptide contains only one Tyr A indica in mass B. We we2.−2− generation are upregulated in type 2 diabetes Since the CAPPP trial reported in 1999 that an ACE inhibitors significantly reduce the risk of developing diabetes as compared to other antihypertensive treatments , numerous other trials have confirmed the unique diabetes-preventive properties of ACE inhibitors and Ang II receptor antagonists as compared to other available blood pressure lowering drugs 2.−- H2O2 - OH. generation cascade, O2−. can also give rise to the generation of RNS such as N2O3, NO+, NO2. and ONOO−, depending upon the NO/O2−. ratio − which can affect DNA, lipids and proteins Whereas oxidative stress usually evokes the activation of the O− generation, we have shown that Ang II stimulates the nitration of MEK 1 and ERK 1/2 Interestingly, Ang II has been shown to stimulate the generation of peroxynitrite 22 in insulin resistance 2.− ratio of 2 to 3 2.−, also increased the inhibition of Akt by the pure nitric oxide donor SNAP 2.− in the presence of NO tends to shift the production of nitrosating species (NO+ and/or N2O3) towards peroxynitrite which selectively nitrates tyrosine residues 2.− generation Hoxidase inhibitor AEBSF and the scavenger myricetin, indicate that increased oxidative stress is involved in this process -oxidases, xanthine oxidase, mitochondrial complexes I and III or iNOS, are activated. This occurs in response to various signals including hormones and cytokines as well as pathological conditions like hypoxia, ischemia, hypo- and probably hyperglycemia As indicated above, S-nitrosation is a modification which is rapidly reversible by reduction and which therefore probably is a fine-tuning mechanism of enzyme activity depending upon small variations of O469-X-X-Phe472-Ser473-Tyr474) of its C-terminal part with the kinase domain appear to play a crucial role 80 or its burying in the hydrophobic groove. Interestingly, this motif contains a Tyr residue at position 474. It is therefore conceivable that if this Tyr were to be nitrated, it may switch the kinase from the active to the inactive state by increasing its hydrophilicity. Moreover, this Tyr has also been reported to be phosphorylated, this modification being required for the full activation of Akt 272 located next to Arg273 which is believed to be in contact with the primary phosphate of ATP and to Asp274 which is assumed to be the active site Although the intramolecular interactions involved in the phosphorylation and activation of Akt are extremely complex, the interactions of its pleckstrin homology (PH) domain and the hydrophobic motif Hoxidase and iNOS and in acute manner by activating them thereby leading to increased nitrative stress. The activation mechanisms of iNOS are obviously very complex and involves interactions with multiple proteins Whereas ARBs and ACEI have demonstrated their efficiency in improving insulin sensitivity and reducing end-organ damage and are now often considered as first-line therapy in hypertension with metabolic syndrome or diabetes l-NAME) from Cayman, insulin and 3-nitrotyrosine were purchased from Sigma. 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) and Chemiluminescence reagent were from Uptima, Interchim. 1400 W and Trolox were from Acros Organics. The MEK inhibitor U0126 was from Cell Signaling Technology. Active recombinant human PKB/Akt1 was from Assay Designs and the Akt activity immunoassay kit from Calbiochem. Dulbecco's modified Eagle's Medium , fetal bovine serum (FBS), horse serum (HS) and other cell culture reagents were purchased from Invitrogen. Cell culture dishes were from Nunc. All other reagents were of analytical grade.Ang II was from Bachem, myricetin, SIN-1 and nitro-l-arginine-methyl-ester  were purchased from Cell Signaling Technology. Anti-ERK1/2 antibody was from Sigma. Anti-GLUT4 antibody was from Biogenesis. Affinity-purified polyclonal anti-nitrotyrosine antibodies were obtained as described previously Antibodies directed against Akt, phosphoSer2 in 37°C until 80–90% confluence. Myoblasts were differentiated into myotubes by replacing FBS with 2% horse serum for 6–8 days. Cells of passages 3–6 were used for experiments.L6 rat skeletal muscle cells (American Type Culture Collection) were grown in DMEM (1 g/L glucose) containing 20% (v/v) of fetal bovine serum (FBS) and gentamycin (10 µg/ml) at 5% COg for 10 min) at 4°C. All subsequent steps were carried out at 4°C. Cells were resuspended in buffer containing 250 mM sucrose, 5 mM NaN3, 2 mM EGTA, 20 mM HEPES pH 7.4, and a protease inhibitor cocktail, as described above, and homogenized using 20 strokes of a Dounce homogenizer. The homogenate was centrifuged at 760 x g for 5 min to remove nuclei and unbroken cells. The supernatant was centrifuged at 30000 x g for 60 min to pellet the crude plasma membrane fraction. Plasma membrane and total cell lysate proteins were resolved by SDS-PAGE, blotted on PVDF membrane and probed with anti-GLUT4 antibody.Subcellular fractionation of myotubes was carried out as described by Yonemitsu et al. ®). Covalent hydrazone bonds were stabilized by reduction with NaCNBH3. For immunoblotting, samples were subjected to 10% SDS-PAGE and transferred to a PVDF membrane (Perkin Elmer Life Sciences Inc®.). Membranes were blocked with TBST  containing 5% milk and probed with the antibodies indicated in the figure legends and appropriate HRP-labeled secondary antibodies. The blots were revealed by enhanced chemiluminescence. Densitometric analysis was performed using Scion Image software.For immunoprecipitation, 50 µg of cell lysate protein was incubated overnight at 4°C with anti-nitrotyrosine antibody covalently coupled to agarose beads according to the manufaturer's protocol  and 100 mM NaCl for 1 hour at 25°C. Active recombinant Akt1 corresponds to Akt1 purified from extracts of Sf9 cells co-expressing full-length human Akt1 and mTOR.++/ATP. Samples were subjected to 12% SDS-PAGE and transferred to a PVDF membrane (Perkin Elmer Life Sciences). Membranes were blocked with TBST  containing 5% milk and probed with an anti-phosphoSer21-GSK-3α followed by an HRP-labeled anti-rabbit antibody. The blots were revealed by enhanced chemiluminescence.Briefly, PKB/Akt was immunoprecipitated and incubated with a GST fusion protein comprising the GSK-3α phosphorylation sequence in the presence of Mg2 and100 µM ATP for 2 hours at 20°C. After nitration, the GST was cleaved with thrombin-agarose (Sigma®) according to the manufacturer's protocol. GST was removed by glutathione-agarose (Pierce®) affinity chromatography. The nitrated ERK samples were digested with trypsin (Promega®) at 25 ng/ml during 2 h. The samples were diluted in 25 mM ammonium bicarbonate (pH 7.4) with 50 mM NaCl and then incubated in the presence of 20 µl agarose-conjugated anti-nitrotyrosine antibodies. The nitrated peptides were eluted with 50% acetonitrile/0.2% trifluoroacetic acid, dried, resuspended in 1.5 µl of matrix suspension , and deposited on a stainless steel target. Mass spectrometry analysis was performed on a Q-TOF Ultima Global mass spectrometer (Micromass®) equipped with a MALDI source. Ionization was achieved using a nitrogen laser . The instrument was externally calibrated using the monoisotopic masses of tryptic and chymotryptic peptides from bovine serum albumin. Acquisitions were performed in a V-mode reflectron position. Microsequencing was performed by argon-induced fragmentation after selection of the parent ion.Recombinant human ERK1-GST (Upstate Biotech) was nitrated with 100 µM SIN-1 in buffer containing 20 mM MOPS (pH 7.2), 25 mM β–glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, with 15 mM MgClStatistical analysis was performed by one-way ANOVA followed by the Holm-Sidak method for comparison between groups. P<0.05 was considered to be statistically significant. Error is represented as standard deviation.


Drosophila embryos is instructed by the morphogen gradient of Bicoid (Bcd). Despite extensive studies of this morphogen, how embryo geometry may affect gradient formation and target responses has not been investigated experimentally.Patterning along the anterior-posterior (A-P) axis in In this report, we systematically compare the Bcd gradient profiles and its target expression patterns on the dorsal and ventral sides of the embryo. Our results support a hypothesis that proper distance measurement and the encoded positional information of the Bcd gradient are along the perimeter of the embryo. Our results also reveal that the dorsal and ventral sides of the embryo have a fundamentally similar relationship between Bcd and its target Hunchback (Hb), suggesting that Hb expression properties on the two sides of the embryo can be directly traced to Bcd gradient properties. Our 3-D simulation studies show that a curvature difference between the two sides of an embryo is sufficient to generate Bcd gradient properties that are consistent with experimental observations.The findings described in this report provide a first quantitative, experimental evaluation of embryo geometry on Bcd gradient formation and target responses. They demonstrate that the physical features of an embryo, such as its shape, are integral to how pattern is formed. Drosophila morphogen Bicoid (Bcd), have overcome such challenges and revealed important new insights into the behavior of this morphogen gradient . Here wDrosophila embryos, is responsible for patterning the anterior structures, including the head [Bcd, a morphogen which forms a gradient along the anterior-posterior (A-P) axis in the head ,12. It ithe head -15. Bcd the head  thought the head ). At thethe head ,19. SincIn this study, we systematically analyze the behaviors of both the Bcd gradient and its target responses on the dorsal (D) and ventral (V) sides of the embryo to investigate the effects of embryo geometry on morphogen gradient formation and action. We sought to address the question, through quantitative studies of Bcd and its direct gap gene targets, of how segmentation gene expression patterns exhibit differences between the dorsal and ventral sides. Previous studies show that the expression boundaries of gap genes and expression stripes of pair-rule genes are not parallel to the D-V axis, but rather present a splay on the ventral side of the embryo -23. How a to point b, which is shorter than the distance along a semi-circle (blue) or a partial ellipse (green). While displacement is simply the length of the line segment between points a and b, more quantities such as curvature are required to determine the distance along a curved path. In developmental biology, how distance is measured  by morphogen gradients is an important question. This question is directly relevant to the fundamental problems of how morphogen gradients are formed and how their encoded positional information is interpreted in a developing embryo or tissue. In early Drosophila embryos, the dorsal and ventral sides exhibit different geometric features. For example, as shown in Fig. CD = 645.4 ± 18.3 μm (standard deviation from 28 wt embryos), is significantly shorter than the contour length measured along the perimeter of the ventral side (red), CV = 674.9 ± 17.2 μm . The difference between the two contour lengths, CV and CD, is a direct consequence of the different curvatures on the two sides of the embryo.In physics and mathematics, the distance along a curved path between two points is distinct from the displacement along a straight line connecting them. For instance, in Fig. x from the anterior pole or contour distance c from the identical anterior pole . In Fig. cembryo/CD (Δcembryo = cD - cV), against fractional embryo length  x/L in wt embryos. This plot reveals the following information regarding the geometric differences between the dorsal and ventral sides of the embryo: 1) cV is longer than cD at same x/L positions in most parts of the embryo, except near the anterior pole (x/L < ~0.12); 2) there are two broad regions of the embryo, ~0.12 <x/L < ~0.36 and ~0.66 <x/L < ~0.96, where cV expands/accumulates its lead over cD; 3) in the mid-embryo section along the A-P axis (~0.36 <x/L < ~0.66), Δcembryo is stable, reflective of a small curvature difference between the two sides of the embryo ; our experimental conditions yielded Bcd intensity profiles with a reproducibility that is comparable to live-imaging studies [x-y coordinates in microns in order to quantify the intensity profiles using two distinct distance measurement methods: x and c as defined above.In our analysis, embryos at early nuclear cycle 14 were triple-stained with anti-Bcd and anti-Hb antibodies as well as DAPI (see Methods for details). We captured high-resolution digital images on the midsagittal plane of the embryos and measured fluorescence intensities along the cortical, nuclear layer Fig. . As furt studies ,8. The hB from the dorsal and ventral sides against either x or c in wt embryos. We binned all recorded positions from all embryos using two distinct methods: 1) either using a constant x interval (11 pixels in this study) between neighboring bins for plotting Bcd intensity profiles as a function of projected distance x from the anterior, or 2) using a constant c interval  between neighboring bins for plotting Bcd intensity profiles as a function of contour distance c from the anterior. Our results show . In contrast, these two average Bcd profiles are much closer to each other when measured as a function of contour distance c . As explained above, these results are supportive of a hypothesis that, at the syncytial stage, the contour distance c, as opposed to projected distance x, represents a more proper measurement of distance during Bcd gradient formation  as either projected distance xHb from the anterior or contour distance cHb. Fig. x or c, respectively. While these two Hb profiles are visually different from each other when measured as a function of x  are significantly different from each other  embryos. To ensure a direct evaluation of the relationship between Bcd and Hb, we extracted Bcd and Hb intensities in wt embryos that were simultaneously stained with both Bcd and Hb antibodies (see Methods for details). Again, we analyze Hb expression profiles in two distinct ways: as a function of either projected distance f x Fig. , they co. c Fig. . Quantit(~6.5 μm ) or the It has been suggested that the A-P and D-V patterning systems can interact with each other during development ,26-28. SB/Kd) for the dorsal and ventral sides of the embryo  on the dorsal and ventral sides of the embryo  nor their n values  exhibit a significant difference. Finally, to evaluate the precision of the Hb readout machinery in response to the Bcd gradient, we calculated the normalized standard deviations of Hb expression (δH/Hmax) on both sides of the embryo as a function of Bcd concentration (B/Kd). Our results . Since lts Fig.  reveal aδB/B  profiles. Previous studies including our own focused primarily on the reproducibility of the Bcd gradient on the dorsal or lateral side of the embryos [x. Here we measure them as a function of c and directly compare the measured Bcd intensity noise on dorsal and ventral sides of the embryo. Our results show that the ventral side has a higher δB/B around cHb than the dorsal side  to its positional errors σc [σc on the ventral side  than the dorsal side (27 ± 0.6 μm at cHb). If Hb expression properties are dictated by Bcd gradient properties as suggested above, Hb expression is expected to be more variable on the ventral side than on the dorsal side. To specifically test this prediction, we compared the precision of cHb on the two sides of the embryos. Our results show that the variability of Hb boundary on the ventral side (σcHb = 11.3 ± 1.3 μm) is higher than that on the dorsal side (σcHb = 9.1 ± 0.9 μm). These results provide further support to our conclusion that the dorsal and ventral sides of the embryo are fundamentally the same in regard to the Bcd-Hb relationship.To further evaluate the Bcd and Hb behaviors on the dorsal and ventral sides of the embryo, we compared their Bcd intensity noise  embryos ,8,30-32.ide Fig. . Althougrrors σc . Our resxHb (see above for definition), effectively depicts a tilt or slant of the Hb expression boundary toward the anterior pole on the ventral side, which is reminiscent of the splay of A-P gene expression stripes at later stages of nuclear cycle 14 [xBcd and ΔcBcd (ΔxBcd = xBcdD - xBcdV and ΔcBcd = cBcdD - cBcdV where BD = BV), as a function of Bcd concentrations on log scale  should have a larger ΔxTarget with an enhanced slanting. Third, the distribution of the lines connecting the boundary positions on the two sides of the embryo should exhibit a global similarity to that of Bcd profiles shown in Fig. xBcd is larger than ΔcBcd except at the highest Bcd concentrations (which correspond to locations near the anterior pole of the embryo), ΔxTarget should be larger than ΔcTarget.Our results shown in Fig. xHb in embryos that have either reduced (1×-bcd) or increased (3×-bcd) Bcd concentrations, thus shifting the xHb position toward the anterior and posterior, respectively. We also analyzed the expression boundary of another direct Bcd target, Orthodenticle (Otd), in wt embryos. Similar to our analysis of Hb expression in wt embryos, we measured the expression boundary positions on both dorsal and ventral sides. Our results  is smaller than ΔxTarget (blue) in all cases tested . Bcd gradient formation is a complex process, which takes place during a developmental period when the embryo itself is undergoing an exponential growth in terms of the nuclear number. There are also migrations of the nuclei to the cortex of the embryo at nuclear cycle 10, leading to the formation of the cortical (nuclear) layer where Bcd activates transcription of its downstream target genes [Drosophila genome. Here we expand this 2-D model to a new 3-D model (see Methods for further details).We now return to the hypothesis that the Bcd gradient is formed in a process where contour distance from the anterior is measured, as suggested by our observed Bcd gradient properties in wt embryos at early nuclear cycle 14  is smaller than their corresponding ΔxBcd .To further study the effects of embryo geometry on Bcd gradient formation, and to specifically determine whether a curvature difference on the two sides of the embryo may be sufficient to explain the observed Bcd gradient properties, we performed simulations in our 3-D model. Fig. lts Fig. , ΔcBcd ax from the anterior pole and the contour distance c along the perimeters of the dorsal or ventral sides of the embryo. Our findings support a hypothesis that the Bcd gradient profile establishes and exerts the positional information by following the contour distance c. Our simulation studies  between the cortical layer and the inner part of the embryo is important in proper Bcd gradient formation.In this study we systematically analyze the Bcd and Hb behaviors on both the dorsal and ventral sides of the embryo. We reasoned that, since these two sides of the embryos have distinct geometric features . These results indicate that simple 1-D diffusion models, even when incorporating the appropriate consideration that the cortical layer is a critical medium for Bcd diffusion, are inadequate to address the geometric issues in such a complex biological process. In contrast, our 3-D simulation with an asymmetric embryo revealed results that are strikingly similar to these experimental findings including a lower Bmax and longer λ on the ventral side than the dorsal side . These differences are also obtained in a 2-D simulation of an asymmetric embryo in which, similar to the simulated 3-D embryo, Bcd molecules can diffuse within the inner part of the embryo and are allowed to exchange between the inner part and the cortical layer (not shown). However, in a 3-D simulation where the inner part of the embryo is impermeable to Bcd molecules , the simulated λV is not larger than λD (not shown). We note that, although our 3-D simulation can replicate our experimentally observed Bcd gradient properties, it is possible that other physical features of the embryo may exert additional effects on Bcd gradient formation. For example, it has been shown that the ventral side of wt embryos has a lower nuclear density than the dorsal side [Our experimental and simulation results suggest that proper Bcd gradient formation may require an interaction between the cortical layer and the inner part of the embryo. In particular, we found that the Bcd gradient profiles on the two sides of the same embryo are not identical even when measured as a function of contour distance c Fig. . Ideallysee Fig.  and 2B asal side ,41. It isal side  but suchThe experimental and theoretical studies described in this report represent a first, systematic investigation of the effects of embryo geometry on the positional information encoded by the Bcd gradient. Our results suggest that the ventral splay of segmentation genes along the A-P axis may be directly traced to the distinct properties of the Bcd gradient (and its gap target gene responses) on the dorsal and ventral sides of the embryo. Our findings also suggest that, while the cortical layer is critical for Bcd gradient formation, Bcd diffusion likely requires an interaction between the cortical layer and the inner part of the embryo. These results demonstrate that modeling a morphogen gradient requires a realistic consideration of the physical properties, including the geometry, of the relevant biological system.bcd embryos were from E1bcd/+ females and 3×-bcd embryos were from CyO, bcd/+ females (gift of Gary Struhl). For Bcd-Hb-DAPI triple staining, we used rabbit anti-Bcd [E1 bcdfemales (thus lacking Bcd). In our study, the mean Bcd intensities of E1 bcdembryos were simply subtracted as background from the intensity data of the experimental embryos. All stained embryos were mounted under cover slips with bridges to avoid geometric distortions, and images were taken on the midsagittal plane. The estimated noise in L measurement caused by focal plane selection is <0.1% [L and CD (~5%) or between CD and CV (~13%) in our current study.In our experiments, embryos were collected, fixed and stained with antibodies as described previously . 1×-bcd anti-Bcd  and guinanti-Bcd  as primaanti-Bcd  and rat anti-Bcd  as primais <0.1% , which iμm2) smaller than that of a nucleus was allowed to slide along the entire nuclear layer, on both the dorsal and ventral sides of the embryo images. The center of the scanning window was kept ~6 μm away from the perimeter of the embryo, with a pixel-by-pixel sliding along the A-P axis projection  were extracted from the images. For each embryo, the contour distance c from the anterior is computed as:All results presented in this study were from embryos at early nuclear cycle 14 with an approximated time window of 6-24 minutes after mitosis, which were selected based on nuclear number, size and shape, and the posterior Hb level as described previously . DAPI imion Fig. . At eachj is the index of the scanning windows. Raw Bcd intensities were obtained by averaging the pixel intensities within the scanning windows, without further adjustments except background subtraction when necessary . In our analysis, the measured fluorescence intensities for both Hb and Otd were normalized by setting the mean of the highest values to 1 and the mean at non-expression regions to 0. See additional file where ary Fig.  and 3C. x/L. To compute the iso-intensity contour lines for Bcd, we choose a series of intensity values within the range of measured raw Bcd intensities as thresholds (kt) to identify positions  where BD = BV = k tby interpolation. Then we plot these positions onto the average embryo frame and connect the positions on the dorsal and ventral sides to visualize the distribution of iso-intensity contour lines  for 1×-bcd, wt and 3×-bcd embryos , whose average frames are calculated separately . For visual comparison, we also plot lines connecting the target boundary positions on two sides of the embryos using normalized average frames of 1×-bcd, wt and 3×-bcd embryos in the same presentation . In our current work, an embryo was selected to resemble the average shape of a wt embryo with its dorsal and ventral curvatures on the midsagittal plane directly extracted from experimental measurements . We tested our model by making reasonable changes in the shape or size of the mRNA distribution and obtained consistent Bcd protein gradient properties (not shown). The parameter value for the aggregate Bcd synthesis rate J for the entire embryo in our current model does not affect the experimentally observed three properties discussed in the main text (not shown), and we chose J = 1000 molecules/s so that at cycle 14, the maximum nuclear Bcd concentration on the dorsal side is 37.1 molecules/μm3, a value consistent with experimental estimates [μm in our current model and was kept unchanged between nuclear cycles 10 to 14; our simulations using thicker cortical layer similarly yielded the three experimentally observed properties (not shown). The simulated developmental time is based on experimental observations [D = 2 μm2s-1; the spatially-uniform degradation rate of Bcd, ω = 0.00005 s-1, and the ratio of DNA-bound to free local Bcd concentrations at nuclear cycle 10, Bbound/Bfree = 0.15. As further detailed in [λ values of the simulated Bcd profiles [3, 23 and 53 μm3 and Δt = 0.1, 0.2, and 0.5 s, and obtained consistent results of Bcd gradient behaviors, indicating that our solutions are numerically stable. Results in Fig. 3 μm3 and Δt = 0.2 s. We also conducted simulations to evaluate the effects of other parameters on simulated Bcd gradient properties. As shown in additional file bcd mRNA sphere's center location along the A-P or D-V axis  or the Bbound/Bfree ratio at nuclear cycle 10, while affecting the specific values of the simulated profiles as expected, yielded results that remain generally consistent with the three experimentally observed properties. Together, these results show that our simulated Bcd properties that are in agreement with those observed experimentally are robust to reasonable variations  that may naturally exist among real embryos.In our model, the anterior pole of the simulated embryo has a coordinate of , and the stimates . The corrvations ,19, and ailed in , these pprofiles ,8, an enprofiles , are adjFH and JM conceived and designed the study. FH, YW and DC performed experiments. FH analyzed data. FH, LJL, RJ, and JM interpreted results. FH and JD developed the theoretical model and conducted simulations. FH generated all figures. FH and JM wrote the paper. All approved the paper.Theoretical 2-D consideration of geometry and positional information.Click here for filebcd embryosFigure S1: Analysis of D-V differences for Bcd and Hb profiles in 1×-. (A and B) Average raw Bcd intensity profiles on the dorsal (blue) and ventral (red) sides of 24 1×-bcd embryos when measured as a function of projected distance x from the anterior (A) or contour distance c (B). In each panel, the inset shows a magnified view of the region surrounding the Hb expression boundary positions (solid arrowheads). Listed below are p values from Student's t-tests at the indicated locations in the insets: 10-5, 10-5, 0.0003, 0.002, and 0.02 for panel A, and 0.004, 0.0004, 0.002, 0.01 and 0.05 for panel B. (C and D) Average normalized Hb intensity profiles on the dorsal (blue) and ventral (red) sides of the same 1×-bcd embryos when measured as a function of projected distance x from the anterior (C) or contour distance c (D). See Additional file bcd embryos analyzed in a scatter plot (E), average input-output relationship (F), profiles of Hb expression noise in response to Bcd concentration (G) and Bcd intensity noise (H). These analyses, together with those obtained in wt embryos , reveal a fundamentally similar Bcd-Hb relationship on both sides of the embryos. See Additional file Click here for filebcd embryosFigure S2: Analysis of D-V differences for Bcd and Hb profiles in 3×-. (A and B) Same as panels A and B of Fig. S1, except the data are from 29 3×-bcd embryos. Listed below are p values from Student's t-tests at the indicated locations in the insets: 0.01, 0.02, 0.01, 0.02, and 0.13 for panel A, and 0.15, 0.39, 0.81, 0.36 and 0.71 for panel B. (C-H) Same as panels C-H of Fig. S1, except the data are from 3×-bcd embryos. See Additional 9- Table S1 for measured values.Click here for filebcd embryosFigure S3: Analysis of distance measurements in 1× and 3×-. (A and C) Profiles of distance differences between the dorsal and ventral sides of the 1×-bcd (A) and 3×-bcd (C) embryos when measured as either projected distance from the anterior  or contour distance . (B and D) Iso-concentration contour lines for Bcd plotted on the average frames for 1×-bcd (B) or 3×-bcd (D) embryos, respectively. The intensity increments between neighboring contour lines (red) are 0.5 (B) and 1.5 (D), respectively. See main text and Fig. Click here for fileFigure S4: Raw Bcd intensity data. (A and B) Raw Bcd intensity profiles on the dorsal (blue) and ventral (red) sides of 24 1×-bcd embryos when measured as a function of projected distance x from the anterior (A) or contour distance c (B). (C and D) Same as panels A and B, except the data are from 28 wt embryos. (E and F) Same as panels A and B, except the data are from 29 3×-bcd embryos.Click here for fileFigure S5: Normalized Hb intensity data. (A and B) Normalized Hb intensity profiles on the dorsal (blue) and ventral (red) sides of 24 1×-bcd embryos when measured as a function of projected distance x from the anterior (A) or contour distance c (B). (C and D) Same as panels A and B, except the data are from 28 wt embryos. (E and F) Same as panels A and B, except the data are from 29 3×-bcd embryos.Click here for fileFigure S6: Simulating an embryo that has an asymmetric geometry. (A) A heat map of local total Bcd concentration on the midsagittal plane of a simulated embryo, which is approximated by two semi-ellipsoids joined on the coronal plane, with a height of 92 μm and 128 μm for the dorsal and ventral sides, respectively, and a length of 560 μm. For consistency, all other parameters here are set the same as those used to generate the results shown in Fig. xOtd/L (panel B) and xHb/L (panel C). Color bar represents normalized local total Bcd concentration only showing the range for the cortical layer. (D and E) Nuclear Bcd concentration profiles from the cortical layer of the dorsal (blue) and ventral (red) sides of the simulated embryo, measured as a function of either projected distance x from the anterior (panel D) or contour distance c (panel E).Click here for filebcd mRNA location and DNA binding on Bcd gradient propertiesFigure S7: Testing the effects of . In these simulations, the center location of the bcd mRNA sphere was systematically moved either along the A-P axis  or along the D-V axis . We also systematically changed the ratio of Bbound/Bfree at nuclear cycle 10 (panels G-I). Here, the shape of the simulated embryo is the same as in Fig. Bmax values , λ values expressed as either c or x , and the values of ΔxBcd and ΔcBcd at target boundary positions of both Hb and Otd . As shown in individual panels, these simulated results remain consistent with the three experimentally observed properties: 1) Bmax is higher on the dorsal side than on the ventral side , 2) λ measured as either x or c is larger on the ventral side than on the dorsal side , and 3) ΔxBcd at target boundary locations is larger than their corresponding ΔcBcd . As further discussed in the theoretical consideration above, the value of ΔxBcd - ΔcBcd in the middle section of the embryo reflects the geometric properties of the embryo. This value is 11.1 μm at xHb/L in our simulation results shown in this figure and, as expected, is insensitive to bcd mRNA location or the Bbound/Bfree ratio. It is also fully consistent with both experimental (10.7 μm as shown in Fig. μm as shown in Fig. Click here for fileTable S1: Experimentally determined parameter values for the Bcd-Hb relationship.Click here for file


Infectious bronchitis virus primarily induces a disease of the respiratory system, different IBV strains may show variable tissue tropisms and also affect the oviduct and the kidneys. Proventriculitis was also associated with some new IBV strains. Aim of this study was to investigate by immunohistochemistry (IHC) the tissue tropism of avian infectious bronchitis virus (IBV) strain M41 in experimentally infected chicken embryos.3 EID50 of IBV M41 at 10 days of age. At 48, 72, and 120 h postinoculation (PI), embryos and chorioallantoic membranes (CAM) were sampled, fixed, and paraffin-wax embedded. Allantoic fluid was also collected and titrated in chicken embryo kidney cells (CEK). The sensitivity of IHC in detecting IBV antigens in the CAM of inoculated eggs matched the virus reisolation and detection in CEK. Using IHC, antigens of IBV were detected in nasal epithelium, trachea, lung, spleen, myocardial vasculature, liver, gastrointestinal tract, kidney, skin, sclera of the eye, spinal cord, as well as in brain neurons of the inoculated embryos. These results were consistent with virus isolation and denote the wide tissue tropism of IBV M41 in the chicken embryo. Most importantly, we found infection of vasculature and smooth muscle of the proventriculus which has not seen before with IBV strain M41.To this end chicken embryos were inoculated in the allantoic sac with 10IHC can be an additional useful tool for diagnosis of IBV infection in chickens and allows further studies to foster a deeper understanding of the pathogenesis of infections with IBV strains of different virulence. Moreover, these results underline that embryonic tissues in addition to CAM could be also used as possible source to generate IBV antigens for diagnostic purposes. Coronaviridae in the order Nidovirales. More than 25 genotypes are distributed worldwide. IBV causes an acute highly contagious viral respiratory disease of chickens which is characterized by respiratory rales, coughing and sneezing [in vitro is restricted to primary chicken cells and depends on the expression of 2,3-linked sialic acids on the cell surface. Thus, these molecules are supposed to serve as receptor determinants for primary attachment of IBV to host cells [Infectious bronchitis virus is the prototype species of the family sneezing . Some IBsneezing ,2. In otsneezing . Most issneezing . Replicast cells .in situ hybridization[The conventional diagnosis of the IBV is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. Since two or three blind passages are often required for successful primary isolation of IBV, this procedure could be tedious and time consuming. Alternatively, IBV may be isolated by inoculation in chicken tracheal organ cultures. This method is sensitive  but highidization. The revidization. Aim of 3 EID50 IBV M41 in SPF ECE resulted in death of embryos at 24 h (3 embryos), 41 h (2 embryos) and single embryonic death at 65, 87 and 120 h after IBV inoculation. Allantoic fluid and CAM were harvested at the times given in Table 2 TCID50 at 120 h PI  were prepared from specific pathogen free (SPF) embryonated chicken eggs (ECE) as described elsewhere . Briefly3 EID50). Inoculated ECE were candled twice daily. At 48, 72, or 120 h dead and/or living whole embryos as well as allantoic fluid and CAM were collected. Embryos died at any time after inoculation, were also sampled accordingly. The allantoic fluid of inoculated eggs was harvested and clarified by centrifugation for 10 min at 925 g. The obtained supernatants were used for titration in CEK.Twelve ten days old SPF ECE were inoculated in the allantoic sac with 200 μl of IBV strain M41(10The presence of IBV antigens was investigated in CAM and embryos using the avidin-biotin complex (ABC) method for immunohistochemistry . To this5 CEK cells per well. After 48 h of incubation, cells were fixed with acetone-methanol, and virus titers expressed as tissue culture infective dose fifty (TCID50) were determined by indirect immunofluorescence using a polyclonal serum raised in rabbits against purified IBV M41 virions.100 μl of tenfold serial dilutions in MEM were added to 10IBV infected CEK cells were fixed with acetone-methanol for 15 min. After washing, cells were incubated with the polyclonal rabbit anti-IBV serum (1:2000) for 1 h and subsequently with FITC-conjugated goat anti-rabbit antibodies (Sigma) (1:2000) for 1 h. Both primary and secondary antibodies were diluted in PBS. Plates were rinsed three times with PBS after each step.The authors declare that they have no competing interests.ASA performed virus inoculation in ECE and cell culture, IFA, contributed to IHC, analyzed the data, and drafted the manuscript. PZ contributed to IHC and histopathological examination, JV performed virus isolation and IFA. GMK and JPT initiated the project, provide continued directions and critically reviewed the manuscript. JPT also performed and supervised histopathology and IHC. All authors read and approved the final manuscript.


This results in the release of Ca2+ from intracellular stores and the activation of protein kinase C. The activation of Group I mGluRs also results in ERK1/2 phosphorylation. We show here, that the proline-rich tyrosine kinase 2 (Pyk2) interacts with both mGluR1 and mGluR5 and is precipitated with both receptors from rat brain. Pyk2 also interacts with GST-fusion proteins corresponding to the second intracellular loop and the distal carboxyl-terminal tail domains of mGluR1a. Pyk2 colocalizes with mGluR1a at the plasma membrane in human embryonic kidney (HEK293) cells and with endogenous mGluR5 in cortical neurons. Pyk2 overexpression in HEK293 results in attenuated basal and agonist-stimulated inositol phosphate formation in mGluR1 expressing cells and involves a mechanism whereby Pyk2 displaces Gαq/11 from the receptor. The activation of endogenous mGluR1 in primary mouse cortical neuron stimulates ERK1/2 phosphorylation. Treatments that prevent Pyk2 phosphorylation in cortical neurons, and the overexpression of Pyk2 dominant-negative and catalytically inactive Pyk2 mutants in HEK293 cells, prevent ERK1/2 phosphorylation. The Pyk2 mediated activation of ERK1/2 phosphorylation is also Src-, calmodulin- and protein kinase C-dependent. Our data reveal that Pyk2 couples the activation mGluRs to the mitogen-activated protein kinase pathway even though it attenuates mGluR1-dependent G protein signaling.Group I metabotropic glutamate receptors (mGluRs) are coupled via Gα InsP3 stimulates the release of Ca2+ from intracellular stores and both Ca2+ and diacylglycerol regulate the activation of protein kinase C (PKC).Glutamate is the predominant excitatory neurotransmitter in the brain and regulates physiological responses that include the development, survival, and death of neurons as well as neural plasticity -6. The rq/11 resulting the displacement of the heterotrimeric G protein from the receptor myo-inositol was acquired from PerkinElmer Life Sciences . The Dowex 1-X8 (formate form) resin with 200-400 mesh was purchased from Bio-Rad. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies, protein G-Sepharose beads, enhanced chemiluminescence (ECL), and mouse monoclonal anti-hemagglutinin (HA) (12CA5) antibody were purchased from GE Healthcare . Anti-GST mouse monoclonal antibody and anti-Gαq/11 rabbit polyclonal antibody were from Santa Cruz Biotechnology Inc. . Anti-mGluR5 rabbit polyclonal antibody was obtained from Upstate . Anti Pyk2/CAKβ mouse monoclonal antibody was from BD Transduction Laboratories . Rabbit polyclonal phospho-p44/44 MAP kinase (Thr202/Tyr402), p44/44 MAP kinase, phospho-Pyk2 (Tyr402) antibody, phospho-Pyk2 (Tyr457), Pyk2, phosphor-Src (Tyr 416) and Src antibodies were all obtained from Cell Signaling Technology. . Lipofectamine Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG were purchased from Invitrogen/Molecular Probes . siRNAs were purchased from Dharmacon RNAi Technologies . Hi-Perfect Transfection Reagent was purchased from Qiagen . All other biochemical reagents were purchased from Sigma , Fisher Scientific  and VWR Scientific .Human embryonic kidney (HEK293) cells and COS-7 cells were obtained from the American Type Culture Collection . Tissue culture reagents were purchased from Invitrogen . Isopropyl-β-D-thiogalactopyranoside (IPTG) was obtained from Bioshop Canada Inc . Quisqualate, DHPG, MPEP and LY367385 were purchased from Tocris Cookson Inc. . myo-inositol in Dulbecco's modified Eagle's medium. Unincorporated [3H]myo-inositol was removed by washing the HEK293 cells with HBSS. HEK293 cells were preincubated for 1 h in HBSS at 37°C and then preincubated in 500 μl of the same buffer containing 10 mM LiCl for an additional 10 min at 37°C. HEK293 cells were subsequently treated in either the absence or the presence of increasing concentrations (0-30 μM) of quisqualate for 30 min at 37°C. The reaction was stopped on ice by adding 500 μl of 0.8 M perchloric acid and then neutralized with 400 μl of 0.72 M KOH, 0.6 M KHCO3. The total [3H]myo-inositol incorporated into the cells was determined by counting the radioactivity present in 50 μl of the cell lysate. Total inositol phosphate was purified from the cell extracts by anion exchange chromatography using Dowex 1-X8 (formate form) 200-400 mesh anion exchange resin. [3H]Inositol phosphate formation was determined by liquid scintillation using a Beckman LS 6500 scintillation system.Inositol lipids were radiolabeled by incubating HEK293 cells overnight with 1 μCi/ml [Figure Legends. Cells were then transferred back on ice and labeled with secondary FITC-conjugated anti-mouse IgG antibody (1:500 dilution) for 45 min. Under these conditions, receptors are able to undergo only a single round of internalization. Receptor internalization is defined as the fraction of total cell receptors lost from the cell surface and thus is not available for labeling with the secondary antibody.Agonist-independent FLAG-mGluR1a internalization was measured by flow cytrometry. Cell surface epitope-tagged receptors were prelabeled with primary anti-mouse FLAG antibody (1:500) on ice for 45 min. Cells were then warmed to 37°C in the absence of agonist for the times indicated in the Figure Legends. The primary antibodies used in the experiments were phospho-p44/44 MAP kinase (Thr202/Tyr402) antibody (diluted 1:1000), p44/44 MAP kinase antibody (1:1000), phospho-Pyk2 (Tyr402) antibody (1:1000), phospho-Pyk2 (Tyr457) antibody (1:1000), Pyk2 antibody (1:1000), rabbit anti-Flag antibody (1:1000). Antibody reactive bands were visualized and quantified as described above.100 μg samples of cell lysate proteins solubilized from either HEK293 cells, COS7 cells or mouse primary cortical neurons were subjected to were subjected to SDS-PAGE, followed by electroblotting onto nitrocellulose membranes for immunoblotting with antibodies described in the The means ± S.E.M. are shown for values obtained for the number of independent experiments indicated in the figure legends. GraphPad Prism software  was used to analyze data for statistical significance, as well as to analyze and fit dose-response and time course data. The statistical significance was determined by either one way analysis of variance or t-test.It was previously reported that both Pyk2 and mGluR1a were components of a protein complex that could be co-immunoprecipitated with the NMDA receptor . MoreoveBecause Pyk2 can be co-immunoprecipitated with mGluR1a, we examined whether Pyk2 was colocalized with Group I mGluRs in both HEK293 cells and primary rat cortical neurons. HEK293 cells expressing FLAG-mGluR1a and HA-Pyk2 were labeled on ice with Alexa Fluor 568-congugated FLAG antibody and treated either with or without 100 μM quisqualate for 45 min at 37°C. Subsequently, the cells were fixed and permeabilized and labeled with Alexa Fluor 488-conjugated HA antibody to stain HA-Pyk2. In the absence of agonist stimulation, FLAG-mGluR1a was localized to the cell surface and although HA-Pyk2 was expressed throughout the cytoplasm, Pyk2 was also localized at the plasma membrane Fig. . In resp50 for quisqualate-stimulated IP formation (EC50 = 26 nM vs 76 nM)  formation in HEK293 cells expressing FLAG-mGluR1a. We found that in the absence of agonist-treatment HA-Pyk2 reduced basal IP formation in FLAG-mGluR1a expressing HEK293 cells to 69 ± 6% of control values Fig. . SimilarnM) Fig. . Previou mGluR1a . Therefolex Fig. . The recate Fig. . MoreoveA previous study demonstrated that mGluR1 regulated NMDA receptor currents via the activation of a Pyk2/Src-family kinase pathway . TherefoTo examine whether mGluR1a-mediated ERK1/2 phosphorylation was mediated by Src/Pyk2-mediated pathway, we pretreated primary mouse cortical neurons with and without the Src family kinase inhibitor PP2 (10 μM) for 60 min prior to treating the neurons with and without 50 μM DHPG. We found that, in the presence of the Src inhibitor, DHPG-stimulated ERK1/2 phosphorylation was effectively inhibited Fig. . We also2+-calmodulin dependent pathway in non-neuronal cells [Tyrphostin A9 was previously demonstrated to be the most effective amongst an array of 51 tyrosine kinase inhibitors in antagonizing Pyk2 activity in neutrophils . Therefoal cells . The preTo further probe the role of Pyk2 in the activation of ERK1/2 by mGluR1, we utilized siRNA to knockdown endogenous Pyk2 expression in primary mouse cortical neurons. Pyk2 siRNA treatment of cortical neurons resulted in a 66 ± 5% decreases in Pyk2 expression Fig. . Howeverq/11, Pyk2 was required for mGluR1-dependent activation of ERK1/2 signaling.To establish a direct role for Pyk2 in mGluR1a-mediated ERK1/2 phosphorylation, we examined the effect of overexpressing wild-type Pyk2, a dominant negative Pyk2 (Y402F) mutant and a catalytically inactive Pyk2 (K457A) mutant on FLAG-mGluR1 signaling in HEK293 cells. We found that FLAG-mGluR1a activation with 100 μM quisqualate resulted in a 2.4 ± 0.4 fold increase in ERK1/2 phosphorylation in the absence of Pyk2 overexpression Fig.  and 10B.q/11 interactions with the receptor resulting in the attenuation of IP formation [q/11 interactions, while simultaneously coupling the receptor to the activation of the mitogen-activated protein kinase (MAPK) signaling pathway.In the present study, we provide evidence that the non-receptor tyrosine kinase Pyk2 interacts with and regulates mGluR1a signaling and desensitization. Pyk2 associates with the second intracellular loop domain and the carboxyl-terminal tail domains of mGluR1a, and similar to what has been described for GRK2, optineurin and CAIN, Pyk2 functions to disrupt Gαormation ,16,17. H2+ concentrations or activate PKC in response to stress signals, such as UV light, hyperosmotic shock and tumor necrosis factor-α [et al. [2+ concentrations and the activation of both PKCδ and Pyk2 [et al. [1B- or α2A-adrenergic receptors stimulate ERK1/2 phosphorylation via a Ca2+-, calmodulin-, Src- and Pyk2-dependent pathway [Pyk2 is a non-receptor tyrosine kinase that is known to be activated by stimuli that either increase intracellular Cafactor-α . Pyk2 ha [et al.  reportedand Pyk2 . Similar [et al.  showed t pathway . In the  pathway . ConsistPyk2 has previously been demonstrated to be autophosphorylated on Tyr402 in response to the activation of the G protein-coupled lysophosphatidic acid receptor resulting in the creation of a docking site for the Src SH2 domain . The recBoth mGluR1 and mGluR5 are expressed in cortical tissue and primary cortical neurons can be positively stained for the expression of both mGluR1a and mGluR5 proteins . However2+-dependent manner and can be regulated by PKC-mediated phosphorylation of the receptor within the calmodulin binding domain [It has previously been reported that Group I mGluRs increase the extent of Pyk2 phosphorylation in mouse neuronal cortical cultures via a mechanism that is both calmodulin- and Src-dependent, but that is independent of PKC activity . We findg domain -37. The We also find that siRNA treatment of cortical neurons results in a significant reduction in Pyk2 protein expression, but unexpectedly this also results in increased basal ERK1/2 phosphorylation. The increase in ERK1/2 phosphorylation is associated with increased phosphorylation of the remaining fraction of Pyk2 protein expressed in the neuronal cultures. The depletion of Pyk2 expression in COS7 cells also leads to increased basal Pyk2 phosphorylation, but mGluR1a expression is required for this Pyk2-mediated increase in ERK/1/2 phosphorylation. The mechanism by which a loss of Pyk2 expression leads to alterations in the phosphorylation of the residual pool of Pyk2 and increased ERK1/2 phosphorylation is unclear. However, it appears to require mGluR1 expression suggesting that constitutive mGluR1 activity underlies the phenomenon and that Pyk2 plays a role in regulating basal mGluR1 activity.q/11 protein, while facilitating ERK1/2 phosphorylation. The direct association of Pyk2 with mGluR1a appears to coordinate the formation of a protein complex that includes Src bound to Pyk2 and potentially calmodulin bound to the carboxyl-terminal tail of the receptor. The formation of this complex may be important for the spatial temporal regulation of Pyk2 activity at post-synaptic densities that is expected to be required for the efficient regulation of NMDA and AMPA receptor function as well as the regulation of other ERK1/2-dependent activities in synaptic transmission. The observation that several signaling proteins, such as Pyk2, optineurin CAIN and GRK2 regulate mGluR signaling via their interaction with the interface provided by the second intracellular loop suggests that peptides which mimic this interface may be useful for the modulation of Group I mGluR signaling.In summary, we show here that Pyk2 interacts with Group I mGluR1a via the intracellular loop2 and the carboxyl-terminal tail domains of mGluR1a and functions to uncouple the receptor from Gαin vitro; CAIN: calcineurin inhibitor protein; COS7: African green monkey cells; CMZ: calmidazolum chloride; DHPG: (S)-3,5-dihydroxylphenylglycine; ECL: enhanced chemiluminescence; ERK: extracellular signal-regulated kinase; GPCR: G protein-coupled receptor; GRK: G protein-coupled receptor kinase; GST: glutathione S transferase; HA: hemagglutinin; HEK293 cell: Human embryonic kidney cell; IP: inositol phosphate; InsP3: inositol-1,4,5-triphosphate; mGluR: metabotropic glutamate receptor; MAPK: mitogen-activated protein kinase; MPEP: 2-methyl-6-(phenylethynyl)-pyridine; NMDAR: N-methyl-D-aspartate receptor; PKC: protein kinase C; Pyk2: proline-rich tyrosine kinase 2.ANOVA: analysis of variance; DIV: days The authors declare that they have no competing interests.AAN, MP, LTF and FMR performed the experiments included in the manuscript. LBD and TC provided technical assistance with cell cultures and confocal microscopy. SSGF conceived of the experiments and wrote the manuscript. All of the authors have read the manuscript.