Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.Protozoan pathogens of the genus  Leishmania parasites. A crucial aspect of Leishmania infectivity is its capacity to sense different environments and adapt for survival inside insect vector and vertebrate host by stage differentiation. This process is triggered by environmental changes encountered in these organisms, including temperature and pH shifts, which usually are sensed and transduced by signaling cascades including protein kinases and their substrates. In this study, we analyzed the regulation of the Leishmania mitogen-activated protein kinase MPK10 using protein purified from transgenic parasites and combining site-directed mutagenesis and activity tests. We demonstrate that this kinase is activated during parasite differentiation and regulated by an atypical mechanism involving auto-inhibition, which is essential for parasite viability.Leishmaniasis is an important human disease caused by  Leishmania, which show two major life cycle stages depending on the host. The extracellular promastigote stage develops inside the midgut of sandflies and is transmitted during blood feeding to a vertebrate host where they are ingested by phagocytic cells, notably macrophages. Inside the host cell phagolysosome, promastigotes develop into proliferating intracellular amastigotes. These developmental transitions are triggered by environmental changes, mainly pH (7.4 to 5.5) and temperature (26°C to 37°C), encountered in insect and vertebrate hosts, respectively, and can be mimicked in vitro Leishmania and thus signaling proteins involved in extra- or intracellular signal transduction are interesting drug target candidates.Leishmaniasis is an infectious disease characterized by a variety of pathologies, affecting more than 12 million people worldwide and ranging from self-healing cutaneous lesions to fatal visceral infection Leishmania, the absence of classical transcription factors and the largely constitutive gene expression suggest that the response to environmental signals occurs mainly post-translationally through the regulation of the level of protein phosphorylation, rather than through modulation of gene expression In eukaryotes, environmental signals are generally sensed and transduced by signaling cascades involving receptors and downstream-regulated protein kinases. The MAPK signaling pathway is a good example of such a phosphorylation cascade Leishmania MAPK pathway comprises two distinct kinase families, the STE family, which includes five putative M2K and M2K-like and 20 putative M3K members, and the CMGC family, including 17 putative MAPK and MAPK-like members Leishmania major and the human kinomes revealed an evolutionary expansion relative to genome size of these two kinase families in the parasite. The STE and CMGC families represent 19% and 25% in the Leishmania kinome, compared to 9% and 12% in the human kinome, respectively Leishmania MAP kinases are required for flagellar development, intracellular survival and viability Leishmania MAPKs could hold the key to the understanding of the mechanisms that allow the adaptation of Leishmania to environmental changes required for extra- and intracellular parasite survival during host infection.As in higher eukaryotes, the Leishmania MAPKs MPK4, MPK7 and MPK10 have been shown to be induced in a stage-specific manner in axenic amastigotes, which occurred concomitant to an increase of their phosphorylation as expected for this class of kinases Leishmania species , whereas only five mammalian MAPKs have such an extension. They are usually regulatory domains implicated in the control of the kinase activity, localization or auto-inhibition et al. provided evidence that recombinant MPK10 adopts an activated conformation, despite the absence of TxY phosphorylation Leishmania DFG motif of MPK10 is replaced by DFN, causing the R and C spines to be aligned thus stabilizing an apparent active conformation in the absence of TxY phosphorylation The activity of the three  species  with a pLeishmania-specific signaling mechanisms, and significantly advance our understanding on parasite-specific protein kinase biology.Here, by utilizing a transgenic approach we unraveled an unusual mechanism of MPK10 regulation that tightly controls kinase activity. Stage-specific increase in MPK10 activity during the pro- to axenic amastigote conversion did not follow the largely constitutive phosphorylation status of the regulatory tyrosine residue in the kinase activation loop, suggesting additional and non-classical mechanisms of MPK10 regulation. Combining limited tryptic digestion with mutagenesis analysis and phosphoproteomics investigation, we uncovered an essential role of the C-terminal domain of MPK10 in limiting stage-specific kinase activity, and identified a novel regulatory phospho-serine residue that is required for axenic amastigote viability. Together our data propose MPK10 as a potential signal sensor of the mammalian host environment whose intrinsic pre-activated conformation is regulated by auto-inhibition. These results shed important new light on in vitro kinase assay at 30°C or 37°C, monitoring the transfer of radiolabeled phosphate from γ-33P-ATP to MPK10 (auto-phosphorylation) or to the canonical MAPK substrate myelin basic protein (MBP) To investigate the biochemical properties of MPK10, we generated non-mutated recombinant MPK10 (NM) and the corresponding MPK10-K51A enzymatically dead mutant, both tagged with GST-Strep. We used the bacterially expressed and purified proteins in an We next varied the pH of the kinase assay in an attempt to improve MPK10 activity. Kinase assays performed with casein and MPK10 or MPK10-K51A at pH 5.5, 6.5, 7.5 and 8.5 revealed different pH optima for auto- and substrate-specific phosphorylation at pH 6.5 and 7.5, respectively  or mutated His6-MPK10 deleted for the last 46 amino acids (ΔC) and monitored their activity towards canonical substrates by performing in vitro kinase assays in the presence of γ-33P-ATP. We changed from the GST-Strep to His6 tag to show that the lack of MPK10 substrate phosphorylation activity is independent from this modification. The auto-radiogram shown in 6-MPK10 NM similar to GST-STREP-MPK10 NM ruling out tag-specific interference with kinase activity. In contrast to His6-MPK10 NM, His6-MPK10-ΔC presented a stronger auto-phosphorylation activity and enhanced phosphorylation of Ets1 and casein. These differences reflect a true increase in phosphotransferase activity as judged by Coomassie staining, which showed equal loading of both recombinant proteins and substrates  as well as T. brucei TbECK1 in situ activated MPK10 isolated from transgenic L. donovani parasites.To investigate the activity of the MPK10 kinase core alone and to test whether deletion of the C-terminal region (46 aa) increases its activity, we generated and purified recombinant non-mutated HisL. donovani promastigotes harvested from logarithmic (log) or stationary (stat) phase cultures, and from cultures at different time points between 12 h to 120 h after induction of axenic amastigote differentiation by pH and temperature shift. Immuno-purified proteins were incubated for 30 min at 37°C in the presence of radiolabeled ATP and MBP, and the kinase reaction was subjected to SDS-PAGE. The phosphotransferase activity was determined by auto-radiography . We first performed a time course experiment to investigate MPK10 activity in promastigotes and during axenic amastigote differentiation. GFP-MPK10 was purified using a monoclonal anti-GFP antibody from iography , and MPKiography . After eThe mean values with standard deviation of three independent experiments are represented by the histogram plot shown in Phosphorylation of both the threonine and the tyrosine residues of the TxY motif have been shown to be required for eukaryotic MAPK activation and dual phosphorylation is correlated with kinase enzymatic activity. As an alternative read out for MPK10 activity we therefore assessed tyrosine phosphorylation using an anti-phosphotyrosine antibody. First, we tested whether phosphorylated Tyr192 was the only residue recognized by the antibody in MPK10. We compared the signals obtained with GFP-MPK10 NM, GFP-MPK10-K51A and the corresponding TxY motif mutants T190A, Y192F and T190A_Y192F purified from respective transgenic promastigotes harvested in logarithmic growth phase  and during axenic amastigote differentiation . Again, Leishmania mexicana using a targeted quantitative phosphoproteomic analysis termed Selected Reaction Monitoring  were found at similar levels in promastigotes and axenic amastigotes, whereas peptides containing pT190-H-pY192  showed significantly increased abundance in the axenic amastigote fraction. Peptides containing a single pT190 phosphorylation were not detected. These results show that phosphorylation of T190 and Y192 occurs independently: while pY192 is identified both in promastigotes and in axenic amastigotes, pT190 is mostly identified in amastigotes and thus this phosphorylation event may be the rate limiting step for MPK10 activation. Altogether, these findings strongly suggest that the phosphorylation of the Y192 residue is largely constitutive and occurs independently from MPK10 activation, which is in contrast to MAPK regulation in most other eukaryotes. This characteristic of MPK10 is likely a conserved feature among Leishmania species as we found similar results with independent experiments performed with L. donovani and L. mexicana.These findings were supported by an independent experiment performed with ing SRM, , from whL. donovani control (UC) and parasites over-expressing GFP-MPK10 NM or mutant forms  to test this possibility. Our analysis identified the expected phospho-peptides encompassing the TxY motif , and at 96 h where on the contrary GFP-MPK10-S395A activity is increased by 75% . No significant difference was observed in promastigotes . These data strongly suggest that in axenic amastigote, GFP-MPK10-S395A is differentially regulated compared to GFP-MPK10 NM, which could cause the observed effect on parasite viability. Parasites expressing GFP-MPK10-S395A presented a delay of 24 h in cell growth compared to GFP-MPK10 NM but thereafter resumed growth with kinetics slightly slower than that of parasites overexpressing GFP-MPK10 NM  observed for parasites expressing GFP-MPK10-S395A at 48 h of axenic amastigote differentiation compared to GFP-MPK10 NM  to validate MPK10 auto-inhibition in situ, (ii) to demonstrate the importance of the C-terminal domain for axenic amastigote survival, and (iii) to identify T190 and S395 as key regulatory residues. We propose that auto-inhibition of active MPK10 provides a fast signaling switch likely involved in environmentally induced pro- to axenic amastigote conversion and stage-specific regulation of MPK10 activity.While the biological processes underlying MAPKs are proline-directed serine/threonine kinases that phosphorylate substrates containing proline in the P+1 site Leishmania MPK10 is mostly constitutively phosphorylated on Y192 in promastigotes and during amastigote development and proliferation. Consequently, this residue seems not to be implicated in regulating the observed stage-specific activation of MPK10. These findings were confirmed by proteomics identification of a mono-phosphorylated T190-H-pY192 activation loop in both pro- and axenic amastigotes, whereas dual pT190-H-pY192 phosphorylation of the activation loop was mainly identified in axenic amastigotes, where MPK10 is most active. However, we observed a decrease in Y192 phosphorylation in axenic amastigotes at stationary phase, which correlated with a decrease in MPK10 activity, suggesting that inactivation of MPK10 at this stage requires Y192 dephosphorylation.Aside these conserved MAPK features, MPK10 regulation presents many non-classical characteristics. We have previously shown that based on its structural conformation, MPK10 appears to be in an active conformation, without the need for dual-phosphorylation of the TxY motif, as the replacement of the DFG motif by a DFN motif results in the alignment of the R and C spines, similar to eukaryotic MAPKs after phosphorylation of the TxY motif by M2Ks cis, a possibility that we rule out since kinase dead MPK10-K51A still shows phosphorylation on this residue. (ii) Classically, the phosphorylation of the TxY motif by M2Ks is sequential, initiating with the tyrosine residue T. cruzi PP2A blocks axenic amastigote differentiation Altogether, these findings indicate that the two residues of the TxY motif are differentially regulated, raising the question on how MPK10 is phosphorylated by M2Ks. There are at least five possibilities: (i) Y192 could be auto-phosphorylated in Dissociation of Y192 phosphorylation from stage-specific induction of MPK10 activation during amastigote differentiation raises the question of its importance for MPK10 function. We showed that similar to mammalian MAPKs, the Y192 residue is essential for MPK10 enzymatic activity. However, the dominant negative effect generated by overexpression of MPK10-T190A on axenic amastigote growth and viability was more detrimental than the one observed by overexpression of MPK10-Y192F, suggesting a more important role for the T190 residue for MPK10 activity. We can only speculate on the role of the largely constitutive phosphorylation of the Y192 residue. As described in the literature, phosphorylation of the tyrosine of the TxY motif is important to form the proline-directed (P+1)-specificity pocket 2+/calmodulin-dependent kinase I) but is a rare feature for MAPKs T. brucei, a hybrid kinase between a CDK (Cyclin dependent kinase) and a MAPK termed TbECK1 is also auto-inhibited by its C-terminal domain A second striking feature of the regulation of MPK10 is represented by its C-terminal domain. Whether we used the recombinant or the transgenic protein, removal of this domain resulted in a significant increase in kinase activity. While this increase translates mainly into higher auto-phosphorylation levels for the recombinant kinase, we observed a dramatic increase in substrate phosphorylation for the transgenic kinase. One main difference between the two kinases is the absence of Y192 phosphorylation of the recombinant protein, which could explain the inability of recombinant kinase to efficiently transfer phosphate onto the substrate . Thus, oOur data show that activating MPK10 via T190 is important for axenic amastigote survival, especially during the first 48 h after induction of axenic amastigote differentiation by temperature and pH shift. However, inhibition of MPK10 activity is equally essential as absence of auto-inhibition caused massive cell death in axenic amastigotes expressing MPK10-ΔC. These results therefore demonstrate the transient requirement for MPK10 during axenic amastigote differentiation and reveal a new mechanism to regulate MAPK activity by auto-inhibition, which is crucial for amastigote viability.Finally, we identified a highly conserved, trypanosomatid-specific serine at position 395 inside the MPK10 C-terminal domain as a novel, stage-specific regulatory residue that is mainly phosphorylated in promastigotes and whose mutation causes an important dominant negative effect on axenic amastigote survival. Because kinases can be released from auto-inhibition by phosphorylation or dephosphorylation Leishmaniaet al. have reported the phosphorylation of MKK5 by ERK5, revealing the existence of a potential feedback loop between a MAPK and its upstream activating kinase Because MPK10 shows a transient peak of activity during the first 48 h of axenic amastigote differentiation, its activity seems to be tightly regulated. The decrease in MPK10 activity after 48 h of induction of axenic amastigote differentiation is concomitant with the decrease of Y192 phosphorylation, suggesting that dephosphorylation of Y192 and probably T190 are required for MPK10 inactivation. We have evidence suggesting that MPK10 could directly or indirectly regulate its activity through the phosphorylation or the dephosphorylation of its TxY motif. Indeed, we have shown that to reduce toxicity caused by the over-expression of the hyperactive MPK10-ΔC, its level of Y192 phosphorylation was decreased. The level of pY192 was restored to wild-type level only after rendering the truncated kinase inactive, suggesting the existence of a feedback loop, where MPK10 could either inactivate the M2K that phosphorylates Y192 or activate the phosphatase that dephosphorylates this residue. This kind of feedback regulation between MAPKs and M2Ks or MKPs has not been extensively studied, as only three types of feedback loop have been described that are linked to regulation of MKPs, involving (i) rescue of MKP from degradation through phosphorylation by MAPK Leishmania to adapt highly conserved signaling proteins to its parasitic life style. Our data propose a model in which MPK10 is not inactivated but partially active in promastigotes as judged by tyrosine phosphorylation and structural conformation , clone LdB was cultured and axenic amastigotes were differentiated as previously described 6 logarithmic promastigotes per mL were grown at 26°C in M199 media ) and differentiated in axenic amastigotes by incubation at 37°C and 5% CO2 with RPMI media . Parasites were harvested at different time points between 12 h to 144 h after induction of axenic amastigote differentiation. For kinetic analysis, one flask (Corning) containing 200 mL of culture medium was used for each time point to avoid internal variations.Leishmania mexicana MNYC/BZ/62/M379 promastigotes and axenic amastigotes were grown as described previously 7 parasites/ml), and either harvested or differentiated into amastigotes by inoculation in Schneider's Drosophila medium  supplemented with 20% heat-inactivated FCS (PAN Biotech), 2 mM L-glutamine, 100 U/ml Pen/Strep, and 20 mM 2-morpholinoethanesulfonic acid monohydrate [MES]  for a final pH of 5.5. Cultures were incubated at 34°C, 5% CO2, for 72 h. Parasites were harvested by centrifugation at 2,000× g at 4°C, and washed consecutively in ice-cold HEPES and ice-cold HEPES with protease and phosphatase inhibitors ). Parasite pellets were snap-frozen in liquid nitrogen and stored at −80°C.L. major MAPK homologue LmaMPK10 (accession number CAJ02415) was PCR amplified from 50 ng of genomic DNA of L. major FVI using primers AGATCTCCACCATGCAGGCCAAGGGC-3′5′-ACC (forward) and AGATCTTCACGACGGGGCCGGCGC-3′5′-GCG (reverse) and cloned into pGEM-T (Promega) to obtain pGEMT-MPK10. This plasmid was subsequently digested by BglII and subcloned into pXG-GFP+2 to generate pXG-GFP-MPK10 . The various MPK10 mutants described in the results section were obtained by site direct mutagenesis of pGEMT-MPK10 using the QuickChange II XL Site-Directed Mutagenesis kit (Stratagene) prior to subcloning into pXG-GFP2+. MPK10-ΔC was PCR amplified with primers AGATCTCCACCATGCAGGCCAAGGGC-3′5′-ACC (forward) and AGATCTTCAGTCGTTGAAGCGCTCC-3′5′-GCG (reverse) containing a stop codon to generate a protein deleted for the last 46 amino-acids (138 nucleotides) from the C-terminal end of LmaMPK10, and was cloned into pXG-GFP2+ as described above to generate the pXG-GFP-MPK10-ΔC construct. All constructs were validated by sequence analysis.For transgenic expression of GFP-MPK10 fusion protein, the gene encoding the For recombinant protein production, the sequences encoding for LmaMPK10 and LmaMPK10-ΔC were amplified by PCR and subcloned into plasmid pQE80 , previously modified to carry a Tobacco Etch Virus (TEV) protease cleavage site for removal of the N-terminal His-tag as previously described TTCGAAAAAGGTGGAATGCAGGCCAAGGGCG-3′5′- (forward) and 5′-CCCGGGTCACGACGGGGCCGGCG-3′ (reverse), and the fragment was cloned into pGEM-T vector to obtain pGEM-MPK10-NcoI-BstBI. Primers GGATCCTTATGTGGAGCCACCCGCAGTTCGAGAAAGGTGGAGGTTCCTGGAGCCACCCGCAGTT-3′5′-CAT and 5′-CGAACTGCGGGTGGCTCCAGGAACCTCCACCTTTCTCGAACTGCGGGTGGCTCCACATAAGGATC-3′ were annealed and subsequently phosphorylated to generate a double stranded oligonucleotide containing the Strep3 sequence with 5′ overhangs. This fragment was cloned into pGEM-MPK10, digested by NcoI/BstBI, to generate the pGEM-Strep3-MPK10 plasmid. pGEM-Strep3-MPK10 was digested by BamHI/NotI and cloned into pGEX-5X-1 (Abbgene) to generate pGEX-GST-Strep3-MPK10. The same strategy as described above was applied to obtain mutated kinase constructs. All constructs were validated by restriction enzyme digestion and sequencing.An alternative expression construct for recombinant MPK10 production was created by PCR amplification of LmaMPK10 with primers 7L. donovani LdB promastigotes from logarithmic culture with 20 µg of plasmid Episomal tranfectants were generated by electroporation of 5×10ex = 488 nm; emission wave length λem = 617 nm). The percentages of cell death and cell growth were calculated using FlowJo (v7.6) software .Cultured parasites were incubated for 15 min with 10 µg/ml propidium iodide (Sigma-Aldrich) and diluted in PBS (Gibco). Cells were analyzed with a FACSCalibur flow cytometer (Beckman Coulter) to determine the incorporation of propidium iodide  and phosphatases . Clear lysates were obtained after sonication and centrifugation at 12 000× g for 10 min and stored at −80°C.10Purified GFP-MPK10 wild-type and mutant proteins were isolated from crude cell extracts of respective transgenic parasites using the μMACS Epitope Tag Protein Isolation Kit, according to the manufacturer's specifications . Briefly, equal amounts of total proteins were incubated with 50 µl of magnetic bead-conjugated mouse monoclonal anti-GFP antibody for 1 hour at 4°C, immuno-complexes were immobilized on the μMACS separator, washed four times with 150 mM NaCl, 1% Igepal CA-630 (formerly NP-40), 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris HCl (pH 8.0), and once with 20 mM Tris HCl (pH 7.5). Bound GFP-MPK10 protein was eluted in 75 µl PBS after removing the columns from the magnetic field.ex = 457 nm and λem = 610 nm. Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce). Proteins were revealed using the following antibodies at the indicated dilutions: i) polyclonal anti-MPK10 antibody, generated by rabbit immunization using recombinant MPK10 protein produced in E. coli transformed with pGEX-Strep3-MPK10 plasmid (Eurogentec), 1∶10,000; ii) anti-phospho-tyrosine antibody 4G10 Platinum from Millipore, 1∶1,000; and iii) secondary goat anti-rabbit-HRP and anti-mouse-HRP antibodies from Thermo Scientific, 1∶20,000. The visualization was performed on X-ray film (Roche) at various exposure times.Purified proteins were separated by SDS–PAGE  and visualized either by Coomassie staining or SYPRO Ruby Protein Gel Stain (Invitrogen) using a Typhoon 9400 scanner (Amersham Biosciences) with λE. coli BL21 Rosetta (VWR) transformed with pQE80-His6-MPK10 or pGEX-GST-Strep3-MPK10 were grown at 37°C and induced with IPTG  overnight at RT. Cells were harvested by centrifugation at 15,000× g for 10 min at 4°C.g for 30 min at 4°C. The supernatant was immediately subjected to GST affinity chromatography . GST-tagged proteins were washed with a buffer containing 50 mM Tris pH 8, 100 mM NaCl, 1 mM DTT and eluted with the same buffer supplemented with 30 mM L-glutathione. Appropriate fractions were pooled and the GST-tag was cleaved by incubation of the fractions with 5 µg/mL Xa factor in presence of 1 mM CaCl2. The reaction was stopped by adding 5 µg/mL of Glutamyl-glycyl-arginine chloromethyl ketone GGACK . A second purification step was performed using a StrepTrap column . Elution was performed with E Strep buffer  and appropriate fractions were collected, and stored at 4°C until used.For the purification of GST-Strep3-MPK10, bacteria were resuspended in a pre-chilled buffer containing 25 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM DTT, 10 µg/mL apoprotein, 1 µM leupeptin, 1 µM pepstatin, 1 mM PMSF. Samples were sonicated  for 2 min at 20 V setting on ice (10 s on/10 s off cycle). After addition of 500 µg/mL of lysozyme and 500 U of benzonase, lysates were incubated on ice for 30 min with 140 µM EDTA, 0.0035% Triton-X100 and centrifuged at 15,000× 6-MPK10 purification, the bacterial pellet was resuspended in PBS containing 60 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mM disodium phenylphosphate, 150 mM sodium chloride, 10 mM imidazole supplemented with protease inhibitor cocktail . The sample was sonicated for 2 min at 20 V setting on ice (10 s on/10 s off cycle). Triton X-100  was added, the sample was incubated for 30 min at 4°C (shaking) and centrifuged at 15,000× g for 30 min at 4°C. The supernatant was purified on Co-NTA agarose (Pierce). The beads were washed with PBS containing 60 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mM disodium phenylphosphate, 300 mM sodium chloride, 30 mM imidazole, 1% Triton X-100 at pH 7.5. Elution was performed with 300 mM imidazole in elution buffer pH 7.5 . The eluate was supplemented with 15% glycerol and stored at −80°C.For His2 and 1 µCi [γ-32P] adenosine-triphosphate (ATP) (3000 Ci/mmol) in final volume of 20 µl. The phosphotransferase reaction was then stopped by adding Laemmli loading buffer. Reaction mixtures were separated by SDS–PAGE, which was stained by Commassie and dried. 32P incorporation was monitored by exposing the dried gel on an X-ray sensitive film (Roche) at −80°C. After exposure, the bands corresponding to MPK10 or MBP were excised from dried gels and radioactivity was quantified by a scintillation counter.Ten percent of the GFP-MPK10 purified protein was incubated on a shaker for 30 min at 37°C with 25 µg myelin basic protein (MBP) substrate, 200 µM of ATP, 50 mM of MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl6-MPK10 and respective mutants were assayed with 36 µg dephosphorylated casein, 12 µg histone H1, 9 µg of Ets1 or 9 µg MBP as substrates in a Tris buffer at pH 7.5  in 20 µl final volume and in the presence of 15 µM [γ-33P]-ATP. After 30 min incubation at 37°C, the reaction was stopped by adding an equal volume of 2× electrophoresis loading buffer to the 20 µl reaction mix. Incorporated 33P was monitored by auto-radiography.Recombinant His50 µg of Strep3-MPK10 were digested with 0.25 µg trypsin at RT. Aliquots were taken at 0, 2.5, 5, 15, 30, 60, and 150 min and the reaction was stopped by adding Laemmli loading buffer. The polypeptides were then separated by SDS-PAGE, transferred to PVDF membrane, stained with amidoblack, and N-terminal sequencing was performed. For the mass determination of cleavage products, pH of the cleavage reaction was lowered to 5.0 and mass determination was performed by SELDI-TOF analysis after immobilizing the samples on a H4 ProteinChip Array (C16 reversed phase surface).Leishmania amastigote cell pellet was submitted for qualitative phosphoproteomic analysis using titanium dioxide phosphopeptide enrichment followed by an iTRAQ labeling experiment for analysis of phosphopeptides by LC-MS/MS. Briefly, 400 µg of Leishmania proteins were reduced with DTT and the free cysteines were alkylated with iodoacetamide for 30 min at 37°C in darkness. Proteins were then digested overnight at 37°C using Porcine trypsin (sample:enzyme ratio of 50∶1). Following the digestion, the peptides were acidified, concentrated and de-salted using a Waters HLB Oasis SPE cartridge. The peptides were then enriched for phosphopepitdes using a TiO2 affinity column and splited into two 100 µL aliquots. Each aliquot was then reduced and alkylated with MMTS followed by iTRAQ labeling . The 116 labeled sample was then treated with FastAPalkaline phosphatase before the samples were combined, fractionated by SCX chromatography and analyzed by ESI-Q-TOF MS/MS. Sample were then analyzed by reversed phase nanoflow (300 nL/min) HPLC with nano-electrospray ionization using a quadrupole time-of-flight mass spectrometer  operated in positive ion mode and a 2 hour gradient.A 7 parasites were lysed in a solution of 7 M urea, 2 M thiourea, 40 mM Tris, 1% n-octyl-β-D-glycopyranoside, 1 mM MgCl2, 1 mM o-phenanthroline, 300 U benzonase, 1 mM Na-pervanadate (Na-orthovanadate activated in 18% H2O2), EDTA-free protease inhibitors (Roche) and phosphatase inhibitor cocktails (P2850 and P5726 from Sigma), and sonicated for 3×15 s on ice. Lysates were incubated at −80°C for 30 min prior to reduction  and alkylation . Proteins were precipitated in 8 fold excess of ice-cold acetone-ethanol  by overnight incubation at −20°C. Proteins were reconstituted in 6 M urea/2 M thiourea and diluted in 50 mM NH4HCO3 for digestion with trypsin at a 75∶1 substrate-enzyme ratio over night. For selected reaction monitoring (SRM) analyses, 3×330 µg digested protein from whole cell lysates of promastigotes, axenic amastigotes were subjected to TiO2 enrichment as described in Rosenqvist et al. 2 eluates were pooled prior to SRM analysis. The discovery analyses were conducted in triplicates for each of the sample types using LTQ Orbitrap XL mass spectrometers . The discovery data were processed with DTASuperCharge  and searched against a customized L. mexicana 6-frame translation library as well as a predicted protein list (GeneDB http://www.genedb.org) using an in-house Mascot server . For SRM analyses, the discovery data were processed in ProteomeDiscoverer, and the MPK10 phosphopeptides identified were imported into Pinpoint, version 1.0.0 (Thermo Scientific).1×10Leishmania spp and Trypanosoma spp MPK10 gene and protein sequences were retrieved from the web databases GeneDB (www.genedb.org) and TriTrypDB (http://tritrypdb.org/tritrypdb/) http://www.rstudio.org/) and R language (R Development Core Team (2005). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL: http://www.R-project.org). Statistical analyses were performed using the t-test on mean values when samples followed a Normal distribution, otherwise the Mann-Whitney Rank Sum Test was used. Differences were considered significant when p value<0.05.Figure S1Leishmania generated with Clustal-X and visualized with BioEdit. Color code: black, identical residues; grey, similar residues; white, no conservation. B) Table of percent of protein sequence identity of MPK10 from L. donovani compared to other Leishmania species. LmjF, L. major Friedlin; LmxM, L. mexicana MHOM/GT2001/U1103; LinJ, L. infantum JPCM5; LdBPK, L. donovani BPK282A1; LbrM, L. braziliensis MHOM/BR/75/M2904; LtaP, L. tarentolae Parrot-TarlI.A) Multiple sequence alignment of MPK10 orthologs from (TIF)Click here for additional data file.Figure S2Effect of pH variation on the activity of recombinant MPK10. Non-mutated GST-Strep-MPK10 NM and kinase dead mutant K/A were incubated with casein for 30 min at pH 5.5, 6.5, 7.5 and 8.5 at 37°C. Recombinant human MEK1 was used as positive control with MBP as substrate at pH 7.5. MW, Molecular Weight.(TIF)Click here for additional data file.Figure S3Trypanosomatidae generated with Clustal-X and visualized with BioEdit. The phospho-serine residue S395 is marked by the grey arrow and the conserved DHMxRTxSxME motif is underlined. Color code: black, identical residues; grey, similar residues; white, no conservation. B) Table of percentage of protein sequence identity of MPK10 from L. donovani compared to other Leishmania species. The following strains were aligned LmjF, L. major Friedlin; LmxM, L. mexicana MHOM/GT2001/U1103; LinJ, L. infantum JPCM5; LdBPK, L. donovani BPK282A1; LbrM, L. braziliensis MHOM/BR/75/M2904; LtaP, L. tarentolae Parrot-TarlI; Tb, T. brucei; Tbg, T. brucei gambiense DAL972; TcIL3000, T. congolense IL3000; TcCLB, T. cruzi CL Brener; TCSYLVIO, T. cruzi Sylvio X10/1; Tc_MARK, T. cruzi marinlellei strain B7; TvY486, T. vivax Y486.A) Multiple sequence alignment of the C-terminal domain of MPK10 orthologs from (TIF)Click here for additional data file.Figure S4p-values for the kinetic analysis of kinase activity plotted in p-value. Dark grey, p-value<0.001; light grey, p-value<0.01; white, p-value<0.05, NS, non significant.Table of the (TIF)Click here for additional data file.Figure S5Recombinant MPK10 is not phosphorylated on Y192. GFP-MPK10 and GST-Strep-MPK10 NM and -K/A proteins were purified from amastigotes at 48 h during axenic differentiation and recombinant bacteria, respectively, and analyzed by western blotting using anti-phospho-tyrosine (α-pTyr) and anti-MPK10 (α-MPK10) antibodies.(TIF)Click here for additional data file.Figure S65 promastigotes were cultured during 8 days and aliquots were taken every 24 h for analysis. Cell number and percent of cell death were assessed by flow cytometry. Upper panel: Untransfected Control, UC ; GFP-MPK10, NM ; GFP-MPK10-K51A, K/A . Lower panel: GFP-MPK10-T190A, T/A ; GFP-MPK10-Y192F, Y/F ; GFP-MPK10-T190A_Y192F, T/A_Y/F . Each point of the line graph represents the mean of three independent triplicate experiments, with standard deviations denoted by error bars.MPK10 mutations do not affect promastigote growth and viability. The analysis represents the combined results of three triplicate experiments. 2×10(TIF)Click here for additional data file.Figure S76 amastigotes were cultured during 8 days and aliquots were taken every 24 h for analysis. Cell number and percent of cell death were assessed by flow cytometry. Upper panel: Untransfected Control, UC ; GFP-MPK10, NM ; pXG-mock, mock . Each point of the line graph represents the mean of three independent triplicate experiments, with standard deviations denoted by error bars.Overexpression of empty vector has only a slight effect on amastigote viability. The analysis represents one triplicate experiment. 1×10(TIF)Click here for additional data file.Figure S8Leishmania total protein extracts from amastigotes. The samples were subjected to phosphoproteomic analysis following titanium dioxide phosphopeptide enrichment and iTRAQ labeling. MS/MS raw data spectra of identified peptides presenting phosphorylation of threonine and tyrosine of the TxY motif (A), and phosphorylation of serine 395 located inside the carboxy terminal extension of MPK10 (B) are shown.LC-MS/MS analysis of (TIF)Click here for additional data file.


Y-O> distances and the (Li + Mn2+ + Cu + Fe2+) content (apfu) at this site with R2 = 0.90. An excellent negative correlation exists between the <Y-O> distances and the Al2O3 content (R2 = 0.94). The samples at each locality generally show a strong negative correlation between the X-site vacancies and the (MnO + FeO) content. The Mn content in these tourmalines depends on the availability of Mn, on the formation temperature, as well as on stereochemical constraints. Because of a very weak correlation between MnO and CuO we believe that the Cu content in tourmaline is essentially dependent on the availability of Cu and on stereochemical constraints.Cu- and Mn-bearing tourmalines from Brazil and Mozambique were characterised chemically (EMPA and LA-ICP-MS) and by X-ray single-crystal structure refinement. All these samples are rich in Al, Li and F (fluor-elbaite) and contain significant amounts of CuO (up to ~1.8 wt%) and MnO (up to ~3.5 wt%). Structurally investigated samples show a pronounced positive correlation between the < XY3Z6(BO3)3T6O18V3W, the individual structural sites can be occupied by the following cations: site X = Na, Ca, K, □ (vacancy); Y = Li, Mg, Fe2+, Mn2+, Al, Cr3+, V3+, Fe3+, Ti4+, Zn, Cu; Z = Al, Mg, (Fe2+), Fe3+, Mn3+, V3+, Cr3+, (Ti4+); T = predominantly occupied by Si, but sometimes also by Al and B; V = OH-, O2-; W = OH-, F-, Cl-, O2-  from the same locality was refined by Ertl et al. , was described for the first time by Nuber and Schmetzer  from Eibenstein an der Thaya, Lower Austria, was described by Ertl et al.  from the island of Elba, Italy.The crystal structure of a Mn-rich tourmaline from Zambia, which contains a relatively high amount of Mnchmetzer . Burns echmetzer  refined l et al. . Bosi etl et al. , 2012 puHere we describe the crystal structure and the chemistry of tourmalines from Brazil and Mozambique, which contain up to 1.78 wt% CuO and up to 3.51 wt% MnO.Blue and green tourmalines from granite pegmatites in the vicinity of the village São José de Batalha near Salgandinho, Brazilian state of Paraiba, became available on the gem market in 1987, exciting considerable interest to gemmologists and gem traders. It turned out that the spectacular colours of these elbaites are due to the combined effect of Mn and Cu; the trace element Cu was hitherto not recorded in tourmaline .In their polarized absorption spectra, the blue Paraiba elbaites reveal strong, dichroic absorption bands with maxima at about 920 and 700 nm, caused by Cu3+ and Cu2+.In an amethyst-coloured Paraiba tourmaline, Schultz-Güttler  recognizSimilar Cu-Mn-bearing, Paraiba type elbaites were recorded in granitic pegmatites at the nearby localities of Quintos de Baixo and Boquerão, in the state of Rio Grande del Norte  with Ti (Kβ). The matrix correction of the EMPA data was done by the PAP program of CAMECA  were analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) at Erlangen University. The measurements were undertaken on a 266 nm Nd:YAG Laser of New Wave Research (Merchantek) Products, connected to an Agilent 7500i ICP-MS quadrupole instrument at 1250 W plasma power. Ar was used as carrier gas (1.28 L/min) as well as plasma gas (14.9 L/min) and auxiliary gas (0.9 L/min). Data acquisition was performed in Time Resolved Mode with measurements on the maximum peak and 25 ms integration time for all chosen isotopes but 10 ms for B and Si; 15 s for measuring the background and also 15 s for acquisition time. 3 single spots with a crater size of 40 μm, a repetition rate of 10 Hz and a laser energy at 0,51-0,74 mJ (energy density 43-52 J/cm2) were ablated on each tourmaline. Data analysis was performed via GLITTER software , using Si as internal standard .In all tourmaline crystals, B., 50 ppm glass, measured as unknown sample, 5 to 11 % relative. The raw values for B2O3 were corrected against the Elbaite monitor crystal containing 10.14 wt% B2O3, an average of 10.10 wt% determined by PIGE, 10.11 wt% by SIMS and 10.20 wt% by PGNAA  and formula occupancies of the tourmaline crystals analysed by EMPA or LA-ICP-MS are presented in Table Kα radiation. Single-crystal X-ray diffraction data were collected at room temperature (up to 80° 2θ), integrated and corrected for Lorentz and polarization factors and absorption correction by evaluation of partial multiscans. The structure was refined with SHELXL-97  values of ~1.4-2.1 % . Although the pink component in some Mn-bearing tourmalines indicates the presence of some Mn3+, we consider the amount of Mn3+ only relatively low , CuO up to ~1.8 wt%) and F (up to ~1.8 wt%)  to 2.676(1) Å  for the tourmalines from Brazil with Ca contents ≤0.06 apfu. A similar correlation  and Li  and Cu2+ (≤0.2 apfu) also occupy this site. A few samples contain minor amounts of Zn (≤0.09 apfu), Mg (≤0.05 apfu) and Fe2+ (≤0.03 apfu). Because Li was calculated, small amounts of vacancies at the Y site cannot be excluded. The <Y-O> distance varies from 1.989(1) to 2.022(1) Å  contents (apfu) for all structurally investigated samples from Brazil and Mozambique. The influence of the Cu content in this correlation is less significant than that of the other cations , because the effective ionic radius of Al is less different to Cu than to the other cations . A negative correlation, which is even better  than Al and the occupancy of the Z site was fixed at Al1.00 (full occupancy) during the final refinement. The <Z-O> distance in all samples is ~1.906 Å within a standard deviation of ±3σ (Table R2 = 0.71) between the <T-O> and the <Z-O> distances for the tourmalines from both localities.In all samples the 3σ Table . NeverthT site is essentially occupied by Si (Table T-site occupancy during refinement did not show a clear evidence for significant amounts of [4]B (>0.10 apfu) in the investigated samples. Hence it was fixed at Si1.00 (full occupancy) during the final refinement. The final T-site occupancy, which was calculated by using the chemical data, gives up to ~0.1 apfu [4]Al  Å; Table R2 = 0.84; Fig. T-O> and the <X-O> distances for all structurally characterized samples (with >0.60 apfu F) from Brazil and Mozambique.In all samples the Si Table . Releasi [4]B >0.0 apfu inThe B site in all samples is completely occupied by B Table  and for V site in all samples is occupied by (OH)3 and the W site shows a relatively high F content in all samples , [4]Al progressively increases up to ~0.25 apfu. Because of a pronounced positive correlation (R2 = 0.99) between (Fe2+ + Mn2+) and [4]Al in tourmalines of the elbaite-schorl series from the Himalaya Mine, Mesa Grande, California, USA  with increasing temperature can be considered. Our elbaitic tourmalines show an excellent negative correlation between (MnO + FeO) and the X-site vacancies  in our samples we believe that the Cu content in tourmaline is essentially dependent on the availability of Cu as well as on stereochemical constraints.Henry and Dutrow  pointed es Figs. , 4. Becaes Figs. , we conces Figs. , 4, becaes Figs. , and b. In the context of running investigations, a larger number of 52 differently coloured tourmalines were analyzed. Colours range from violet, pink-violet and reddish-violet to violet-blue,blue, blue-green, pale blue-grey, pale blue-green and yellow-green. No obvious correlation of the colour and the content of Cu and/or Mn is recognized. Instead of that, the Cu-content seems to depend primarily on the provenance, with Cu-contents <4,000 ppm in tourmalines from Mozambique and 7,000 – 14,000 ppm in tourmalines from Brazil. Additional spectroscopy is planned to investigate the question of tourmaline colours more in detail.R2 = 1.00) between X-site charge and F content in tourmaline, first described by Ertl et al.  content . Similar correlations were already described in tourmalines of the elbaite-schorl series for the Cruzeiro Pegmatite, Minas Gerais, Brazil , R2 = 0.98; Ertl et al. The <T-O> and <X-O> distances for our samples from Brazil and Mozambique which contain ≥0.65 apfu F , which could be a possible explanation for such a correlation. A relationship with the F content is evident through crystal-chemical reasoning. The W site, located on the three-fold axis central to the pseudohexagonal ring of tetrahedra, is bonded to three Y-site cations. In cases OH occupies the W site, the H atom points toward the X site. Crystallographic studies as well as extensive analytical data on tourmaline establish that F is found exclusively at the W site –1 exchange vector) and of F contents, and a decrease of X-site vacancies via the X□Al(NaR2+)–1 exchange vector . The positive correlation between F contents and <Y-O> distances , exhibit a larger <Y-O> distance. Hence, such tourmalines would be enriched in cations with a larger effective ionic radius  and depleted in Al2O3.A positive correlation has been observed between <dy Henry  and Henrdy Henry  showed ices Fig.  is perhaCu-bearing tourmalines from Brazil exhibit relatively low Pb contents  and Pb  have also been observed. Cu-bearing tourmalines from Mozambique, compared with samples from Brazil, can have higher amounts of Pb, while tourmalines from Brazil can contain higher contents of YMg.Blue, bluish green, yellowish green, green and violet-pink tourmalines from Brazil and Mozambique have been characterized chemically and structurally. All these samples can be classified as Mn


These purine cyclic nucleotides activate cAK and cGK, respectively. So far, there was no evidence of further cyclic nucleotides acting as second messengers. Meanwhile the existence of cCMP was described ,2. cCMP 5-24, suggesting a stimulatory function for PKA in cCMP-mediated MAPK phosphorylation. To elucidate the role of cGK in murine tissues in this process, we used cGKII knockout (cGKII KO) and cGKI knockout (cGKI KO) mice. We detected stimulation in the jejunum tissues from cGKI KO and cGKII KO mice. It is interesting to note that the phosphorylation in the jejunum cGKII KO tissue was significantly increased when compared with the WT and cGKI KO tissues, suggesting an inhibitory role for cGKII in cCMP-induced MAPK phosphorylation in the jejunum.To elucidate propable functions cCMP-binding and -activated proteins were identified using different methods. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins in murine tissue lysates, using 4-AH-cCMP agarose. cCMP (200 µM) was added (+) or omitted (-) during the affinity chromatography experiments to investigate the specificity of the binding. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. Interestingly, no specific interaction of MAPK with 8-AET-cGMP agarose was detected. Moreover, DB-cCMP (100 µM) was also able to stimulate the phosphorylation of p44/p42 MAPK. The phosphorylation of MAPK was inhibited by the addition of the PKA inhibitor ASThese results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum.


Taken together, these results indicate that DHGA-D may be a promising anti-inflammatory agent that mediates suppression of both COX-2 expression and the MLK3 signalling pathway through direct binding and inhibition of MLK3.Dehydroglyasperin D (DHGA-D), a compound present in licorice, has been found to exhibit anti-obesity, antioxidant and anti-aldose reductase effects. However, the direct molecular mechanism and molecular targets of DHGA-D during skin inflammation remain unknown. In the present study, we investigated the effect of DHGA-D on inflammation and its mechanism of action on UVB-induced skin inflammation in HaCaT human keratinocytes and SKH-1 hairless mice. DHGA-D treatment strongly suppressed UVB-induced COX-2 expression, PGE2 generation and AP-1 transactivity in HaCaT cells without affecting cell viability. DHGA-D also inhibited phosphorylation of the mitogen-activated protein kinase kinase (MKK) 3/6/p38, MAPK/Elk-1, MKK4/c-Jun N-terminal kinase (JNK) 1/2/c-Jun/mitogen, and stress-activated protein kinase (MSK), whereas phosphorylation of the mixed-lineage kinase (MLK) 3 remained unaffected. Kinase and co-precipitation assays with DHGA-D Sepharose 4B beads showed that DHGA-D significantly suppressed MLK3 activity through direct binding to MLK3. Knockdown of MLK3 suppressed COX-2 expression as well as phosphorylation of MKK4/p38 and MKK3/6/JNK1/2 in HaCaT cells. Furthermore, Western blot assay and immunohistochemistry results showed that DHGA-D pre-treatment significantly inhibits UVB-induced COX-2 expression  Cox-2 knockout mice are protected against UV-induced skin carcinogenesis , after which luciferase activity was measured by using a luminometer .HaCaT cells  were cultured in a 10 cm dish for 48 hrs, followed by starvation in serum-free DMEM for 24 hrs. Cells were then treated with DHGA-D (2.5 or 5 μM) for 1 hr and irradiated with UVB (0.05 J/cm2). The protein concentration was determined by using a dye-binding protein assay kit (Bio-Rad Laboratories) following instructions in the manufacturer's manual. Lysate protein was subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). After transferring, the membranes were incubated with specific primary antibodies at 4°C overnight. Protein bands were visualized by using a chemiluminescence detection kit (Amersham Pharmacia Biotech) after hybridization with a horseradish peroxidase-conjugated secondary antibody.For Western blot assay, cells . Exactly 20 ng of MLK3 protein was added to a mixture containing myelin basic protein, 5× assay buffer, and diluted [γ-32P]ATP solution with or without DHGA-D. Reactions were carried out at 30°C for 10 min., and incorporated radioactivity was determined by using a scintillation counter. The kinase activity data represent the mean of three independent experiments.CNBr-activated DHGA-D beads were produced as described in a previous study . For theFor knockdown of MLK3, HaCaT cells were transfected with scrambled sequence or 3 μg human MLK3 siRNA  plasmids by using Lipofectamine 2000 , following the manufacturer's suggested protocols. The transfected cells were then exposed to UVB irradiation and used in subsequent experiments.Sections (5-μm thick) of 10% neutral formalin solution-fixed, paraffin-embedded tissues were cut on silane-coated glass slides. Deparaffinized sections were then incubated in proteinase K (20 μg/ml) for 20 min. at room temperature. In addition, the sections were heated for 15 min. in 10 mM citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. For the detection of target proteins, slides were incubated with affinity-purified primary antibody in a refrigerator overnight in 1% BSA solution and then developed by using the HPR En VisionTM System  for anti-rabbit and antimouse antibodies. Peroxidase-binding sites were detected by staining with 3,3′-diaminobenzidine tetrahydrochloride (Dako). Finally, counterstaining was performed by using Mayer's haematoxylin.anova. A probability value of P < 0.05 was used as the criterion for statistical significance.Where appropriate, data are expressed as the mean ± SEM, and significant differences were determined by using one-way Ultraviolet irradiation has been reported as inducing abnormal COX-2 expression –5 and uncox-2 is a target gene with an AP-1-binding site in its promoter region, while AP-1 activity is regulated by activation of MAPK signalling pathways [Published studies have reported that pathways ,27. To fPrevious studies showed that MLK directly regulates MKK3/6 and MKK4 phosphorylation ,29. AddiKnockdown of MLK3 significantly reduced UVB-induced COX-2 expression when compared with scrambled sequence controls Fig. A. Additiin vivo (Fig. The SKH-1 mouse model is well suited for studying skin inflammation and chemoprevention using phytochemicals –5,30. Weivo Fig. A. Our imivo Fig. B.The regular consumption of natural phytochemicals has been closely linked to chemopreventive effects . Both thIn vitro and genetically modified animal study results have clearly shown that COX-2 is a critical player in inflammation and carcinogenesis, and COX-2 signalling is a promising target for chemoprevention [evention ,7,23,31.evention ,12. Hereevention , DHGA-D evention ,17,32.Mlk3 gene has been shown to result in selective reduction of JNK phosphorylation in response to TNF-α [AP-1 is a core transcription factor in UV-induced inflammation and carcinogenesis. UVB irradiation is known to induce AP-1 expression ,27. Therto TNF-α . Additioto TNF-α . We therAlthough our results showed that DHGA-D suppressed UVB-induced COX-2 expression by directly inhibiting MLK3 activity and subsequent signalling including MAPKK/MAPK, to date, there has been no evidence to suggest that MLK3 regulates UVB-induced COX-2 expression. We found that knockdown of MLK3 induced down-regulation of UVB-induced MKK4/JNK1/2 and MKK3/p38 phosphorylation, resulting in suppression of COX-2 expression. These results indicate that MLK3 plays an important role in UVB-induced COX-2 expression by regulating MKK4 and MKK3 signalling. We further clarified that DHGA-D inhibited UVB-induced COX-2 expression in hairless mouse skin. Analysis of tissue samples confirmed that DHGA-D was able to inhibit UVB-induced COX-2 expression in mouse skin.in vivo. This inhibition occurs primarily through inhibition of the MLK3 kinase, leading to suppression of COX-2 expression through reduced MAPK and AP-1 activity. The therapeutic inhibition of MLK3 kinase by DHGA-D may provide clinical benefits for the better treatment of skin inflammation. This represents the first report to elucidate the molecular basis of the anti-inflammatory activity of DHGA-D.Taken together, our results suggest that DHGA-D significantly inhibits UVB-induced COX-2 expression


Both the neurons with orientation-selective and with non-selective surround inhibition have been observed in the primary visual cortex (V1) of primates and cats. Though the inhibition coming from the surround region  has been considered playing critical role in visual perception, the specific role of orientation-selective and non-selective inhibition in the task of contour detection is less known. To clarify above question, we first carried out computational analysis of the contour detection performance of V1 neurons with different types of surround inhibition, on the basis of which we then proposed two integrated models to evaluate their role in this specific perceptual task by combining the two types of surround inhibition with two different ways. The two models were evaluated with synthetic images and a set of challenging natural images, and the results show that both of the integrated models outperform the typical models with orientation-selective or non-selective inhibition alone. The findings of this study suggest that V1 neurons with different types of center–surround interaction work in cooperative and adaptive ways at least when extracting organized structures from cluttered natural scenes. This work is expected to inspire efficient phenomenological models for engineering applications in field of computational machine-vision. At the level of individual neurons and neuronal population, center–surround interactions across the receptive fields (RFs) of neurons are regarded as the underlying physiological bases of visual information processing  in V1 has been also found by many physiological studies  properties of simple cells in V1 can be well-described by a family of Gabor filters Daugman, . In thisA two-dimensional (2D) Gabor filter can be written asx cos θ + y sin θ, ỹ = −x sin θ + y cos θ, in which θ is the preferred orientation of an orientation-selective V1 neuron. Standard deviation σ defines the spatial size of CRF. γ is the spatial aspect ratio determining the eccentricity of the Gaussian envelope. λ is the wavelength and σ/λ represents the spatial frequency bandwidth. In this study we set σ/λ = 0.56 and γ = 0.5, which are physiologically based , there is a model V1 hypercolumn composed of cells whose CRFs are centered at  and tuned to Nθ different orientations θi spanning 180°:According to the physiological finding of Hubel and Wiesel , we defif, the CRF response of a complex V1 cell to the stimulus placed at location  is computed according to the Gabor energy model, which is written asFor an input image e0 and eπ/2 are the responses of symmetric (or even) and asymmetric (or odd) Gabor filters at orientation θi, respectively.where * denotes the convolution operation. To quantify the neuronal behavior that the modulation strength coming from nCRF decreases non-linearly with the increased distance from the center of CRF, a distance related weighting function is defined as Grigorescu et al.  and Zeng1 denotes the L1 norm. H (s) is used to guarantee that neuronal responses should not be negative. DOGkσ, is the commonly used difference of Gaussian (DOG) function written aswhere ||·||k is the size ratio of nCRF to CRF. In this study we set k = 4 based on the physiological finding that the spatial size of nCRF is typically 2–5 times (in diameter) larger than that of CRF  strategy is used to select the neuron with the maximum CRF response across the Ẽ is called the maximum Gabor energy map, and the corresponding optimal orientation map is given bywhere In order to describe the influence of the orientation contrast between CRF and nCRF on the inhibition strength, an orientation contrast based weighting function is defined as Tang et al. .CRF and θnCRF are the orientations of the stimuli placed in CRF and nCRF, respectively, θΔ is the orientation contrast between θCRF and θnCRF. The standard deviation σΔ establishes a non-linear decreasing of the inhibition strength with the increasing of orientation contrast. We experimentally set σΔ = π/6 in this study.where θosI is computed at each locationThe orientation-selective (OS) inhibition x, y) that meet  ∈ nCRFR, where nCRFR represents the nCRF region.Note that on the right-hand-side of above equation, we take the summation across all the locations around  is independent of the orientation contrast, which can be modeled by convoluting the Gabor energy Ẽ with the spatial weighting function dWIn contrast, the non-selective (NS) inhibition term Therefore, the final neuronal responses produced by the orientation-selective and non-selective model can be respectively written asH(·) is defined as in Equation (5), i.e., H(s) = s while s > 0 and H(s) = 0 while s ≤ 0. α is a parameter used to control the strength of surround inhibition.where We schematically draw in Figure The models mentioned above have been tested with synthetic and natural images  when computing the CRF response  to the stimuli in Figure P1 in Figure P3 in Figure The results shown in Figure P2 in Figure On the other hand, the texture in region (iii)  Li, , the salBased on the computational analysis mentioned above, we speculate that V1 cells with orientation-selective and with non-selective surround inhibition work cooperatively when they extract visual features such as salient contours from complex natural scenes. To verify this prediction from a computational point of view, we propose two new integrated models for contour detection by combining the two surround inhibition mechanisms in two different ways. (i) Model 1: a binary orientation-saliency (BOS) map is defined to determine the surround inhibition type at each location, i.e., two types of neurons with different surround inhibition mechanisms are selectively activated according to the local orientation saliency. (ii) Model 2: a real-valued orientation-saliency (ROS) map is defined as a spatial weighting function to control the contribution of orientation-selective and non-selective inhibition to the neuronal responses at each location, i.e., neurons with both types of surround inhibition work at each location, but with different contributions.In Model 1, we suppose that two types of neurons with different surround inhibition mechanisms are selectively activated according to the local visual patterns. Figure ros, the real-valued orientation-saliency (ROS) at spatial location , as a ratio of the maximum CRF response across all Nθ cells with different preferred orientations within a hypercolumn divided by the sum of CRF responses of these cells, which is given byIn order to distinguish the different texture patterns based on local orientation features, we define μThe computation of ROS according to Equation (15) could be realized by a neural network of divisive normalization shown in Figure ros, and the whole ROS map is normalized to make μros ∈  for convenience. Then, a BOS map is calculated by binarizing the ROS map according toAn operation of Gaussian filtering is used to smooth μth is a threshold, and we experimentally set th = 0.4 in this study. Based on the BOS map, an input image is divided into orientation-salient regions  = 1) and non-orientation-salient regions  = 0). In the orientation-salient regions, only the neurons with orientation-selective surround inhibition are activated, and in contrast, neurons with non-selective inhibition work only in the non-orientation-salient regions.where The final neuronal response produced by Model 1 is given byH(·) is an operator defined as in Equation (5), i.e., H(s) = s while s > 0 and H(s) = 0 while s ≤ 0. α1 and α2 are weighting factors to control the strengths of orientation-selective and non-selective surround inhibition.where th (see Equation 16) seems non-biologically plausible and is not easy for each natural image; (2) most natural images are so complicated that uniformly and randomly oriented features always coexist in many local regions  is defined as in Equation (5) to restrict neuronal responses non-negative. ROS map μros is used as a spatial weighting function. α1 is a parameter used to control the synaptic strength of the orientation-selective surround inhibition. Equation (18) indicates that neurons at local regions with a higher orientation saliency would receive greater orientation-selective surround inhibition.where nsR is easily computed by convoluting osR with the spatial weighting function dW:The output produced by Equation (18) contains randomly oriented features, which is further processed by the neurons with non-selective inhibition. The non-selective surround inhibition term The final response of the neurons simulated in Model 2 is given by2 is a parameter controlling the synaptic strength of the non-selective surround inhibition. Equation (20) indicates that in the regions with more randomly oriented features, neurons with non-selective inhibition would receive higher surround inhibition, which is scaled by 1 − μros.where αIt should be clarified that the present Models 1 and 2 are computationally implemented using the Gabor-based phenomenological framework and the operation of convolution, but in fact, these models can be biologically plausibly explained and implemented using the form of neural circuits. The convolution of Gabor energy map with a weighting template  is in nature a simple implementation of summing the synaptic strength weighted modulation coming from the activated neurons in the nCRF region  and (iii) and the boundary between the regions (iii) and (iv) can not be extracted and connected into clear and smooth contours by Models 1 or 2. The reason is mainly that these two boundaries are texture defined, and more visual features like frequency and phase contrasts and even higher cortical areas like V2 may be required to effectively detect such kind of edges . The performance of our models was compared with that of the typical orientation-selective and non-selective inhibition models proposed by Grigorescu et al.  is E = DE ∩ (GTE ⊕ T) (Note that ⊕ is the dilate operator and T is a 5 × 5 square template). The pixel set of false positives (denoted by FPE) is determined by eliminating the correctly detected contour pixels from all pixels detected by model, i.e., FPE = DE − E. In contrast, the pixel set of false negatives (denoted by FNE) is determined by eliminating those ground truth pixels, which present in the 5 × 5 square neighborhood of correctly detected contour pixels, from all ground truth contour pixels, i.e., FNE = GTE − (GTE ∩ (DE ⊕ T)). Then the percentage of false positives FPe, the percentage of false negatives FNe and the overall performance measure P could be computed according to Grigorescu et al.  represents the number of elements of the set S. It is obvious from the definitions that a lower FPe, caused by the lower false positives and more correctly detected pixels, indicates a better suppression of textured background. Similarly, a lower FNe means a better integrity of salient contour, and as a whole, a better overall performance results in a higher P.where card by balancing the rate of false positive and false negative (lower FPe and lower FNe).Table P  among the 80 performance measures obtained with the 80 groups of parameter combinations. In our Model 2, the best performance Pmax is substantially higher in comparison to that of the orientation-selective and non-selective inhibition models. In addition, our models produce a consistently higher median performance Pmed than the two models with orientation-selective or non-selective inhibition alone.Figure Pmax and Pmed achieved by each model, computed on the all 40 images of RuG dataset. Our Model 2 outperforms the models with non-selective or orientation-selective inhibition alone in terms of both Pmax and Pmed statistics. Meanwhile, we also analyzed the distribution range of P over all parameter combinations. The average values of the length of boxes and whiskers were computed on each model, as shown in Figure Figure Pmax could reflect a model's capacity to achieve best performance P on each image with optimal parameter settings. In contrast, the statistics of Pmed and the length of boxes and whiskers could reflect a model's robustness with various non-optimal parameter settings. The obvious superior statistics of Pmed and the length of boxes and whiskers produced by our modes, especially Model 2, indicates that suitable combining of orientation-selective and non-selective inhibition mechanisms endows a computational model with a higher chance to obtain a better contour detection result on each image. We believe that the statistics of Pmed and the length of boxes and whiskers are more critical for a contour detection model due to the fact that for a real-time computer vision application under varying scenes, it is impracticable to use an exhaustive searching method to find the optimal model parameters for each input image.The statistics of Taken together with Figures This work focuses on the functional roles of two specific types of surround inhibition of V1 cells. From a physiological point of view, the goal of this study is not to develop a contour detection model incorporated with the full V1 mechanisms including surround inhibition, spatial facilitation, feedback modulation from higher levels, temporal dynamics, etc.  for salient contour detection by combining the two types of surround inhibition in different ways. The results on a natural image dataset with contour ground-truth showed that both our Models 1 and 2 outperform substantially the models with orientation-selective or non-selective inhibition alone, which implies a cooperative way among different types of V1 neurons at least when extracting salient contours from cluttered natural scenes. Furthermore, the substantial superiority of Model 2 over Model 1 may at least suggest a reasonable hypothesis that regularly and randomly oriented background patterns co-exist with different amounts at most spatial locations of natural scenes, which may provide meaningful implications for natural stimulus based physiological experiments and computer vision oriented applications. Note that it is beyond the scope of this work to compare the performance of our new models with that of the state-of-the-art methods developed mainly for the purpose of engineering applications . Hence, we suggest that a V1 model explicitly taking into account the two types of surround inhibition will become more physiologically plausible and computationally feasible.Though discriminating the different roles of the two types of surround inhibition mechanisms is of no doubt meaningful, and the two possible solutions proposed in this study for combining the two inhibition types seem to work well, there is still no direct evidence for (or against) our two proposals of integrating different surround inhibition types. Even so, we can see many interesting experimental findings that may provide indirect support to the physiological plausibility underlying our models. For example, as for the biological counterpart of BOS or ROS map, a most possible explanation is that the BOS or ROS map is a certain kind of “gist” extracted very rapidly via a nonselective pathway from very brief visual presentations  in Figure Finally, it is necessary to comment on some possible future improvements of the proposed models. Considering the special role of collinear facilitation in visual processing and its successful applications in computational modeling, one of our further research directions is to integrate facilitatory surround modulation into our models, which is not only helpful to objectively evaluate the functional role of different V1 elements, but also beneficial to the application goal of reconstructing incomplete contours and extracting Gestalt edges. Furthermore, incorporating top-down feedback mechanisms into computational models with appropriate ways is of no doubt helpful to substantially improve the performance of contour extraction in cluttered scenes, which is an especially challenging but engrossing future direction since only little knowledge about feedback from higher cortical areas has been experimentally discovered (Gilbert and Li, In conclusion, much remains to be investigated about the functional role and the underlying mechanisms of surround inhibition of V1 neurons in visual processing of natural scenes (Fitzpatrick, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Serum specific igE levels, serum total igE levels and serum total eosinophil counts in asthma bronchiale patients were compared with healthy controls and the relation between serum specific igE levels, serum total igE levels and serum total eosinophil counts were evaluated.5 specific igE level measurements  and serum total igE levels, serum total eosinophil counts of a total 60 patients with Asthma  aged 19 to 52  and 34  healthy persons were compared.60 percent of patient study group was determined to have positive specific igE, it is obtained that 70 percent of this positive specific igE group has sensitiveness to house dust mite antigen.Asthma bronchiale cases have specific igE positiviness level and total igE level and total eosinophil counts were significantly higher than the control group.Patients with asthma and allergic rhinitis revealed significant correlation with specific igE Df, total igE and eosinophil counts, furthermore significant correlation was behold between specific igE Dp, total igE and eosinophil counts.In our study compared to other allergens, sensitivity against house dust was much more higher and it is coherent with other studies in literature.The usefullness of evaluating total serum igE or allergen specific igE for reasons of diagnosis and management is mutable however serum igE level is an important indicator of the intensity of inflammatory processes in allergic diseases and serum igE, total eosinophil counts and specific igE are helpful for diagnosis and developing therapeutic strategy in allergic diseases.


In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the  The solution was replaced after every 10 minutes. Subsequently, the gel was incubated in 50 mL of renaturation buffer for another 3 hours at 4°C without agitation. The solution was replaced, and the incubation was continued for 16 hours with the same conditions.In-gel kinase assays were performed as described by Wooten et al. . Two mil2, 2 mM β-mercaptoethanol, 0.1 mM EGTA) for 1 hour with gentle agitation. For the kinase reaction, the gel was incubated with 50 μM of 5 μCi/mL [γ_32P]ATP in 3 mL of kinase buffer for 4 hours with gentle agitation. For inhibition assays, 15 μg/mL of PKI (Promega) were included in the kinase buffer. The reaction was terminated by washing the gel five times with 50 mL of stop buffer  for 75 minutes. The gel was dried on cellophane membrane for 16 hours at room temperature, and finally the radioactive signal was detected using X-ray film (Kodak).Prior to the kinase reaction, the gel was washed twice with 50 mL of kinase buffer  or 20 μg of cell lysate in 50 mM KH2PO4, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 20 mM MgCl2, 10 mM β-mercaptoethanol, 3 mM of [γ_32P]ATP (1 μCi) or unlabeled ATP, 1× complete protease (Roche) and 1× phosphatase inhibitor cocktail  in a total volume of 40 μL at 30°C for 1 hour. The reaction was terminated by heating at 95°C for 5 minutes in 2× SDS loading buffer. In vitro phosphorylation of other human and yeast CKs was performed as described above using 80 U of PKA. Samples containing 5 or 10 μg of phosphorylated protein per well were analyzed by 10% SDS-PAGE. The phosphoprotein was detected by either autoradiography or Pro-Q Diamond phosphoprotein gel stain (Invitrogen). The phosphoprotein gel staining was performed according to the manufacturer’s protocol. The level of protein loading was determined by Coomassie blue stain or SYPRO® Ruby stain (Invitrogen). For inhibition assays, 15 to 45 μg/mL of PKI (Promega) were added to the reaction mixture. To investigate the effect of PKA and ATP concentrations on the level of CKβ phosphorylation, 20 to 120 U of PKA and 25 to 100 μM of ATP were used in the in vitro CKβ phosphorylation reactions.2 chromatography procedure for phosphopeptide enrichment . GFP-CKβ protein was immunoprecipitated with CKβ antibody : Here, 0The phosphoprotein was detected by phospho-(Ser/Thr) PKA substrate (PhosphoPKAS) antibody  diluted at 1:1000 ratio with blocking buffer . The primary antibody was incubated with the membrane at 4°C for 16 hours followed by detection with rabbit IgG-horseradish peroxidase (HRP)-conjugated secondary antibody . The signal was developed by Supersignal West Pico chemiluminescent substrate (Thermo Scientific) and detected by using the FUSION-FX chemiluminescence documentation system (Vilber Lourmat). After imaging, the antibody bound on the membrane was removed with stripping buffer , and the membrane was reprobed with rabbit anti-GFP antibody (Abcam) at 1:1000 dilution.For the detection of CKβ-phosphorylating kinase in the in-gel kinase assay, a replicated gel was blotted onto nitrocellulose membrane followed by Western detection with 1:5,000 dilution of PKA catalytic subunit-recognizing polyclonal antibody (Abcam) according to similar procedures as described above.2, 0.5 mM phosphoenolpyruvate, 0.25 mM NADH, 2 mM ATP, 4 U of pyruvate kinase, 5 U of lactate dehydrogenase, 1 to 2 μg of purified CK protein and 4 mM choline chloride or 4 mM ethanolamine in a final volume of 1 mL. The reaction was performed at 37°C and initiated by adding choline or ethanolamine. Any background activity before the addition of choline or ethanolamine was subtracted from the total activity. The purified CK protein used in such assays was either unphosphorylated, in vitro phosphorylated (as described above), or the S39D/S40D phosphorylation mimic mutant. The in vitro phosphorylated CKβ was used immediately for enzymatic assay after the 1 hour PKA treatment. The unphosphorylated CKβ wild type and the S39D/S40D CKβ variant were either freshly purified proteins or purified proteins stored at −80°C. All the kinetic parameters were obtained from nonlinear regression curve fitting of the Michaelis-Menten plot by using GraphPad Prism version 5.0.Choline kinase activity was determined by using the pyruvate kinase (PK)-lactate dehydrogenase (LDH) coupled-enzyme assay as previously described  using a 50) values of the unphosphorylated, PKA- phosphorylated, and the S39D/S40D phosphorylation-mimic mutant CKβ. IC50 was determined by the standard CK assay as described above in the presence of saturating substrate concentration (4 mM choline) and increasing HC-3 concentrations, and fitting the data plotted in a dose-response curve by using GraphPad Prism 5.0.The effect of PKA-induced phosphorylation on HC-3 inhibition of CKβ was investigated by comparing the half-maximal inhibitory concentration Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.


However, it is not clear how well the data from slice recordings match the inhibition in the intact brain and how it contributes to complex phenomena such as non-monotonic rate-level functions (RLF). Therefore, we determined the input-output relationship of a model SBC with simulated endbulb inputs and a dynamic inhibitory conductance constrained by recordings in brain slice preparations of hearing gerbils. Event arrival times from in vivo single-unit recordings in gerbils, where 70% of SBC showed non-monotonic RLF, were used as input for the model. Model output RLFs systematically changed from monotonic to non-monotonic shape with increasing strength of tonic inhibition. A limited range of inhibitory synaptic properties consistent with the slice data generated a good match between the model and recorded RLF. Moreover, tonic inhibition elevated the action potentials (AP) threshold and improved the temporal precision of output functions in a SBC model with phase-dependent input conductance. We conclude that activity-dependent, summating inhibition contributes to high temporal precision of SBC spiking by filtering out weak and poorly timed EPSP. Moreover, inhibitory parameters determined in slice recordings provide a good estimate of inhibitory mechanisms apparently active in vivo.Spherical bushy cells (SBCs) of the anteroventral cochlear nucleus (AVCN) receive input from large excitatory auditory nerve (AN) terminals, the endbulbs of Held, and mixed glycinergic/GABAergic inhibitory inputs. The latter have sufficient potency to block action potential firing  Animals were attached to a custom stereotactical device via a headmount glued to the skull. The rostral AVCN was accessed with low-impedance patch electrodes  using angles described by Frisina et al.  was conducted for 30 min at 37°C. Recordings were made at nearly physiological temperature (33 ± 0.5°C).All experimental procedures were approved by the Saxonian district Government Leipzig  and conducted according to the European Communities Council Directive (86/609/EEC). Coronal slices (170 μm) containing the rostral AVCN were cut with a vibratome (Microm HM 650 V) from P22–P33 gerbils of either sex. At this stage, the development of inhibitory transmission onto SBCs was shown to be completed . The recordings of synaptically evoked IPSCs (eIPSCs) on SBCs were conducted as reported earlier  filled with bath solution and placed at distances of 30–60 μm from the recorded cell. The stimulus intensity was slowly increased until obtaining stable IPSC amplitudes within a train. A stimulator (Master 8) was used to trigger single- or trains of pulse stimuli (100 μs), delivered via an isolated stimulus unit (AMPI Iso-flex). In all experiments, glutamate receptors were pharmacologically blocked . To rule out the possible effects of presynaptic GABAB receptors located on inhibitory terminals . The average series resistance was 11.33 ± 1.66 MΩ , which was compensated by 50% to a remaining Rs of 3–7 MΩ. During experiments the series resistance changed on average by 1.5% (n = 34). Cells with series resistance changes >10% were excluded from analysis. There was neither a correlation between the IPCS amplitudes and decay time constants nor between the IPSC amplitudes and rise times  to have resistances of 4–5 MΩ when filled with (mM): 140 CsMeSOpost hoc biocytin-labeling with Cy2-conjugated streptavidin /(Afast + Aslow), where Afast and Aslow are amplitudes at t = 0 and τfast and τslow are the fast and slow time constants, respectively. IPSC conductance was calculated from the Cl−-driving force of 20 mV, determined by the Vhold−EIPSC = −71 – −50.8 mV . To exclude possible errors due to variations in series resistance, EIPSC was controlled at the beginning of each experiment.SBCs were verified according to their morphology revealed either on-line by intracellular labeling with ATTO 488 or by All simulations were performed with the NEURON simulation environment , an axon hillock/first segment compartment  containing all voltage-activated sodium conductance and a stretch of passive axon  were included in the model. Reversal potential for the leak conductance  was −65 mV. Axial resistance was 150 Ω·cm for all compartments. Voltage-activated ion channel conductances were Nav 1000 nS, LVA-K 200 nS, Ih 40 nS (Erev −43 mV) and HVA-K 175 nS. Basic somatic parameters of the model SBC therefore resulted in a total membrane capacitance of 20.1 pF, a total input resistance (at rest) of 69.2 MΩ, and a resting membrane potential of −65.1 mV.A somatic compartment  were used. In the respective signals, the peak of the EPSP component was defined as the inflection point in the rising part of the waveform. This inflection point was calculated as the minimum between the local maxima in the first derivative (maximum EPSP and AP rising slope). The EPSP amplitude was defined as the difference (in mV) between the local baseline at the beginning of the analysis window and the peak of the EPSP component. In cases when APs failed, the EPSP amplitude was defined as the maximum membrane potential deflection in the analysis window and timing of the peak as well as upward and downward slopes were calculated.Simulations were run at a temporal resolution of ≤20 μs. The membrane potential of the model SBC was calculated and spikes were timed on the peak of the postsynaptic APs. The amplitude and relative timing of the EPSP and AP components were routinely analyzed using a waveform analysis procedure similar to the one described in Section rev = 0 mV) and on the other hand the inhibitory inputs (Erev = −75 mV) were connected to the somatic compartment of the SBC model. The Erev for inhibitory current in our model matches the EGABA experimentally determined with gramicidin perforated patch recordings  or generated by a simple AN model (Section Auditory Nerve Model). The spike times driving the inhibitory point-process were delayed by 1 ms.Two conductance point-processes representing on the one hand the excitatory endbulb of Held synapse (Eτ = 200 μs) of the excitatory postsynaptic conductance (EPSG) template were fitted to EPSP waveforms recorded in vivo  and decay time-constant . Note: the time-constants applicable for this state-model differ from recovery time-constants derived from paired-pulse experiments. The maximal EPSG amplitude was multiplied with the state variable at the time of the event resulting in the instantaneous maximal excitatory conductance. We tested a wider range of endbulb conductance for the dynamic endbulb models because both are expected to lower the excitatory efficacy. However, as shown by Pliss et al. . Finally, we also implemented a phenomenological depression model, as described before Nerlich et al. . Brieflys et al. , endbulbn = 42), while the decay time-constant was 23.9 ± 5.5 ms (n = 41). In a number of experiments, the decay time-constant was systematically varied between 1 and 24 ms.The template waveform of the inhibitory postsynaptic conductance (IPSG) was fitted to IPSC data from slice recordings presented in this study: The rise time was 455 ± 170 μs  model was created to generate synthetic input spike trains for the SBC model. The AN model was implemented as a gamma-tone filterbank driving leaky integrate-and-fire neurons with noise and refractoriness  using the tools provided by the spiking neural-network simulator “Brian”  at higher sound pressure levels , with only one unit ranging below 0.9, whereas the mean MI of the SBC output was 0.69 ± 0.26 (n = 37). From the latter, 30% (11/37) showed MI above 0.9  and 46% (17/37) indices below 0.75  were calculated by dividing the mean response rates at the respective three highest stimulus levels by the maximum response rates Figure . Thus, pdark gray trace) evoked in naïve synapses had an average conductance of 23.8 ± 5.4 nS (n = 41), a rapid 10–90% rise time (0.46 ± 0.18 ms) and a slow decay time constant (tau weighted) of 23.9 ± 5.5 ms (n = 41). Repetitive electrical stimulation of inhibitory inputs  led to summation of the overall current , while individual phasic events (inset) were subject to activity dependent changes in amplitudes. A prolongation of the stimulus train  yielded a characteristic temporal profile of the total inhibitory current described by a transient temporal summation of individual events and progressive activity dependent amplitude decrease . The activity dependent changes also included the prolongation of the decay time constant of the last IPSC . In the course of a pulse train, phasic IPSC amplitudes (Ipeak) showed a depression that followed a double-exponential course when the recordings were performed using the standard extracellular solution containing 2 mM Ca2+ . During the first 5–6 events of a 100 Hz pulse-train, the phasic IPSC amplitude rapidly depressed below 50% of the initial value, with a more moderate reduction thereafter towards a steady-state phasic current of about 20% (18 ± 2%) of the initial values. Yet, the depression of phasic IPSC amplitudes was markedly different in extracellular calcium condition (1.2 mM), which is thought to more faithfully reflect the in vivo condition   Borst, .2+, the total inhibitory conductance was smaller at the stimulation onset, while towards the end of the train the baseline IPSCs were not different for the different calcium concentrations . In fact, failures only occurred with short intervals, presumably when approaching the refractory period of the SBC. Under these conditions, the output of the SBC model without inhibition closely resembled the input . Thus, we concluded that for the non-monotonic SBC output the overall level of summed inhibition was more important than the precise temporal structure of inhibitory events.Initial simulations utilized a fixed temporal structure of inhibition delayed by 1 ms to the excitation. This was however only a first approximation of the actual in vivo data (MI = 0.54) as a contour in the plot revealed a wide range of parameter combinations that yield such results. However, the sum of squared errors between measured RLF and model RLF  was used. A wide range of MI was encountered Figure  includinThese simulations show that, given a specific and confined range of excitatory and inhibitory synaptic properties are used, the SBC model with dynamic inhibition can closely reproduce RLF measured in the intact gerbil brain.In an earlier study . The synaptic mechanisms of the SBC model were driven by the phase-locked spiketimes Figure . With miper se reduces the temporal precision of the SBC model mainly by causing AP initiation close to threshold, which introduces a non-linear temporal delay to the events. As shown in Figure The cycle histograms and vector-strength values presented here were calculated by using the peak of the AP as spiketime of the SBC output. This was considered the most relevant metric for the SBC function. Further analysis of the temporal precision between the peak of the EPSP component (successful EPSPs only) and the following AP revealed the cause of the phase-locking deterioration. The EPSP timing had vector strength of 0.81 ± 0.01, i.e., on average 0.15 ± 0.02 better than the phase locking of the AP. Moreover, the precision of the EPSP peak was not affected by the inhibitory strength and the mean phase delay was only slightly increased . AP initiation close to threshold, presumably occurring under the influence of strong inhibition, was thus the major cause of poor temporal precision in the SBC model. Thus, these data suggest that the interaction of excitation and inhibition in vivo recordings indicating that inhibition alone is not sufficient to improve the temporal precision of SBC. Kuenzel et al. , will provide a comprehensive picture of inhibitory contribution to the SBC output function.In summary, this study reveals the functional importance of dynamic inhibitory filtering in sensory neurons and determines the inhibitory parameters putatively active The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P3DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in  Arabidopsis, seed germination is driven largely by the metabolism of storage products other than lipids, whereas seedling establishment involves the mobilization of seed oil reserves. Of  Of ArabiArabidopsis thaliana seedlings [pTLLFGEKPVTVFGIR in both isoforms indicates that both are phosphorylated at T70. However, a second phosphopeptide SDLDIVpSNASCTTNCLAPLAK, which had previously been detected in Arabidopsis seedlings [Of the identified phosphoproteins previously reported in eedlings   compareeedlings,,16,32 sueedlings, By enablhttp://www.genome.jp/kegg/pathway.html). The two largest groups were those involved in carbohydrate/energy metabolism (22%) and oxidative stress/redox regulation (20%), which together with photosynthesis and respiration (11%) accounted for more than half of the identified phosphoproteins , for example, plays a central role in chloroplast development [Arabidopsis TPI , a major chloroplast protein, is involved in photosynthesis  and the elopment.Arabidopsis young seedlings  during seed germination and seedling development.[2O2 from the oxidative breakdown of oxalate,[Although there is growing evidence of crosstalk between redox signaling and hormonal response pathways during seed germination, the moleelopment. Germin-l oxalate, was alsoArabidopsis [Arabidopsis seedlings and identify sites of phosphorylation in HSP60 (T80) and chaperonin 20  that, to the best of our knowledge, have not been reported before (Heat shock proteins (HSPs) are involved in bud dormancy  and phosbidopsis ,49 and pbidopsis  Our resud before  Fig.Arabidopsis leaf extracts  were also observed  in young seedlings , spot 80is seeds  shows this seeds  althoughArabidopsis young seedlings. These included enzymes involved in chloroplast development, mobilization of TAG, and other processes known to be important during the early stages of plant development. Comparison of our results for young seedlings with those obtained previously for Arabidopsis seeds [Arabidopsis young seedlings. By purifying and enriching phosphorylated proteins under non-denaturing conditions our approach also lends itself to the study of phosphorylation in endogenous protein complexes and during protein-protein interactions.Seedling establishment involves the efficient utilization of endogenous protein reserves and external resources, requiring that developmental and metabolic programs adapt to the prevailing environmental conditions. Using a is seeds  and matuis seeds  shows thS1 Figm/z) ratios, as obtained by trypsin digestion of proteins selected from the 2-DE gel shown in m/z 701.377, showing phosphorylation of FBA (spot 17) at S84; (B) m/z 878.968, showing phosphorylation of GAPC-2 (spot 18) at T70; (C) m/z 619.981, showing phosphorylation of cytosolic TPI (spot 64) at S178; and (D) m/z 721.369, showing phosphorylation of PMDH1 (spot 25) at T138.The MS/MS spectra correspond to phosphopeptides with the following mass-to-charge ((PDF)Click here for additional data file.S2 Figm/z) ratios, as obtained by trypsin digestion of proteins selected from the 2-DE gel shown in m/z 745.425, showing phosphorylation of ATGST10 (spot 47) at T4; (B) m/z 612.269, showing phosphorylation of ATGST6 (spot 49) at T15; (C) m/z 549.250, showing phosphorylation of HSP60 (spot 78) at T80; and (D) m/z 615.308, showing phosphorylation of chaperonin 20 (spot 76) at S61.The MS/MS spectra correspond to phosphopeptides with the following mass-to-charge ((PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.


Dear EditorMacroautophagy (hereafter referred to as autophagy) is a cellular adaptation to starvation, involving the vesicular sequestration of the long-lived cytoplasmic proteins and organelles such as mitochondria  , a natural product isolated from the Electron microscopy, which is believed to be one of the most convincing instruments to detect autophagy, was used to investigate the autophagy induced by C42 , suggesting that C42 increases the formation of autophagosome.To further measure autophagosome formation in the C42-treated cells, HCT116 cells were transfected with a fusion protein of green fluorescent protein (GFP) and LC3 (a specific marker of autophagosomes) and visualized by confocal microscopy , indicating that C42 induces autophagy in the cells. To detect autophagic flux, the level of LC3-II was measured in the presence of bafilomycin A1 (Baf A1), which acts as a specific inhibitor of the vacuolar type HMammalian target of rapamycin (mTOR) inhibits autophagy and its kinase activity can be detected by measuring the phosphorylation of its substrates, such as p70S6 kinase S6K1). As shown in Figs. . As showInterestingly, we found that C42 promoted autophagy concomitantly with increase in Tyr279/216 phosphorylation of GSK3 at both the 1 and 3 h time points  and GSK3 phosphorylation at Tyr279/216 caused by C42 . Moreover, addition of chloroquine (CQ), an agent often used in autophagic flux detection  or GSK3β  or both  , Beijing Natural Science Foundation (5111003), and the National Program of Drug Research and Development (2012ZX09301-003).S. N. and D. Y. performed the experiments; Y. C. and X. J. designed the experiments.Shubin Niu, Dongdong Yuan, Xuejun Jiang and Yongsheng Che declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by the any of the authors.Supplementary material 1 (PDF 229 kb)Below is the link to the electronic supplementary material.