Multinucleated giant cells, formed by the fusion of monocytes/macrophages, are features of chronic granulomatous inflammation associated with infections or the persistent presence of foreign material. The tetraspanins CD9 and CD81 regulate multinucleated giant cell formation: soluble recombinant proteins corresponding to the large extracellular domain (EC2) of human but not mouse CD9 can inhibit multinucleated giant cell formation, whereas human CD81 EC2 can antagonise this effect. Tetraspanin EC2 are all likely to have a conserved three helix sub-domain and a much less well-conserved or hypervariable sub-domain formed by short helices and interconnecting loops stabilised by two or more disulfide bridges. Using CD9/CD81 EC2 chimeras and point mutants we have mapped the specific regions of the CD9 EC2 involved in multinucleated giant cell formation. These were primarily located in two helices, one in each sub-domain. The cysteine residues involved in the formation of the disulfide bridges in CD9 EC2 were all essential for inhibitory activity but a conserved glycine residue in the tetraspanin-defining ‘CCG’ motif was not. A tyrosine residue in one of the active regions that is not conserved between human and mouse CD9 EC2, predicted to be solvent-exposed, was found to be only peripherally involved in this activity. We have defined two spatially-distinct sites on the CD9 EC2 that are required for inhibitory activity. Agents that target these sites could have therapeutic applications in diseases in which multinucleated giant cells play a pathogenic role. The tetraspanins are a family of transmembrane glycoproteins, with thirty-three members identified in mammals The EC2 domain has been shown to be critical for many of the interactions with partner proteins Mycobacterium tuberculosisThe tetraspanins have been reported to be involved in a number of cell-fusion processes such as sperm∶egg fusion, muscle cell fusion, and virus-induced syncitial formation CD9 and CD81 EC2 are expected to have a similar structure because they are of a similar length, have the same number of cysteine residues E. coli (Merck Biosciences), culturing at 37°C for 4 hours after IPTG induction. Cells were pelleted and lysed by sonication in the presence of a protease inhibitor cocktail. Recombinant protein was purified in a single step by affinity chromatography on glutathione beads (Amersham-Pharmacia). Protein purity was analysed by Coomassie staining of SDS-PAGE gels and total protein concentration determined by Bradford assay. As it was not possible to separate the full-length EC2 fusion protein from the smaller fragments produced, the percentage of full length material in each sample was measured by densitometry. This was plotted against the inhibitory activity of each sample to ensure that inhibition of MGC formation was not a simple function of the concentration of the full length fusion protein . After overnight culture, adherent cells were cultured in RPMI containing 10% (v/v) foetal bovine serum in the presence or absence of 10 µg/ml Concanavalin A (Con A)  for 72 h at 37°C. The recombinant tetraspanin EC2 proteins were added at the stated concentrations at the same time as the Con A. In some cases 200 nM E. coli lipopolysaccharide  was used to determine if contaminants from the production process were responsible for effects observed. The cells were washed with PBS, fixed and permeabilised with acetone (5 min at room temperature), rehydrated with PBS then labelled with FITC-anti-CD63 (clone H5C6) and the nuclei counter-stained with propidium iodide . Fusion indices (number of nuclei within MGCs)/×100) were determined by counting the number of nuclei in fused cells (>5 nuclei per cell) and unfused cells in 6 randomly chosen fields  using a Nikon Eclipse E400 immunofluorescence microscope. The numbers of nuclei per MGC were recorded and the average nuclei per MGC calculated. Counts from each chamber are presented as separate data points.Peripheral blood monocytes were derived from peripheral whole blood of healthy volunteers by Ficoll-Hypaque density centrifugation as described elsewhere The study was approved by the South Sheffield Research Ethics Committee (protocol number SSREC/02/299). Participants provided written consent and records have been retained by the named researchers on the Ethics Protocol, as required by the Research Ethics Committee.The sequences of human CD9 and CD81 EC2 are shown in E. coli system was used to express the GST-fusion proteins, was tested for any effect on MGC formation. No effect was observed on fusion index or the average number of nuclei within a giant cell. Co-incubation of CD9 and CD81 EC2 proteins, at either 250 nM or 500 nM of each protein, caused significantly less inhibition relative to CD9 EC2 alone  at 500 nM, a dose of human CD9 EC2 previously shown to inhibit MGC formation As mouse CD9 EC2 is inactive in the MGC formation assay, the sequences of mouse CD9 and human CD9 EC2 D2 and D4 regions were compared . In the Other point mutants tested in the fusion assay included the four cysteine residues and the G154 residue in the ‘CCG’ motif, characteristic of the mammalian tetraspanins . The mutConsistent with previous findings, GST-CD9 EC2 significantly inhibited size and fusion rates of MGC  and a number of residues  aligned on the outer face of CD81 EC2 helix B (flanking the position of Y148 in CD9 EC2) Schistosoma japonicum, sjc23, can bind human IgG at a single site immediately preceding the CCG motif and synthetic peptides carrying the sequence KIQTSFHCC (most of helix B) were found to block binding Using CD9/CD81 EC2 chimeras, we have identified two distinct regions of CD9 EC2 that are essential for inhibition of MGC formation . These rMutation of a number of residues in the D2 and D4 sites of CD9 EC2 resulted in the loss of inhibitory activity to varying extents. Residues in the D4 region of CD9 have previously been implicated in sperm∶egg fusion The functional sites on human CD9 EC2 that are required for the inhibition of MGC formation have been mapped to two separate regions, both which are required for activity. Compounds that interfere with the activity of these sites may be useful therapeutic agents that can block the formation of MGC in pathological conditions such as giant cell arteritis.S1 FileS1 Fig. The relation between percentage of full length fusion protein and ability to inhibit MGC formation. Fig S1A is a graphical representation of the level of protein present in the major 35–36 kDa tetraspanin band on SDS-PAGE plotted against the percentage inhibition of MGC fusion at 500 nM total protein concentration. Fig. S1B is a representative SDS-PAGE experiment, showing the full-length GST fusion protein indicated by the arrow on the right and with the percentage of each chimera at full length, measured by densitometry, shown in each lane. S1 Table. Details of chimeras.Contains the following files: (DOCX)Click here for additional data file.


Moreover, 3 functional motifs associated with O-GlcNAc/Phos sites were extracted. We further used sequence features and GO features to predict O-GlcNAc/Phos cross talk sites based on phosphorylated sites and O-GlcNAcylated sites separately by the use of SVM model. The AUC of classifier based on phosphorylated sites is 0.896 and the other classifier based on GlcNAcylated sites is 0.843. Both classifiers achieved a relatively better performance compared with other existing methods.Reversible posttranslational modification (PTM) plays a very important role in biological process by changing properties of proteins. As many proteins are multiply modified by PTMs, cross talk of PTMs is becoming an intriguing topic and draws much attention. Currently, lots of evidences suggest that the PTMs work together to accomplish a specific biological function. However, both the general principles and underlying mechanism of PTM crosstalk are elusive. In this study, by using large-scale datasets we performed evolutionary conservation analysis, gene ontology enrichment, motif extraction of proteins with cross talk of O-GlcNAcylation and phosphorylation cooccurring on the same residue. We found that proteins with  Reversible posttranslational modification (PTM) can determine a protein's localization and activity state and interaction with other molecules by adding a modifying group or proteolytic cleavage . Single in situ cross talk) or one PTM masks the recognition site of the other PTM. Recent application of new mass spectrometric methods for O-GlcNAcylation and large-scale identification of phosphorylated sites show extensive and dynamic cross talk between O-GlcNAcylation and phosphorylation. Studies show that the level of O-GlcNAcylation within cells decreased after the treatment with phosphatase inhibitors and kinase activators  significantly enriched in endomembrane system organization and GTPase binding, Txxx[S] enriched in cytoskeleton organization, and [T]xxxxxxxxxP to take part in regulation of cell cycle. After that, we evaluated whether proteins containing the same motif are part of a single protein network. As shown in After using motif-X and filtering our result, we finally discovered three potential functional motifs  data source –20. in situ O-GlcNAc/Phos sites; our results showed that proteins having in situ O-GlcNAc/Phos sites were significantly enriched in nuclear transport, cytoskeleton, and structure molecular activity. It not only substantiates the overall functional role of O-GlcNAc/Phos cross talk but also enlightened us that the GO terms hold the potentiality to improve the performance in site prediction, which drove our investigation on site prediction method in the second part and made a great contribution to prediction performance.From the GO enrichment analysis, we obtained the functions specific to the fact ofOur evolution analysis showed no additional evolution pressure on sites having O-GlcNAc/Phos cross talk, implying that the conservation of cross talk sites may depend more on PTM types. Combined with the result in GO enrichment analysis, we can conclude that the functional role of O-GlcNAc/Phos site may not directly lead to an increase in site conservation. It makes sense as previous study shows that the O-GlcNAc/Phos cross talk tends to play roles in chronic diseases rather than fatal diseases. in situ O-GlcNAc/Phos sites may not follow a stringent recognition motif, which also suggests that a traditional site prediction strategy using primary sequence may have a poor predictive outcome.Then, we extracted 3 functional motifs. The workflow we used reduced the possibility of false discoveries and literature finding also proved the reasonability of the outcomes. Our following investigation of these motifs gave us more understanding of the underlying mechanism. Although it should be noted that the statistical significance cannot guarantee a biological importance, our results provide sensible hypotheses for follow-up experiments, which may lead to a further confirmation of our predictions. On the other hand, as only 3 motifs were extracted, it is suggested thatSite prediction is an intriguing and significant topic in the study of PTMs because it is a prospective way to overcome the high-cost and labor-intensive shortcomings of PTM site identification using experimental techniques. However, the development of computational approaches in identifying O-GlcNAc/Phos sites is still immature. Current approaches only take the advantages of primary sequences and the predictive performances are usually not ideal , 33. in situ O-GlcNAc/Phos cross talk sites.Our bioinformatics analysis gave us important clues to the feature selection and led to satisfying predictive performance. In this work, we integrated primary sequence features and GO features by using MRMR criterion and then adopted statistics learning method to predict O-GlcNAc/Phos cross talk sites. The following performance evaluation showed that our method achieved relatively high sensitivity comparing with other existing tools. Due to the development of mass spectrometry, a large amount of phosphorylated sites was experimentally verified. In addition, the computational prediction of phosphorylation sites and O-GlcNAcylated sites is continuously improving –36. ThusIn this work, by using bioinformatics tools, we found that proteins having O-GlcNAc/Phos were significantly enriched in nuclear transport, cytoskeleton, and structure molecular activity. Although no additional conservation was found in O-GlcNAc/Phos sites, 3 functional motifs were extracted. For one thing, these findings give us more understanding on general characteristics and potential mechanism of O-GlcNAc/Phos cross talk and also provide sensible hypotheses for follow-up wet experiments. For another thing, the results drove our further research on site prediction. The integration of GO terms with primary sequence features and the introduction of MRMR criterion into feature selection workflow lead to a significant promotion in predictive performance of O-GlcNAc/Phos cross talk sites. It is undoubtedly a progress in the development of sensitive computational tools to discover O-GlcNAc/Phos cross talk sites. Thus, it is anticipated that the classifiers constructed in this work could be useful for biomedical research and guide the related experimental validations. in situ O-GlcNAc/Phos cross talk on Ser and Thr. Currently, lots of publications also demonstrate the common collaboration of PTMs near each other [ in situ O-GlcNAc/Phos cross talk. In addition, many publications show that cross talk of O-GlcNAcylation and phosphorylation in close proximity play roles in several signaling pathways [In this study we only focused onch other , 37, 38 ch other , 40, butpathways , 41–43. Table S1: It lists the extracted motifs from motif analysis and relevant information of sites containing the motif. Enriched GO terms obtained from GO enrichment analysis and GeneMANIA analysis are also contained.Table S2: Top 10 GO terms associated with phos-based classier ranked by MRMR and top 20 GO associated with O-GlcNAc-based classier are listed.Table S3: Top 10 potential in situ O-GlcNAc/Phos cross talk sites that were annotated with only phosphorylation are listed.


Background: Melanopsin is a retinal light-sensitive receptor involved in non-image forming functions including circadian timing.Results: The intracellular C-terminal part of melanopsin was shown to be phosphorylated at distinct functional sites in response to light and dark.Conclusion: Phosphorylation status of melanopsin influences its functions.Significance: Distinct sites of phosphorylation are important in determining the signaling properties of melanopsin. in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca2+ response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca2+ response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed  Melanopsin is a unique light-sensitive receptor that is expressed in the membrane of a subset of retinal ganglion cells of the mammalian eye, rendering these cells intrinsically photosensitive. These intrinsically photosensitive ganglion cells (ipRGCs),1–2+   showed high phosphorylation potential (prediction score 0.93–0.99). Four of these were by sequence homology found to be conserved in mammalian and other vertebrate species . Custom phospho-site-specific antibodies against rat melanopsin phosphorylated at Ser-381 (abMel-P381) or Ser-398 (abMel-P398) were subsequently produced by Pacific Immunology Corp., Ramona, CA. The sequences of the two peptides containing the phosphorylated version of either Ser-381 and or Ser-398 were C-RSHPSLpSYRSTHRSTLS and C-SSQSSDLpSWISGQKR (pS is phosphoserine), respectively. The peptides were conjugated through the N-terminal cysteine thiol to keyhole limpet hemocyanin. The antigens were emulsified with complete Freund's adjuvant for the initial immunization and with incomplete Freund's adjuvant for booster injections. The immunogen emulsions were injected subcutaneously into New Zealand White rabbits, and antibody generation against the specific peptide was monitored by competitive ELISA. Antibodies present in serum from the last bleed were affinity-purified using a gel cross-linked with the appropriate synthetic phosphopeptide. The specificity of the antibodies was demonstrated in cells expressing melanopsin or melanopsin mutated in either Ser-381 or Ser-398 (see below). For detection of non-phospho-site-specific melanopsin in immunoblotting, a commercial antibody raised against a C-terminal peptide was used (abMel-WB) code no. PA1-781 , whereas immunostaining was performed with an in-house-produced C-terminally directed rabbit anti-melanopsin antibody code no. 41K9 (abMel) (The amino acid sequence of rat melanopsin (NP_620215.1 from the NCBI Protein Data Bank) was analyzed by the bioinformatics web-based tool NetPhos 2.0, which scans protein sequences and calculates a theoretical probability of an amino acid residue to be phosphorylated . Using tFig. 1 (abMel) .GCCTACCGCTCTACC and GGTAGAGCGGTAGGCGAGGGAGGGGTGG and primer set CAGTCCTCAGACCTCGCCTGGATCTCTGGGC and GCCCAGAGATCCAGGCGAGGTCTGAGGACTG were used to generate the amino acid substitution at positions 381 (prMelS381A) and 398 (prMelS398A), respectively, the nucleotides changed are underlined. Generation of a plasmid carrying mutations in both codons (prMelS381 + 398A) was made using prMelS381A as template and the primer pair used for the S398A mutation. All plasmids were verified by sequencing (Eurofins MWG Operon).The full coding sequence of rat melanopsin cDNA was amplified by PCR using Pfu polymerase , AGCATGAACTCTCCTTCAGAA and CTACATCCTTCGGTCCAG as primers, and a previously described plasmid containing the cDNA of rat melanopsin as template . The obtFlp-In T-Rex-293 cells (Invitrogen) derived from the human embryonic kidney cell line HEK-293 were used for transfection. Flp-In T-Rex-293 cells stably express a Tet repressor and contain a single integrated Flp recombination target (FRT) site. The cells were grown in DMEM (Invitrogen) supplemented with 10% tetracycline negative fetal calf serum (Invitrogen), 100 units/ml penicillin, and 0.1 mg/ml streptomycin , 100 μg/ml zeocin (Invitrogen), and 15 μg/ml blasticidin . The cells were co-transfected with one of the above described plasmids  and pOG44 (Invitrogen) expressing Flp recombinase (Invitrogen) according to the supplier's protocol. The day after transfection, cells were split, and the subsequent day 100 μg/ml hygromycin B (Invitrogen) as opposed to zeocin was added to the medium to select for Flp-mediated integration in the host cell genome. Due to the single FRT-site, the transfected cells are isogenic, rendering clonally selection unnecessary. The pcDNA5/FRT/TO-TOPO vector used for the melanopsin plasmids  contains a tetracycline-regulated promoter resulting tetracycline inducible expression of either WT melanopsin (rMel-WT) or melanopsin harboring single or double mutations rMel-S381 + 398A in the transfected cells.5 cells were seeded on Lab-TekTM PermanoxTM Chamber Slides (Thermo Fisher Scientific), whereas ∼3 × 106 cells were seeded in T25 flasks for immunoblots evaluating the time course of phosphorylation/dephosphorylation. The cells were either grown in the presence of tetracycline for induction of melanopsin or in absence of tetracycline. The latter served as negative controls. A tetracycline concentration-dependent increase in the expression of melanopsin was found, and 1 μg/ml tetracycline was optimal and chosen. The day after seeding, the medium was changed with medium containing 10 μm retinal (Sigma) and tetracycline. On the subsequent day the cells were either light-stimulated (white light >300 lux) or kept as dark controls. Cells for immunocytochemistry were fixated for 15 min in Stefanini's fixative and stored at −20 °C until further processing. After removal from the dark or light incubator, cells for immunoblotting were immediately placed on ice, washed twice in ice-cold phosphate-buffered saline (PBS), and extracted for 30 min on ice using Cell Extraction Buffer (Invitrogen) supplemented with 10 mm EDTA, Halt Phosphatase Inhibitor Mixture (Thermo Fisher Scientific), and Protease Inhibitor Mixture (Sigma). The whole-cell lysates were cleared by centrifugation for 10 min at 15,000 × g, 4 °C before storage at −80 °C.For immunocytochemistry, 2 × 10m H-89 (PKA), 1 μm Gö 6850 (PKC), 0.1 mm βARK1 (β-adrenergic receptor kinase 1)/GRK2 (G protein receptor kinase 2), 25 μm D4476 (casein kinase 1), 10 μm PF-4708671 ), 10 μm DG2 (S6K1), and 5 μm BI-D1870 . 1.5 × 106 cells were seeded in each well of 6-well plates, and individual inhibitors were added 30 min before light stimulation. Otherwise experiments were as described above. Three to five experiments were performed with each inhibitor, which also included both vehicle and non-vehicle controls. Induction of phosphorylation in the presence or absence of inhibitor was compared.Kinase inhibitors from Merck Millipore  were dissolved in DMSO. The inhibitors (targeted kinases are in parentheses) and their concentrations were: 5 μin situ in the eyecup in Stefanini's fixative overnight. The retina was then removed for flat mount immunohistochemistry and placed in cryoprotection until staining for melanopsin (see below) or the eye was cut in a cryostat  in sections of 12–14 μm and mounted on glass, frozen, and stored at −80 °C until processed for immunohistochemistry.24 Wistar male rats  weighing 150–200 g housed with free access to food and water and under regulated temperatures entrained to 12 h light:12-h dark cycle (zeitgeber (ZT) 0, lights on; ZT12, lights off) were used. 12 rats were decapitated in dim red light at ZT24 and had their eyes removed. Another 12 rats were light-exposed for 5 min (>300 lux of white light) after which they were decapitated, and their eyes were removed. Between decapitation and eye removal the rat head was placed in wet ice (0 C°), and the eyes were removed within 10 min. One eye from each animal was dissected and immersion fixated in Stefanini's fixative over night. The other eye was dissected, and the anterior chamber was removed whereby the retina was fixed SDS-PAGE and blotting were performed using NuPAGE electrophoresis and blotting systems (Invitrogen) as previously described . Melanop2  in combination with avidin-biotin-peroxidase complex (ABC)  followed by biotinylated tyramide  and streptavidin-Alexa 488 . The phospho-site-specific melanopsin antibody directed against the phosphorylated Ser in position 398 (abMel-P398) was visualized as either the melanopsin antibody abMel or when using two antibodies raised in the same species by the tyramide amplification system described above. The phospho-site-specific melanopsin antibody directed against the phosphorylated Ser in position 381 (abMel-P381) was visualized by EnVisionTM  followed by Alexa Fluor® 488 tyramide . As a control the primary antibodies were omitted, which eliminated all staining. Furthermore, no melanopsin staining was found in retinal sections from melanopsin deficient mice (OPN4−/−) (Single and double antigen immunohistochemistry/cytochemistry were performed as described in detail previously . BrieflyOPN4−/−) . The abs2+]i measurements in HEK-293 cells expressing tetracycline-inducible melanopsin (rMel-WT) or the following melanopsin mutants: rMel-S381A, rMel-S398A, and rMel-S381 + S398A using the Ca2+-sensitive dye, Rhod-2 (Invitrogen). 24 h before experiments, cells were seeded on 1.76-cm2 HCl- and EtOH-washed coverslips in 28-cm2 Petri dishes in the absence or presence of 1 μg/ml tetracycline. Cells were incubated for 1 h in the dark with retinal (10 μm) and 5 μm Rhod-2 before measurements. From the addition of retinal, the cells were exclusively visualized in red light. After incubation, coverslips were mounted in a Warner RC-26G perfusion chamber and mounted on top of the iMIC microscope system (see below). Cells were continuously perfused with 37 °C Krebs solution  using a SH27A inline heater with TC324B temperature controller. To analyze melanopsin expression, cells remaining in the Petri dish were washed 2× in ice-cold PBS and collected in 150 μl of radioimmune precipitation assay buffer  and stored at −20 °C. Harvested cells were lysed by sonication and cleared by centrifugation , and protein concentration was measured by DC assay (Bio-Rad). Melanopsin expression was analyzed by immunoblotting as described above using the non-phospho-site-specific antibody (abMel-WB). There was no significant difference in the expression levels between WT and the various mutants (data not shown). Fluorescent signals were visualized using an Ixon 885 camera  through a 40× oil immersion objective  on an iMIC2000 inverted microscope with Polychrome V monochromator . Microscope control, signal visualization (binning 2 × 2), and analysis were performed in Live Acquisition software (Till Photonics). Rhod-2 signals of >590 nm were visualized through a 49005-ET filterset  every 10 s after 50 ms of 550-nm (bandwidth 10 nm) excitation at low intensity 0.005 watt/cm2. Melanopsin was blue light stimulated using an RL5-B2545 5-mm LED  with an intensity of 0.16 watt/cm2. Cross-talk between stimulation diode and Rhod-2 excitation was very limited. Autofluorescence in non-loaded cells was subtracted from all signals, and these were further linearly corrected for signal rundown.To elucidate the possible functional significance of Ser-381 and Ser-398, we performed i measurements in HEK-293 cells expressing WT melanopsin or the mutants rMel-S381A and rMel-S398A using the Ca2+-sensitive dye, Rhod-2. Emission spectrum for light stimulus did not overlap with the excitation spectrum for Rhod-2 (data not shown). In HEK-293 cells expressing rat melanopsin, we observed a fast and robust increase in cytosolic Ca2+ when cells were light-exposed as opposed to cells without melanopsin expression in which no Ca2+ response was observed . Furthermore, the time from stimulus initiation to peak response was significantly reduced as compared with the response in WT melanopsin-expressing cells (data not shown).To investigate if mutation of Ser-381 and Ser-398 in melanopsin affects light-induced Caobserved . All cel mutants C. Furthein vitro and in vivo is phosphorylated in this position during dark and is dephosphorylated when light is turned on. We consider the finding to be a genuine dephosphorylation at Ser-381. Another interpretation is however possible, namely that a light-induced phosphorylation of the nearby Ser-379 could disrupt the antibody binding, but the Ser-379 is not conserved among either mammalian or vertebrate species. In contrast to Ser-381, melanopsin at Ser-398 was unphosphorylated in dark and became phosphorylated after light stimulation. The latter finding is in accord with a previous study in which a general anti-phosphoserine antibody was used to provide evidence that melanopsin is phosphorylated in the C-terminal tail after 10 min of light exposure (Drosophila TRPC (The predicted secondary structure of melanopsin indicates a long cytoplasmatic tail with many potential phosphorylation sites suggesting that this effector domain of melanopsin could be highly regulated, and recently a phosphorylation cassette was identified in mouse and zebrafish melanopsin . It was exposure . It is iila TRPC . It is lThe changes in phosphorylation status at both Ser-381 and Ser-398 when light was turned on were rapid and lasted throughout the 4-h experimental period. The light-induced dephosphorylation of Ser-381 seemed to be somewhat faster than the phosphorylation at Ser-398, which may pose the question of whether the phosphorylation of Ser-398 depends on prior dephosphorylation of Ser-381. We address this question by examining cells in which the individual phosphorylation sites were eliminated by mutation and clearly showed that changes in phosphorylation at Ser-381 and at Ser-398 were independent of each other.2+ response in cells treated with siRNA directed against GRKs, and double immunostaining, some evidence was provided that GRK2 could be an endogenous kinase in melanopsin phosphorylation (The activity of G protein-coupled receptors is often rapidly attenuated after activation typically accomplished by phosphorylation on the C-terminal tail by members of the GRK family of serine/threonine kinases . A possirylation . We did rylation . Using p2+, albeit the Ca2+ response was significantly reduced in cells expressing melanopsin mutated at Ser residues. Kumbalasiri (et al. previously showed that Ca2+ responses in HEK-293 made photosensitive by melanopsin expression is exclusively mediated by Ca2+ release from intracellular stores. This may not be representative of the Ca2+ signaling in ipRGCs in which Ca2+ from intracellular stores is not released during photostimulation (2+ response could be accounted for by influx via voltage-gated Ca2+ channels with the remaining 10% associated with melanopsin associated activation of TRPC7 (2+ release from intracellular stores, whereas in ipRGCs diacylglycerol accumulation activates TRPC7, which in turn depolarizes the membrane and activates voltage-gated calcium channels. Hence, store-operated Ca2+ release in melanopsin-expressing HEK-293 cells is a representative measure for melanopsin photoactivation in ipRGCs.When expressed in HEK-293 cells, all melanopsin constructs mediated robust light-induced elevations in intracellular Cabalasiri  et al. pmulation , 25. In of TRPC7 . Howeverof TRPC7  cells anof TRPC7 , leading2+ in HEK-293 cells expressing mouse melanopsin, phosphorylation of a cluster of serines and threonines in the region amino acid 386–396 was identified to control the deactivation response (2+ response. We found that both the Ser-381 and Ser-398 mutation facilitated a reduced Ca2+ response when compared with WT melanopsin-expressing cells without showing reduced melanopsin protein expression. This is in contrast to previous studies in which C-terminal phosphorylation of mouse melanopsin was correlated to reduced G protein activation (et al. (2+ signaling when investigating melanopsin truncated after amino acid 396. However, data were normalized to the peak value to investigate deactivation kinetics, and it is, therefore, not possible to see whether this truncation affected melanopsin activation (2+ response was in both cases less than in control cells, thus removing the basal phosphorylation at Ser-381, and removing light-induced phosphorylation at Ser-398 had the same effect. Furthermore, in the double mutant the effect on the Ca2+ response did not seem to be additive, which could indicate that both phosphorylation events are linked to the same mechanism of Ca2+ regulation.In a previous study examining light-induced kinetics of Caresponse . No efferesponse . Here wetivation , 15, in tivation . Certain


Spatial tuning properties of retinal ganglion cells (RGCs) are sharpened by lateral inhibition originating at both the outer and inner plexiform layers. Lateral inhibition in the retina contributes to local contrast enhancement and sharpens edges. In this study, we used dynamic clamp recordings to examine the contribution of inner plexiform inhibition, originating from spiking amacrine cells, to the spatial tuning of RGCs. This was achieved by injecting currents generated from physiologically recorded excitatory and inhibitory stimulus-evoked conductances, into different types of primate and mouse RGCs. We determined the effects of injections of size-dependent conductances in which presynaptic inhibition and/or direct inhibition onto RGCs were partly removed by blocking the activity of spiking amacrine cells. We found that inhibition originating from spiking amacrine cells onto bipolar cell terminals and onto RGCs, work together to sharpen the spatial tuning of RGCs. Furthermore, direct inhibition is crucial for preventing spike generation at stimulus offset. These results reveal how inhibitory mechanisms in the inner plexiform layer contribute to determining size tuning and provide specificity to stimulus polarity. Many of the complex processing properties of neural networks arise from the precise arrangements of excitatory and inhibitory connections. In the retina, the spatio-temporal patterns of excitation and inhibition are critical for determining response strength, centre-surround organisation, adaptation, edge detection and directional selective responses of RGCs among other functional properties1245Current knowledge about the sources of lateral inhibition derives from experimental approaches that are only able to show the contribution of the OPL and/or IPL in an indirect way for the following reasons. Firstly, bath application of pharmacological agents leads to changes in multiple targets in the retinal network, thus this method is unable to precisely isolate the sites of action and their contribution by measuring the spike output of RGCs. Secondly, mapping and quantification of excitatory and inhibitory synaptic inputs onto RGCs provide critical information about the spatial organisation of the inputs but do not report quantitatively on the impact of the inputs on spike output. Gain control is known to take place in the outer retina at the photoreceptor level79103In the primate retina recordings from parasol ganglion cells initially suggested that lateral inhibition was mediated purely via feedback mechanisms in the OPL12In the present study, we used conductances from an OFF narrow thorny RGC whose visual-evoked responses consisted of excitation and feedforward inhibition at stimulus onset and crossover excitation and inhibition at stimulus offset to determine the relative contributions of tetrodotoxin (TTX) -mediated presynaptic and direct inhibition onto RGCs to spatial tuning. First, we recorded responses to current injections composed of excitatory and inhibitory conductance waveforms derived from visually-evoked responses to stimuli of different sizes, and confirmed that these current injections recreated the classical centre-surround organisation of RGCs receptive fields. Second, we studied the responses to injections of excitatory and inhibitory conductance waveforms measured in the presence of a blocker of action potential signalling in the IPL. We found that inhibitory processes in the IPL sharpened the spatial tuning of RGCs. Inhibitory inputs onto the excitatory afferents as well as direct inhibitory input were shown to contribute to surround inhibition. Furthermore, inhibitory inputs were shown to be critical for preventing otherwise occurring spikes at stimulus offset. These results provide critical information about the extent of inner plexiform processing in shaping and controlling the retinal output.We recorded light-evoked responses from a narrow thorny RGC in the marmoset retina  in ‘loosexcctl and Ginhctl, into RGCs produced depolarising responses and action potentials. Response strength varied as a function of the spot size used to generate the conductance waveforms. Injection of conductances derived from stimulus-evoked responses generated with small spots produced relatively weak responses. Injection of conductances obtained with medium size spots produced stronger responses. Injection of conductances derived from stimulation with large spots resulted in a reduction in response strength. We recorded responses from nine marmoset RGCs, to current injection of different combinations of excitatory and inhibitory conductance waveforms measured from the above described marmoset RGC. Responses to current injections were obtained from different cell types, including midget, parasol and wide-field RGCs. Injection of control excitatory and inhibitory conductances, GThe normalised area-response function of the spike output for injection of control conductances peaked for a spot diameter of 150 μm , matching the peak amplitude of the excitatory conductance. Injection of excitatory and inhibitory conductances measured with larger spots resulted in the characteristic roll off of the area-response curve, indicating that they faithfully reproduce the lateral inhibition phenomenon . The supexcctl and inhibitory conductances recorded in the presence of TTX (Ginhttx). To gain insight into the contribution of the inhibitory activity generated in the IPL to spatial tuning, we co-injected different combinations of excitatory and inhibitory conductance waveforms recorded in control conditions and during bath application of TTX, which abolishes the activity of those amacrine cells that signal via action potentials. The role of TTX-mediated direct inhibition onto RGCs was examined by injecting Gexcttx) and Ginhctl. To determine the impact of the modulation of bipolar cell output by presynaptic inhibition arising from spiking amacrine cells on RGC output, we injected currents composed of excitatory conductances obtained in the presence of TTX . The change in response strength as a function of stimulus size is shown in excttx of the smallest spot size there was a reduction. This clearly provides evidence that wide field spiking amacrine cells spatially modulate excitatory inputs and provide direct inhibitory input, thus, contributing to sharpen the spatial tuning of RGCs. The change in surround inhibition was quantified with SI, which was significantly reduced in all test conditions  as shown in The effects of removing TTX-mediated presynaptic and direct inhibition on spatial tuning are illustrated in the area-response functions for each condition normalised to the maximum spike response in each condition  and quanDynamic clamp experiments injecting currents generated with visually-evoked conductances recorded from a marmoset RGC into marmoset RGCs showed proof of the principle that the use of size tuned conductances mimics the spatial properties of RGC receptive fields. We also carried out similar current injection experiments in ganglion cells from the mouse retina to establish whether or not the response properties of mouse RGCs to different combinations of current injections showed behaviour and spatial tuning properties similar to those described above for marmoset RGCs. If established, this would provide an exciting opportunity to carry out many more experimental manipulations due to easier accessibility to mouse tissue.et al.’s classificationet al. are shown in Given that different types of RGCs display distinct morphological, functional and intrinsic properties1617In the following analyses we combined measurements from all RGC types, as their responses to current injections followed a similar pattern in terms of their degree of surround inhibition. The effect of abolishing the activity of spiking amacrine cells was quantified in the area-response function of responses to injection of different combinations of conductances. The observed changes in RGC output upon removal of TTX-mediated inner plexiform inhibition were clearly shown to depend on the relative strength of excitatory and inhibitory inputs. The underlying mechanisms that lead to increased responses in each of the conditions tested, however, are not necessarily the same: integration of these inputs might lead to an increase in the amplitude of the EPSPs, in the rate of rise of the EPSP or in both. To determine how the removal of TTX-mediated presynaptic inhibition and direct inhibitory input affect RGC output, we quantified the effects of injections of different combinations of conductances on the amplitude and rate of rise of the EPSPs. In the following we present only data obtained from mouse RGCs, although analyses of marmoset RGC recordings yielded similar results for responses to injection of conductances of large size (data not shown). Abolishing TTX-mediated direct inhibitory inputs did not modify the amplitude of EPSPs for conductances of optimal stimulus sizes and produced a small but significant  increase in the amplitude of EPSPs for conductances of large stimulus sizes, as seen in Injection of excitatory conductances measured during bath application of TTX together with control inhibitory conductances produced no change in the amplitude of the EPSPs for conductances measured with optimal size spots but caused a significant increase in the EPSP amplitude for conductances measured with large size spots  as shown in excttx + Ginhttx is not consistent with the lack of change at the spike level in this condition.Removal of TTX mediated inhibition onto bipolar cells (excitatory conductance) and directly on RGCs (inhibitory conductance) resulted in no change in the amplitude of responses evoked by injection of conductances measured with optimal spot size. For conductances recorded with spots of larger sizes, however, it led to significant increases in response amplitude  produced small amplitude EPSPs but did not lead to spike responses . When thWe quantified the effects of removing TTX-mediated inner plexiform inhibition onto bipolar cells and directly onto RGCs at non-preferred contrast stimulus on the output of RGCs. Removal of TTX-mediated inhibition on the excitatory input did not produce any significant change in spike output, as shown in the area-response functions for the injection of different combinations of conductances in marmoset  and mousIn this study we showed that inhibition in the inner plexiform layer, when present, can strongly contribute to the generation of lateral inhibition. We specifically tested the impact of inhibition originating from spiking amacrine cells, which modulate bipolar cell output and provide inhibition to ganglion cells, on the spatial tuning of RGCs. Our results demonstrate that these two loci of inhibition can independently alter the spatial tuning properties of RGCs and when acting in concert, can further enhance lateral inhibition. We also showed that crossover inhibition in RGCs with crossover excitation at the offset of preferred contrast stimulation, prevents firing in response to an otherwise non-preferred stimulus contrast.Most previous studies have examined the sources of surround inhibition either by characterising the area-response function of the spike output and/or of the excitatory and inhibitory synaptic inputs onto RGCs before and after targeting synaptic transmission in the outer or inner plexiform layer with pharmacological agents312exc by TTX for this spot size. Narrow thorny RGCs stratifying in the OFF sublamina of the marmoset retina are potential targets of DB3a bipolar cellsexc for small spot sizes by suppressing bipolar cell signalling whilst it increases Gexc for larger stimuli by blocking wide field, spiking amacrine cells that inhibit bipolar cells. TTX effects on the injection of conductances elicited with larger sizes, had a significant impact for all conditions tested, namely when TTX-mediated inhibition was removed from excitatory inputs and from direct inhibitory input. As expected, an even stronger impact was observed when TTX-mediated inhibition was removed from both conductances. The mechanisms by which TTX-mediated inhibition contribute to surround inhibition seem to differ when acting via inhibition of the excitatory input and via direct inhibition.The effects of TTX on the injection of conductances obtained with optimal size stimuli had negligible impact on RGC output. Interestingly, injection of currents generated with excitatory conductance measured for the smallest spot (60 μm) under TTX produced a reduction in spike response. This can be explained due to the 28% reduction in peak amplitude of GInjections of currents, generated with stimulus-evoked conductances recorded from an OFF marmoset RGC with feedforward inhibition, into different types of marmoset RGCs recreated their classical receptive field organisation with centre-surround antagonism. Interestingly, the same current injections into mouse RGCs also produced typical area-response functions with similar degrees of surround inhibition regardless of the cell type. This suggests that for cells with synaptic inputs with transient temporal properties, like those used in our study, the intrinsic properties of RGCs do not seem to be a critical factor in determining the receptive field properties. A different outcome could be expected if synaptic conductances with more sustained kinetics  were used in cells with a complement of membrane channels that can only account for transient responses. In this case, interpretation of the area-response function might be ambiguous due to the additional constraints of the response attributable to the intrinsic properties.The conductances used in this study were recorded from an RGC whose synaptic inputs were spatially modulated by amacrine cells in the IPL, as shown by their sensitivity to TTX. This opens the question about how general these findings are. Several lines of evidence show that most bipolar cells show spatial tuning due to inner plexiform inhibition31531527Amacrine cell mediated inhibition from the receptive field surround is likely to modify RGC output via multiple mechanisms. Removal of TTX-mediated direct inhibition speeds up the rate of rise of postsynaptic potentials and produces a moderate yet significant increase in their amplitude. A quicker rate of rise of EPSPs is likely due to the lack of fast action potential-dependent inhibition, given that TTX produces a delay of approximately 20 ms in the onset of the inhibitory conductance. This suggests that direct inhibition most likely reduces excitability by slowing down the rising time of the postsynaptic depolarisation in addition to reducing its amplitude. A slower rising phase might also lead to stronger inactivation of sodium channels, which combined with a reduced EPSP amplitude leads to a reduction in spiking output, as we observed. When presynaptic and direct inhibition were removed, both the amplitude and rate of rise of the EPSPs were strongly enhanced. The stronger increase in EPSP amplitude indicates that presynaptic inhibition acts to reduce the output of bipolar cells whilst direct inhibition acts to slow down the EPSP.et al.’s results and provides further information about the function of the size dependent magnitude of inhibition on receptive field organisation. Moreover, our study provides additional information about the functional impact of presynaptic inner retinal inhibition on the spatial organisation of RGC receptive fields.The impact of inhibitory inputs in primate ON midget and parasol RGC spiking output has also been studied with a focus on the temporal properties of the responses using a dynamic clamp approach141529In the visual system, lateral inhibition occurs first in the retina and it is later found in downstream areas such as the lateral geniculate nucleus3234Crossover inhibition between ON and OFF signals has been shown in different ganglion cell types using GABA blockers that target GABA receptors in bipolar cells and/or in RGCs2021381425et al. (2013) in which properties of the excitatory and inhibitory synaptic conductances can be artificially manipulated, such as their relative magnitude and/or time course, to gain further understanding of the characteristics of the synaptic inputs critical for the generation of RGC responses.Overall, our studies provide direct evidence of the contribution of inner plexiform inhibition originating from spiking amacrine cells to the receptive field organisation and response selectivity of RGCs. In addition, they demonstrate how dynamic clamp experiments allow the dissection of the contribution of different conductances to RGC output. They also provide support for the design of experiments such as those in Cafaro Callithrix jacchus) obtained at the end of electrophysiological experiments unrelated to this study, generously donated by Dr. Sam G. Solomon . Eyes were removed from the animal at the time of euthanasia under deep anaesthesia and under dim red light. The cornea, lens and vitreous were removed and the eyecup containing the sclera and retina were transferred to a dish containing carboxygenated Ames medium. A small piece of retina was isolated using a dissection microscope under white light. The piece of retina was then mounted in a recording chamber with the ganglion cells layer facing upwards. The tissue in the chamber was transferred and mounted on an upright Olympus microscope (BW50WI). The tissue was continuously perfused with carboxygenated Ames medium at a flow rate of 3–5 ml/min at 35 °C using an inline heater . Recordings from the ganglion cell somas, visualised through the microscope under 40x magnification using infrared illumination, were made under low ambient light conditions.Recordings were made from retinae of nine adult marmosets  and euthanased by cervical dislocation. Eyes were rapidly enucleated and transferred to a dish containing Ames medium where the cornea and anterior part of the eye were removed and the retinae were isolated. The remainder of the dissection procedure is similar to that described above.All animal procedures followed the guidelines for animal experiments issued by The University of Sydney and the Australian NHMRC Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Ethics Committee of The University of Sydney.+-gluconate: 140, MgCl2:4.6, EGTA: 10, HEPES: 10, ATP-Na+: 4 and GTP-Na+: 0.4. Recordings were done initially in voltage clamp mode using an EPC8 patch clamp amplifier . Only cells that displayed large sodium currents (>2 nA) were recorded from and final confirmation of their identity was the presence of a long axon in the fluorescent and/or confocal images. Recording mode was then switched to fast current clamp mode and cells were held at -65 to -70 mV  to carry out dynamic clamp recordings.Whole-cell recordings were obtained from RGCs as previously describedet al.Conductance waveforms were obtained from voltage clamp recordings from a narrow thorny OFF-RGC in the marmoset retina see . ExcitatThe spatial profile of the excitatory conductance recorded at stimulus onset in control conditions displayed surround inhibition, with a peak response for a stimulus diameter of 150 μm, which decreased as stimulus diameter was increased. The suppression index  of the excitatory conductance was 61%, indicating that surround stimulation produced a strong inhibitory effect on the centre response. The inhibitory conductance measured at stimulus onset increased with stimulus size up to 300 μm and then reached saturation. SI for the inhibitory conductance in control conditions was 8%, indicating that inhibitory inputs mostly displayed spatial summation and weak surround suppression. Bath application of TTX modified the amplitude and spatial profile of both excitatory and inhibitory stimulus-evoked conductances. The peak amplitude of excitation was reduced by 18% and the SI of the excitatory conductance during bath application of TTX was 31%. A smaller SI in this case indicates that TTX reduced inhibitory inputs onto bipolar cell terminals elicited by surround stimulation. Peak amplitude of inhibitory conductance was reduced by 46% and the SI of the inhibitory conductance was 42%. The increase in SI of the inhibitory conductance indicates that TTX removed direct inhibitory inputs onto RGCs triggered by surround stimulation.Changes in excitatory and inhibitory conductances were also present at the offset of stimulus presentation, namely this cell displayed crossover excitation and inhibition. In control conditions excitatory and inhibitory conductances at stimulus offset were spatially tuned and showed a peak magnitude for stimuli of 300 μm and 150 μm respectively. The magnitude of the inhibitory conductance was 3.6 fold larger than that of the excitatory conductance at optimal size and on average 3.5 fold larger for all stimulus sizes. The SI of the excitatory and inhibitory conductances at stimulus offset was 59% and 57% respectively. TTX also modified the strength and spatial tuning of visually-evoked excitatory and inhibitory conductances at stimulus offset: their magnitudes were reduced by 26% and 50% respectively. The SI of the excitatory conductance was reduced to 9% and the inhibitory conductance had a SI of -48%, indicating that the magnitude of the inhibitory conductance elicited by a large spot was larger than that elicited with a spot of 300 μm.et al.A set up was used where a hybrid ganglion cell-computer circuit was created. Dynamic clamp was executed via a field-programmable gate arrays board  using a custom-written program in Labview as described in detail in Huang t; Gexc is the excitatory conductance at time t, Ginh is the inhibitory conductance at time t, Vm is the membrane potential at time t, Vexc is the reversal potential of excitatory currents and Vinh is the reversal potential of inhibitory currents.where I is the total current injected at time The magnitudes of the excitatory and inhibitory synaptic conductances were scaled by a common factor but their ratio was kept constant in order to evoke responses consistent with those evoked by visual stimulation. A small constant current (<- 200 pA) was injected to keep resting potential between −65 and −70 mV if required.At the end of each experiment cells were visualised with fluorescent light for initial morphological identification. Subsequently, tissue was fixed with paraformaldehyde (4% in 0.1 M phosphate buffer) and processed for immunocytochemical staining of Lucifer Yellow. Fixed retinae were incubated in primary anti-Lucifer Yellow rabbit IgG  for five days, then in secondary goat anti-rabbit IgG  overnight. Retinae were then wet mounted with Fluorescent Preserving Media, coverslipped, and sealed with nail polish. Ganglion cells were then visualised under a Leica Spec-II confocal microscope and morphologically reconstructed see a and 3a.et al.Data analysis was carried out using custom written routines in IGOR  and MATLAB . Spikes were quantified by detecting the location of maxima calculated from the smooth first and second derivatives of the membrane potential signal and comparing it to a threshold. If the amplitude of that point was larger than threshold , it was considered to be a spike. Spontaneous activity (0.9 ± 0.3 Hz) was observed in 14 out of the 33 RGCs recorded from and, when present, was subtracted from the stimulus-evoked responses. The amplitude of the membrane potential depolarisation  was calculated after removing spikes by linear interpolation of the membrane potential signal 4 ms before each spike and ~10 ms after each spike (as in Demb A suppression index (SI) was calculated to estimate the degree of inhibition on the peak response, achieved by injecting currents composed of conductances measured with the largest spot:Max is the response elicited with current injection based on conductances measured with the largest spot and RPeak is the response elicited with current injection based on conductances that produce the peak response . An index of 100 indicates that injection of currents based on conductances measured with large spots abolished response completely and an index of 0 indicates that they did not reduce response.where RA non-parametric Mann-Whitney U-test was used to assess significant differences between two sets of data for individual cells. Wilcoxon matched-pairs signed-rank test (Wilcoxon test) was used for pair-comparisons of populations of cells in different conditions. A two-tailed criterion was used for these tests and significance was taken when P ≤ 0.05. Statistical analyses were made using MATLAB . Values in the text represent mean ± SD (standard deviation), whereas data and error bars on plots represent mean ± SEM (standard error of the mean). Comparison of these responses allowed us to establish the relationship between the integrated input that the cell receives and its output response, providing critical information about the functional role of inhibition in spatial tuning and gain control.How to cite this article: Huang, J. Y. and Protti, D. A. The impact of inhibitory mechanisms in the inner retina on spatial tuning of RGCs. Sci. Rep.6, 21966; doi: 10.1038/srep21966 (2016).


Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis.Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium  Bacillus subtilis  could be regulated by complex signaling cascades, similar to the ones found so far only in eukarya. This study constitutes the most comprehensive view of a bacterial signaling network based on protein phosphorylation to date.To address these crucial questions on the physiological relationships of the kinases, their substrates and alternative kinase modulators, we set out to construct a high confidence protein-protein interaction network centered on known BY-kinases, activators and phosphatases in B. subtilis 168 genomic DNA as template and primers as listed in Table minD mutant, a PCR-amplified internal fragment of the minD gene was inserted into pMUTIN2  to get the 6xHis-tag fusion proteins. Strep-tagged versions of proteins were obtained from a pQE-30 vector with His6-tag replaced by the strep-tag  plates supplemented with 0.5 mM 3-aminotriazole. After 7–10 days at 30°C, the His+ colonies were transferred on SC-LUA  plates for 3 days at 30°C. All the His+ Ade+ colonies were subjected to PCR amplification targeting AD prey-inserts were sequenced and compared with the B. subtilis genome.The bait vectors were used to screen the pGAD-expressed http://www.imexconsortium.org)  auto-activation of the reporter genes independently to the ability of the targeted protein to bind to a protein partner, (ii) proteins with stickiness properties and (iii) modification of the basal reporter gene expression in the yeast diploid cell. This last category mostly gives non reproducible growth phenotypes. False positive were eliminated experimentally by rescuing the protein-encoding prey plasmids from the His+ Ade+ colonies and reintroducing in PJ69–4α strain in order to perform binary interaction test assays. The rescue of the prey plasmid from diploid cells was carried out using a “gap-repair” cloning procedure as described elsewhere  were used for vector construction and protein purification, respectively. Cells were routinely grown in LB medium supplemented with appropriated antibiotics. Protein synthesis and purifications were carried out as described previously . Cells were harvested 3 h later and sonicated. From crude extracts, the 6xHis- or Strep-tagged proteins were purified using Ni-NTA (Qiagen), or Strep-Tactin affinity chromatography (Novagen), prior to desalting by PD-10 columns . Protein purity was estimated by scanning densitometry of coomassie stained SDS-Page gels (Table S5).Proteins were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes. Proteins were then renatured  containing 50 μM ATP and 20 μCi/mmol [γ−32P]-ATP, for 1 h, prior to migration on an 8–12% SDS-polyacrylamide gel. The radioactive signal was captured by a FUJI phosphoimager  and RecA (5 μM) in the absence or presence of TkmA (2.5 μM). Phosphorylation and dephosphorylation of RecA by YabT and SpoIIE, was performed by incubating RecA (1 μM) with YabT (0.5 μM). Then SpoIIE (5 μM) was added to the reaction. Phosphorylation and dephosphorylation of RacA by YabT and SpoIIE was performed by mixing RacA (0.8 μM) with YabT (0.15 μM) at 37°C for 1 h. Centricon was used to remove ATP from the sample, prior to addition of SpoIIE (2 μM) and DnaK (0.8 μM). Phosphorylation of DnaC by PrkD was performed by mixing 1 μM of DnaC/I stable co-purified complex with 0.1 μM PrkD. Only DnaC (50.6 kD vs. 30.1 kD for DnaI) was found to be phosphorylated by PrkD. MinD potential kinase activity was assayed by mixing equimolar concentrations of TkmA, PtkA K59M, and MinD (2 μM). In vitro phosphorylation of DivIVA was assayed by mixing equimolar concentrations of PtkA and TkmA (2 μM) with DivIVA (3.5 μM). MinD-mediated activation of PtkA was tested by incubating PtkA (2 μM) with 0.5, 1.0, 2.0, 4.0, and 8.0 μM of MinD. All the in vitro phosphorylation assays were reproduced at least three time.Proteins were incubated at 37°C in a reacting buffer . In this case PtkA-dependent phosphorylation stimulated DNA-binding of SalA, which in turn repressed the transition state regulator scoC (Derouiche A. and Mijakovic I. unpublished result). Furthermore, we found that TkmA interacted with the N-terminal domain of the general DNA recombinase RecA  and dnaB exhibit a high pairwise correlation throughout ~100 different physiological conditions . We first validated the interaction between MinD and PtkA by far Western blotting , compare to the wild type cells (<1%)  as well as cells containing asymmetric septa (3%), sometimes leading to anucleated cells (Figures Transcription profiles of s Figure . A close Figures . We inve Figures . These ominD also abolished the polar localization of PtkA foci, indicating it is MinD-dependant. This result provides in vivo validation of the MinD/PtkA interaction and hints at a role of MinD in holding PtkA at the cell pole during cytokinesis.To further understand the role of MinD in PtkA activity, we examined their subcellular localization. MinD is known to exhibit a very specific localization pattern at the cell poles as well as at mid cell. When fused to CFP, PtkA was found to localize as a single focus, preferentially at the old cell poles of elongating cells Figure . The absB. subtilis. Combined with a systematic and experimental elimination of false positives, our approach has proved effective for identifying numerous substrates of BY-kinases and phosphatases, as well as new proteins that modulate their activity. This PPi network not only provided a kinase context for already identified phosphoproteins, but also identified new protein partners for kinases, including other kinases and phosphatases. This network can be considered of high biological relevance, with numerous proteins being contacted by both a kinase and cognate phosphatase, strongly suggesting they are true substrates or they belong to the same regulatory assembly. Our examination of kinase/substrates and kinase/modulators from the tyrosine but also the serine/threonine phosphorylation pathways corroborated these assumptions. This study highlights the role of protein phosphorylation in the regulation of various aspects of the B. subtilis cell cycle as DNA replication, transcription, cell division and sporulation.A hallmark of BY-kinases from Gram-positive bacteria is that the cytosolic kinase and its transmembrane activator are individual proteins encoded by separate adjacent genes. This differs from their Gram-negative counterparts where the two activities are encoded by the same gene. A functional consequence of this particular architectural feature could be that it enables the kinases from Gram-positive bacteria to interact with alternative activators, which may provide different substrate specificities. We presented here a protein-protein interaction landscape of the tyrosine phosphorylation network of B. subtilis, the best characterized example is the two-component histidine kinase DegS, which was shown to be phosphorylated by the two Ser/Thr kinases PrkD and YabT in vitro (Jers et al., In vivo, DegS phosphorylation at serine 76 was shown to affect various aspects of cellular behavior such as competence, swarming and motility. Our B. subtilis interaction network also revealed that other proteins can be phosphorylated by more than one kinase (Table B. subtilis cells, where multiple and sequential phosphorylation events could mediate integration of several input signals to better adjust the cellular response to environmental changes, similar to what is described in eukarya.Common signaling mechanisms often involve multisite protein phosphorylation to regulate the various activities of a protein whether carried out by the same kinase or by the sequential action of several kinases. In se Table . A notabse Table . Here, ose Table . WhetherB. subtilis BY- and Ser/Thr kinases to phosphorylate each other in vitro was explored. This assay revealed many trans-phosphorylations at functionally critical residues indicative of kinase regulation by phosphorylation (Shi et al., M. tuberculosis also provided strong evidence for a hierarchical and spatially organized crosstalk among Ser/Thr kinases in this bacteria (Baer et al., in vivo as a kinase remains to be determined. An alternative hypothesis could be that PtkB acts as a pseudokinase to mediate interaction with protein substrates for subsequent tyrosine modification by PtkA. Our data also hints at a potential a role of modulators in recruiting some substrates. In agreement with this notion is the finding that the transcriptional regulator FatR, which interacts with TkmA, is regulated by PtkA (Derouiche et al., Remarkably, our network reveals important crosstalk among different classes of kinases. Interactions were observed between components of the Tyrosine and Ser/Thr phosphorylation pathways, as well as among Ser/Thr-kinases. These connections strongly suggest that kinases might phosphorylate each other, generating signaling cascades similar to what was observed in eukarya. This hypothesis has been explored in a separate study, in which the ability of all soluble in vitro, as well as in its in vivo localization as a focus at the cell poles. It has been previously reported that PtkA co-localizes with its transmembrane adapter TkmA at the cell membrane in exponentially growing cells (Jers et al., ptkA and tkmA genes were simultaneously overexpressed which may account for this discrepancy. In our present study the localization was detected with wild type expression levels from the ptkA natural promoter, without overexpression of tkmA. Based on these results, we propose that MinD could act as a platform protein that would tether PtkA at a specific cellular area. Additionally MinD would also act as an activator of PtkA for signal transmission to DivIVA. The biological significance of DivIVA phosphorylation by PtkA during cystokinesis in B. subtilis remains to be investigated, but our data clearly points to a role of PtkA in the regulation of cytokinesis. This is in agreement with the cell cycle phenotype of the ptkA mutant which was reported previously (Petranovic et al., A remarkable feature of this PPi network is the central position of the cell division regulator protein MinD which appears as a hub protein densely connected with many members of the tyrosine phosphorylation pathways. MinD shares sequence and structural homology with BY-kinases, but lacks crucial tyrosine residues involved in phosphoryltransfer activity (Mijakovic et al., Streptomyces coelicolor, the polarization machinery is regulated by AfsK, a Ser/Thr protein kinase which localizes at hyphal tips and phosphorylates DivIVA on its C-terminus (Hempel et al., S. pneumoniae, StkP localizes at the division site and specifically phosphorylates DivIVA on threonine 201 (Fleurie et al., Mycoplasma pneumoniae, phosphorylation of the DivIVA homolog Wag31 by S/T kinases PknA/PknB affects cell shape control (Kang et al., B. subtilis, this regulation may involve the phosphorylation of DivIVA on a tyrosine residue.In several bacteria, the phosphorylation of DivIVA was shown to be a key step in the regulation of cytokinesis. So far, this regulation is known to be mediated by Ser/Thr Hank-type kinases. In In conclusion, this study shows that bacterial regulatory network based on protein phosphorylation is considerably more complex and inter-connected than previously believed. The BY-kinase sub-network is inextricably connected to the Ser/Thr-kinase sub-network. Together with the separate study (Shi et al., The Associate Editor, Christophe Grangeasse, declares that, despite having collaborated with authors Ivan Mijakovic and Lei Shi, the review process was handled objectively and no conflict of interest exists. 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.


We assessed rising CO2 effects on metabolism and development of a nesting wrasse by reciprocal transplant experiments in the field. Offspring brooded under different CO2 conditions exhibited similar responses. However, embryos from High-CO2 site were resilient to a wider range of pCO2 levels than those belonging to current-day conditions. 2 seeps provide opportunities to investigate the effects of ocean acidification on organisms in the wild. To understand the influence of increasing CO2 concentrations on the metabolic rate (oxygen consumption) and the development of ocellated wrasse early life stages, we ran two field experiments, collecting embryos from nesting sites with different partial pressures of CO2  and reciprocally transplanting embryos from ambient- to high-CO2 sites for 30 h. Ocellated wrasse offspring brooded in different CO2 conditions had similar responses, but after transplanting portions of nests to the high-CO2 site, embryos from parents that spawned in ambient conditions had higher metabolic rates. Although metabolic phenotypic plasticity may show a positive response to high CO2, it often comes at a cost, in this case as a smaller size at hatching. This can have adverse effects because smaller larvae often exhibit a lower survival in the wild. However, the adverse effects of increased CO2 on metabolism and development did not occur when embryos from the high-CO2 nesting site were exposed to ambient conditions, suggesting that offspring from the high-CO2 nesting site could be resilient to a wider range of pCO2 values than those belonging to the site with present-day pCO2 levels. Our study identifies a crucial need to increase the number of studies dealing with these processes under global change trajectories and to expand these to naturally high-CO2 environments, in order to assess further the adaptive plasticity mechanism that encompasses non-genetic inheritance (epigenetics) through parental exposure and other downstream consequences, such as survival of larvae.Volcanic CO This species was chosen because it was previously observed spawning along the CO2 gradient and its eggs are laid on benthic nests. In the first experiment, we used a factorial design to assess the oxygen consumption of three embryonic stages  reared in nesting sites in present-day ambient conditions  and end-of-century high-CO2 conditions . Embryos were also transplanted from the high-CO2 nesting site to the ambient site and vice versa  or replaced into the original nesting site (HH and AA) to control the translocation effect, and then exposed for 30 h before testing. This enabled us to assess the effects of elevated CO2 on egg size and oxygen consumption of the S. ocellatus embryos and their responses when developed in different CO2 conditions from the original nesting site. In the second experiment, using the same factorial design, we assessed how different pCO2 levels may affect the yolk consumption and length of newly hatched larvae.Here, we performed two experiments in a natural COSymphodus ocellatus is a Mediterranean wrasse widespread in shallow rocky areas. This species is a benthic spawner, and the females lay eggs in nests built by territorial males . Other gases potentially toxic for cell respiration (e.g. H2S) are found at the main seeping site but do not extend to the nesting sites we considered in the present study, which are located at a distance of >400 m from the main degassing area (pCO2 and pH levels were identified for the experiments: ambient CO2 (A) in present-day conditions  and high CO2 (H), mimicking projected end-of-century CO2 conditions  in the Baia di Levante of Vulcano Island . In this area, a submersed CO2 conditions, we identified 10 nests where the dominant nesting male was in the spawning phase, and we collected portions of the nests for subsequent 30 h exposure. Specifically, fragments of nests with eggs were moved from the high-CO2 nesting site to the ambient-CO2 site and vice versa  or replaced into the original nesting site  to control the translocation effect. Translocations were performed by placing the portions of each nest in 100 ml plastic containers with four sides opened and covered with a mesh (0.1 mm) to ensure water flow-through and oxygenation of the embryos. The containers were fixed to the bottom for 30 h at the same depth as the nests of provenience (3–4 m). After the 30 h exposure, eggs from the A and H nesting sites were also collected from eight additional nests.In each nesting site exposed to different COEmbryos belonging from the six different treatments  were classified on the basis of their development stages, as follows: the first stage  included embryos at the end of epiboly phase (∼6–8 h after fertilization); the second stage (middle) included embryos from the end of the epiboly phase to the formation of the last somite (∼10–30 h after fertilization); and the last stage (late) started with the exhibition of body and eye pigmentation and progressed until the pre-hatching phase (∼60–80 h after fertilization). As expected, only embryos belonging to the middle and late stage of development were found in the AH, AA, HA and HH treatments after the 30 h exposure.2. As the difference in size of eggs could influence the amount and the capacity of gas exchange of the embryos tested for the respirometry, we standardized the oxygen consumption to the average egg surface calculated for each replicate (10 embryos) and thus reported oxygen consumption in micromoles per hour per square millimetre.The oxygen consumption rate (OCR) was measured for groups of 10 embryos from each treatment and developmental stage. To measure the OCR, we used the electrode microrespiration System, MRS-8 , provided with eight microrespiration glass chambers containing ∼0.5 ml . When th2 conditions may affect the yolk consumption (i.e. yolk area at hatching) and the length of larvae at hatching. We used the same experimental design and the same methods as in the first experiment. Portions of nests were collected from A and H nesting sites (five nests per site), and portions of five nests each containing some hundreds (appoximately 500–700) of embryos were exposed to each of the translocation treatments . In this case, we chose to focus collections on nests in the last stage of the nesting cycle  to be sure to obtain enough hatchlings for subsequent measurements. After the 30 h exposure, the nest portions were collected from each CO2 treatment and transferred to a laboratory facility close to the field site, where they were placed in six different rearing aquaria (10 l each) and maintained in aerated seawater from the site of origin. The temperature was monitored using a 556 MPS YSI  multiprobe.In June 2013, a second experiment was performed to evaluate whether exposure to different CO2 and 0.01 mm, respectively, using the digital photographs and the image analysis software ImageJ.Hatching is synchronous in this species and occurs at dusk, probably to avoid predation . HatchliT) were measured in situ for 3 days consecutively in early and late May 2012, using a 556 MPS YSI multiprobe positioned at 2 m depth and previously calibrated using Tris–HCl and 2-aminopyridine–HCl buffer solutions (n = 6 for each nesting site). At both sites, 100 ml water samples (n = 3) were also collected, passed through a Whatman GF/F, poisoned with 0.05 ml of 50% HgCl2  and stored in the dark at 4°C for subsequent analyses of total alkalinity (TA). Three replicate 20 ml sub-samples were analysed at 25°C using a titration system composed of a pH meter with a Methrom pH electrode and a 1 ml automatic burette (Methrom). The pH was measured at 0.02 ml increments of 0.1 N HCl. Total alkalinity (in micromoles per kilogram) was calculated from the Gran function applied to pH from 4.2 to 3.0, as milliequivalents per litre from the slope of the curve for pH vs. HCl volume. Results were corrected against TA standards provided by A.G. Dickson (batch 99 and 102). For the first experiments, parameters of the carbonate system  were calculated from pHT, average TA, temperature and salinity using the free-access CO2SYS package  was continuously measured by deploying a HidroCtmCO2 II sensor for dissolved CO2  at 2 m depth at each nesting site for 20 h, whilst salinity and seawater temperature (in degrees Celsius) were monitored on several visits using a 556 MPS YSI.For the second experiment, pCO2 measured during the second experiment are presented as The seawater carbonate chemistry of the first experiment and the pCO2 levels on eggs laid at different nesting sites, or ‘period’ as a fixed factor with two levels (early May and late May) to assess potential differences across the breeding season.Potential differences in egg size, OCR, yolk area and length at hatching were tested by permutational univariate analysis of variance  for the initial developmental stage, five levels for the middle developmental stage  and with six levels  for the late developmental stage. Differences in length at hatching were determined using ‘treatment’ as a fixed factor with six levels . Data for each experimental trials were not transformed, and a triangular matrix based on Euclidean distance was calculated for each data set. One-way and two-way PERMANOVAs were run using 9999 unrestricted permutations of the raw data. Pairwise t-test comparisons of significant terms in PERMANOVAs were used to assess differences between levels. A linear regression analysis was also run to assess the relationships between the three development stages of S. ocellatus embryos from ambient- and high-CO2 nesting sites and their oxygen consumption (in micromoles per hour per square millimetre).Differences in the overall oxygen consumption of embryos among the three developmental stages were then determined by a two-factor design, with ‘embryonic stage’ as a fixed factor with three levels  and ‘nesting site’ as a fixed orthogonal factor with two levels (H and A). Within each embryonic stage, differences in OCR were tested using ‘CO2; H, 0.408 ± 0.003 mm2; PERMANOVA, pseudo-F1,174 = 0.55209, P = 0.4476; Fig. 2 sites  for 30 h exposure  and the smallest in the H treatment  living at a temperate CO2 seep are tolerant to CO2 concentrations expected by the end of this century. Specifically, the oxygen consumption of embryos and their size at hatching were similar between ambient- and high-CO2 nesting sites. However, we found an altered respiration rate in embryos collected from an ambient-CO2 and moved to a high-CO2 nesting site for a 30 h exposure, implying that embryos from ambient-CO2 conditions could be less able to cope with higher pCO2 levels. Likewise, hatchlings showed an impaired development in these conditions. On the contrary, embryos belonging to the high-CO2 nesting site showed no such response when translocated to the ambient-CO2 treatment, therefore suggesting that offspring from high-CO2 nesting sites could be resilient to a wider range of pCO2 values than those belonging to the site with present-day pCO2 levels.We show that embryos of the ocellated wrasse  reared in aquaria at pCO2 levels comparable with those considered in the present study  can compensate for the effects of high CO2 on embryonic metabolic performance by conditioning the offspring to specific CO2 levels through epigenetic transgenerational plasticity, allowing offspring to develop efficient physiological pathways for the high-CO2 environment  led to an increase of OCR, which was more evident during the late developmental stage (with embryos showing an average 33.8% increase in OCR relative to embryos exposed to other treatments). As oxygen consumption may be considered a proxy of energetic demand for basal maintenance and development, our results could suggest that embryos from parents living in ambient-CO2 conditions increase their oxygen requirement when they are exposed to high-CO2 conditions in order to support the increased acid–base regulatory activity  were significantly smaller than larvae hatched from the control treatment, suggesting an additional energetic cost to cope with altered pCO2 levels  through parental exposure.In conclusion, our study reveals that This work was supported by FFR-A (2012) from the University of Palermo to M.M. and C.C., and contributes to the COST action ‘Conservation Physiology of Marine Fishes’ (FA1004).Supplementary DataClick here for additional data file.


Descurainia sophia seeds have a variety of pharmacological functions and been widely used in traditional folk medicine. However, their effects on human drug metabolizing enzyme (DME) activities have not been elucidated. The present study investigated the inhibitory effects of an ethanol extract of D. sophia seeds (EEDS) on human Phase I/II (DMEs) and P-glycoprotein (p-gp) in vitro.i) value of EEDS were graphically determined with Lineweaver-Burk double reciprocal plots and secondary plots, respectively.The enzyme activities of human Phase I , Phase II  DMEs, and the drug transporter P-gp were determined in the presence of various concentrations of EEDS using commercially available luminogenic assay systems. The mode of enzyme inhibition and the inhibitory constant (K50) of 47.3, 25.8, and 38.7 μg/mL, respectively. Graphical analyses with Lineweaver-Burk double reciprocal plots and secondary plots indicated that EEDS competitively inhibited CYP2C9 with a Ki value of 19.8 μg/mL; however, it inhibited CYP2C9 and CYP2C19 in a mixed mode with Ki values of 5.2, and 11.9 μg/mL, respectively. Other Phase I  and Phase II (UGT1A1 and UGT2B7) enzymes as well as P-gp were weakly or negligibly affected by EEDS with concentrations up to 500 μg/mL.The enzyme activity assays showed that EEDS moderately inhibited the CYP1A2, CYP2C9, and CYP2C19 isoforms with half maximal inhibitory concentrations (ICEEDS is a selective inhibitor of CYP1A2, CYP2C9, and CYP2C19 with moderate enzymatic inhibition. Clinically, full consideration should be given to a potential toxic adverse effect from a herb-drug interaction when drugs that are particularly susceptible to CYP1A2, CYP2C9, or CYP2C19-mediated metabolism are taken together with EEDS. Characterization of metabolic profiles of specific herbal drugs could help consumers and medical specialists to use them safely as a complementary and alternative medicine. In the past few decades, complementary and alternative medicine (CAM) has been combined with conventional medicine to treat diverse diseases. Approximately 38 % of American adults (83 million) are using some form of CAM for their healthcare, and their expenditures on CAM reached $34 billion in 2007 . In East50 of warfarin in wild-type vitamin K epoxide reductase complex subunit 1 (VKORC1) in patients [in vitro [max) of co-administered kawain in vivo [Herbal medicines comprise an important part of CAM and are used worldwide. Consumers of herbal medicines consider them safe and sometimes forget the fact that herbal extracts contain many phytochemicals and that they can act as substrates, inhibitors, or inducers modulating human metabolic enzymes . The incin vitro  and incr in vivo . These sDescurainia sophia (L.) Webb ex Prantl (Flixweed), which belongs to the family Brassicaceae (Cruciferae), originates from Southern Europe and North Africa, and is widely distributed in Northeastern China [D. sophia seeds are to relieve coughing and asthma, treat jaundice, reduce edema, promote urination, etc. [D. sophia seeds relieved chronic functional constipation in children which was statistically significant [D. sophia seeds. They are cardiac glycosides, sinapoyl glycosides, flavonoids, lactones, lipids, coumarins, nor-lignans, lignans, and phytosterols [D. sophia seeds regulated diverse genes which are closely associated with several biological functions like cell survival and death [rn China . The ethon, etc. . A recennificant . Some reosterols , 16–21. osterols , and antnd death .D. sophia seeds increases, the clinical applications and medicinal requirements of D. sophia seeds may increase gradually. However, no scientific study is currently available showing the effects of D. sophia seeds on the activities of human DMEs. The goal of the present study was to elucidate the inhibitory effect of D. sophia seeds on major human DMEs including Phase I  and Phase II  enzymes and a drug transporter .As evidence for the ethnopharmacological use of D. sophia were purchased from Kwangmyungdang Medicinal Herbs Co. . D. sophia seeds were collected in the Shandong Province, China in June 2010 which was written on the batch label. They were identified by Dr. Go Ya Choi at the K-herb Research Center, Korea Institute of Oriental Medicine, Daejeon, Republic of Korea. A voucher specimen (KIOM-CRC-5) was deposited at the KM-Convergence Research Division, Korea Institute of Oriental Medicine. Whole extract of the dried seeds of D. sophia was prepared by repeated maceration in 80 % (v/v) ethanol. The solid phase of the ethanol extract (EEDS) was further concentrated and dried. Finally, the EEDS was stored at 4 °C. The EEDS was dissolved in 100 % dimethylsulfoxide  to make a stock solution and then stored at -80 °C until used. A detailed procedure on preparing EEDS was described previously [The dried seeds of eviously .In vitro enzymatic inhibition assays of human Phase I (CYPs) and Phase II (UGTs) DME, and P-gp were done with commercially available luminogenic assay systems. The assay systems were purchased from Promega , and the microsomes containing the recombinant human CYPs were purchased from Life Technologies . The microsomes containing human UGT and P-gp were supplied in the luminogenic assay systems (Promega). The details on the assay systems and the microsomes are presented in Table 50) was determined with the SigmaPlot program  with a 4-parameter logistic equation.i values were further determined for the enzymes with IC50 values less than 100 μg/mL. They were determined by incubating a series of EEDS concentrations and different concentrations of respective substrates for each enzyme using the luminescence-based method described earlier. The enzyme reaction conditions are summarized in Table i value was calculated with the secondary plot representing the slope rate of the Lineweaver-Burk plot versus the EEDS concentrations  [The modes of enzyme inhibition and the K0 μg/mL) .Table 2SThe data are presented as the means ± standard deviation (S.D.) for at least duplicate experiments. The means and S.D. were determined with the SigmaPlot program.in vitro luminescent assay described earlier. The calculated IC50 of the EEDS and specific inhibitors against each DME are presented in Table 50 ≤ 20 μg/mL for crude extract or IC50 ≤ 10 μM for a single compound), moderate (20 < IC50 < 100 μg/mL or 10 < IC50 < 50 μM), or weak (IC50 ≥ 100 μg/mL or IC50 ≥ 50 μM) [50 of 47.3, 25.8, and 38.7 μg/mL, respectively. However, CYP2C8 (IC50 > 100 μg/mL), CYP2D6 (IC50 = 137.5 μg/mL), and 3A4 (IC50 = 134.2 μg/mL) were weakly inhibited by EEDS. The effect of EEDS on UGT1A1 was very weak (IC50 = 335.1 μg/mL) and on UGT2B7 and P-gp negligible (IC50 > 500 μg/mL).The inhibitory effect of EEDS on human DME activities were evaluated with the ≥ 50 μM) . Accordii values and the mode of enzyme inhibition for the CYP1A2, CYP2C9, and CYP2C19 isoforms with an IC50 less than 100 μg/mL for the EEDS. From the results, the EEDS competitively inhibited CYP2C9 but showed mixed inhibition for CYP1A2 and CYP2C19 seen in the Lineweaver-Burk double reciprocal plots  of 5.5 μM and 10.0 μM, respectively. Several research groups have reported on the biological activities of D. sophia seeds. Khodarahmi et al. evaluated the cytotoxic effects of volatile oil from D. sophia seeds on the MCF-7 and HeLa cell lines with an IC50 of 70 μg/mL and 180 μg/mL, respectively [et al. reported on the in vivo anti-asthmatic, anti-tussive, and expectorant activities of D. sophia seed oil supporting the ethnopharmacological use of D. sophia seeds to relieve respiratory diseases [D. sophia seeds on human DMEs and P-gp. Biochemical luminogenic inhibition assays showed that human CYP1A2, CYP2C9, and CYP2C19 were moderately inhibited by EEDS based on their pharmacokinetic parameters (IC50 and Ki). Other tested CYP isotypes including CYP2C8, CYP2D6, and CYP3A4, were weakly inhibited by EEDS. These results show that the bioavailability of drugs, which are metabolic substrates of CYP1A2, CYP2C9, or CYP2C19, can be affected when these drugs are concomitantly administered with EEDS, causing potential adverse effects by elevating the plasma levels of cytotoxic drugs, or leading to therapeutic failure by inhibiting the metabolic processing of inactive prodrugs. Among approved drugs, the substrates for CYP1A2 include amitriptyline (anti-depression) and erlotinib (anticancer). The substrates for CYP2C9 include ibuprofen , warfarin (anticoagulant), and tamoxifen (anticancer). The substrates for CYP2C19 include diazepam, mephenytoin (anti-epileptic), methadone  and bortezomib (anticancer)  [D. sophia seeds as an herbal formulation and a NSAID like celecoxib, diclofenac, ibuprofen, or naproxen are co-administered to treat cold, the two drugs will compete with each other to bind with CYP2C9 [D. sophia seeds has been used as an antipyretic for measles and smallpox [D. sophia seeds. For more information of cytochrome P450 drug interactions, please refer to the following site (http://medicine.iupui.edu/clinpharm/ddis/).In our previous study, we reported on the anticancer effects of EEDS against various human cancer cell lines and identified various bioactive constituents from EEDS , 22. Fouectively . Gong etdiseases . To the h CYP2C9 , 30. The50 of 134 μg/mL. Therefore, EEDS may only slightly affect the bioavailability of anticancer drugs, such as dasatinib for chronic myelogenous leukemia and gefitinib for locally advanced or metastatic non-small cell lung cancer , whose primary administration route is oral. These drugs are metabolized and eliminated mainly by CYP3A4.Due to the fact that CYP3A4 metabolizes more than 50 % of the clinically prescribed drugs and represents the major metabolic enzyme in the human liver (40 %) and intestinal mucosa (80 %), CYP3A4 could contribute significantly to the metabolism of herbal drugs because most of the herbal drugs including EEDS are orally administered and metabolized primarily by the drug metabolizing enzymes in the liver and intestinal mucosa , 31, 32.in vitro and clinical settings [Graphical analysis with the Lineweaver-Bulk and secondary plots showed that the effect of EEDS on CYP2C9 is a competitive-type of inhibition. On the other hand, the effects of EEDS on CYP1A2 and CYP2C19 were best fitted to a mixed-type of inhibition. Competitive inhibition of EEDS implies that EEDS competes with a substrate (Luciferin-H used in this study) to bind to the active site of CYP2C9. Therefore, the inhibition of CYP2C9 by EEDS can be overcome by increasing the substrate concentration. The competitive inhibition of CYP is reversible which is most commonly observed in both settings . In a noFructus schisandrae, which has been used as a tonic in traditional Chinese medicine, potently inhibit the UGT1A3 isotype [50 = 335.1 μg/mL) and UGT2B7 (IC50 > 500 μg/mL) in our biochemical assay system; thus, its effects on these two UST isotypes were negligible.UGTs catalyze the glucuronidation reaction through which the glucuronic acid moiety of UDP-glucuronic acid is transferred to substrates or xenobiotics, which are eliminated from the human body . Althoug isotype . In the The P-gp protein is an ATP-dependent drug efflux transporter and also known as multidrug resistance 1 (MDR1) or ATB-binding cassette subfamily B member 1 (ABCB1). When the distribution of P-gp was determined with a monoclonal antibody raised against P-gp, the distribution of human P-gp in normal tissues was concentrated in cells found in the intestinal epithelium, liver, proximal tubule of the kidney, and capillary endothelium, where it pumps back toxic materials into the intestinal lumen, bile and urinary ducts, and capillaries, respectively –38. ManyD. sophia seeds were evaluated for their effects on human DMEs. Quercetin, a flavonoid, is a potent competitive inhibitor of CYP1A2, CYP2C, CYP2C19 and CYP3A4  [et al reported that enhanced doxorubicin absorption in the gastrointestinal tract is attributed to quercetin-induced inhibition of P-gp and that the reduced first-pass metabolism of doxorubicin is attributed to quercetin-induced inhibition of CYP3A in the small intestine [D. sophia, exhibited inhibitory effects on P-gp in adriamycin-resisitant human breast cancer cells, MCF-7/ADR [Some constituents of ctively) , and inhctively) . Additiontestine . SyringaCF-7/ADR .In vitro enzyme activity assays showed that EEDS modulates the activities of human Phase I/II DMEs and P-gp to varying degrees of potency indicating the potential risk of herb-drug interactions. CYP2C9 was competitively inhibited; however, CYP1A2 and CYP2C19 were inhibited in a mixed manner by EEDS with Ki values of 19.8, 5.2, and 11.9 μg/mL, respectively. CYP2C8, CYP2D6, CYP3A4, UGT1A1, UGT2B7, and P-gp were not weakly or negligibly affected by EEDS. In the present study, however, the bioactive constituent(s) responsible for affecting the enzyme activities were not identified. Further studies will have to be done to identify the responsible phytochemical constituents and their enzyme modulating properties. In addition, we should keep in mind that various in vivo factors could influence in vitro to in vivo extrapolation.