Ipomoea murucoides. Xylophagy may modify the branch chemical constitution, which also affects the liberation of allelochemicals with defense and allelopathic properties. We evaluated the bark chemical content and the effect of extracts from branches subjected to treatments of exclusion, mechanical damage and the presence/absence of epiphytes, on the seed germination of the epiphyte Tillandsia recurvata. Principal component analysis showed that branches without any treatment separate from branches subjected to treatments; damaged and excluded branches had similar chemical content but we found no evidence to relate intentional damage with allelopathy; however 1-hexadecanol, a defense volatile compound correlated positively with principal component (PC) 1. The chemical constitution of branches subject to exclusion plus damage or plus epiphytes was similar among them. PC2 indicated that palmitic acid  and squalene, a triterpene that attracts herbivore enemies, correlated positively with the inhibition of seed germination of T. recurvata. Inhibition of seed germination of T. recurvata was mainly correlated with the increment of palmitic acid and this compound reached higher concentrations in excluded branches treatments. Then, it is likely that the allelopathic response of I. murucoides would increase to the damage  that may be caused by a high load of epiphytes than to damage caused by the xylophages.Herbivory activates the synthesis of allelochemicals that can mediate plant-plant interactions. There is an inverse relationship between the activity of xylophages and the abundance of epiphytes on  Ten seeds of T. recurvata were placed in a Petri dish using filter paper as substrate. Twenty-four Petri dishes were used for each treatment along with four Petri dishes with distilled water but without extract (control). In order to exclude the effects of factors other than allelopathy (e.g. non-viable seeds or dormancy) from the calculation, percentage of inhibition of seed germination was calculated relative to that recorded in the control. Percentage inhibition of seed germination was therefore calculated according to the following equation:Xi is the number of germinated seeds under a specific concentration and n is the number of seeds (n = 10) in the Petri dish. Positive and negative values indicate inhibition and promotion of germination, respectively, while zero indicates a complete absence of bark host effect.We hypothesized that extracts obtained from the artificially damaged branches of As with the dichloromethane extracts, dimethyl sulfoxide (DMSO: (CH3)2SO, Fermont) was used, at a maximum concentration of 1% (v/v), in order to prepare the solutions . In all −1. All extracts were analyzed using the following temperature program: 1–45°C 1 min, with an increase of 10°C min−1 until reaching 250°C and then 260°C was maintained for 30 min. The database used for comparison of the results was NIST-MS version 1.7a.In order to know the chemical constituents among extracts obtained from the branch treatments 21 mg of each of the eight samples (one sample per branch treatment), were solubilized in ethyl acetate (1 mL). Solid phase extraction was done using 1 mL cartridges SUPELCO® with a normal phase (LC-SI). 1 mL of ethyl acetate was recovered from the sample loaded in the cartridge. Just 400 μL were evaporated. Six mL of ethyl acetate was recovered from washing cartridge. Solvent was evaporated. The eight samples were reconstituted in dichloromethane (1 mL) and injected in GC-MS. Aliquots (150 μL) from these solutions were injected into a gas chromatograph (Agilent 6890) coupled to a mass detector , equipped with an HP5-MS capillary column . Helium was the carrier gas at a flow rate of 1 mL minThe variables total length of branch, diameter, percentage of non-intentional damage, number of orifices and number of xylophages were analyzed using an analysis of variance with three factors , where tT. recurvata seeds caused by the dichloromethane extracts was analyzed with an analysis of variance of three factors with two levels per factor. To normalize the data and make the variances uniform, the data were transformed using the arcsin function [The percentage of inhibition of germination of function . The facfunction . All anafunction .T. recurvata seed germination. In order to test this hypothesis we performed a principal components analysis (PCA) [T. recurvata exerted by these extracts. PCA analysis was based on the correlation matrix, for each eigenvector , we considered as significant, those variables that contribute with at least 10% to the eigenvector normalized length and that at the same time were highly correlated with it (r ≥ 0.6) [We hypothesized that bark extracts would form associations according to their chemical constituents and that those extracts obtained from branches under intentional damage would have allelopathic properties on is (PCA)  using asr ≥ 0.6) .F1,48 < 2.3, all P > 0.05).In three of the ten trees in which the experiment was conducted, mesh bags used to achieve exclusion were lost, for this reason only the data derived from the branches (n = 56) of seven trees were considered. General measurements of branches were: length of 188.62 cm ± 13.60 cm, diameter of 17.24 mm ± 0.53 mm, 0.82 ± 0.23 orifices, 1.28 ± 0.30 xylophages and non-intentional damage of 8.30 cm ± 1.21 cm. There were no significant differences of length of branches, diameter, number of xylophages, number of orifices and natural damage between levels of the main factors . FurtherBrachymyrmex sp. (Hymenoptera: Formicidae) and the other was a bark-bettle (Curculionidae: Scolytinae). Due to the fact that the majority of individuals found were larvae, identification was limited. Of the total number of arthropods, the majority (83.52%) belonged to the order Coleoptera, followed by Lepidoptera (12.94%), Hemiptera (1.17%), Hymenoptera (1.17%) and by the class Arachnida (1.17%). Four families of the order Coleoptera were identified, with Cerambicidae accounting for most individuals (92.95%), followed by Buprestidae (4.22%), Curculionidae (1.40%) and Tenebrionidae (1.40%). The analysis only included the insect xylophages; in this case, Coleoptera.A total of 85 arthropods were found, of which 83 were larvae and two were adults one belonged to T. recurvata seeds was different in the exclusion treatments , with lower germination recorded on application of the extract derived from the excluded (51.77% ± 1.68% inhibition) than non-excluded  branches. There were no differences  in the inhibition of T. recurvata seed germination on application of extract derived from branches with intentional damage (39.58% ± 1.98% inhibition) and from those without intentional damage . The percentage of inhibition of T. recurvata seed germination was equal  when tested with extracts derived from branches with (40.31% ± 2.09%) and without  epiphytes.The percentage of inhibition of T. recurvata seeds were equally inhibited by the dichloromethane extract obtained from excluded branches and from those with damage  on the germination of T. recurvata seeds, with greater inhibition found in the seeds treated with extracts derived from excluded branches, regardless of the presence/absence of epiphytes ; however, multiple comparisons testing did not establish significant differences between the percentages of inhibition of germination, for which reason this result may be considered spurious. None of the interactions among the three main factors on inhibition of T. recurvata seed germination were statistically significant  contributed with 82% of the total length of the eigenvector . While the extracts obtained are from individuals of I. murucoides from the same study zone, it is probable that the differences found are due to the temporal variability of the climate, to the availability of resources that affect the composition and concentration of secondary metabolites [T. recurvata seeds from different years.Previous evidence shows that dichloromethane bark extract of bout 58% . We founbout 58% , the extabolites  in brancU = 6.00, P = 0.018) between the weighted means of a) xylophages/length (cm) of branches of this study (1.75 ± 0.56 xilophages/cm) and b) data of Valencia-Díaz et al. [I. murucoides, this comparison must be considered with caution because of the temporal variability of xylophages populations.As stated previously, herbivory induces the synthesis of allelochemicals that can perform multiple biological functions , 7 and tT. recurvata seed germination, but according to the PCA chemical content analysis, those extracts with intentional damage contain more trans-9-hexadecen-1-ol, cis-9-eicosen-1-ol and 1-hexadecanol; of these it has been shown that 1-hexadecanol may increase their concentration due to plant injury [T. recurvata does not preclude the activation of direct defenses against the herbivore. It is possible that the chemicals constituents of the branches that suffered damage can have a direct detrimental effect on the xylophages, but confirmation of this would require studying the insecticidal activity of extracts of I. murucoides on this guild.We did not find evidence that extracts obtained from intentionally damaged branches provoked a greater allelopathic effect in t injury . When het injury , 43, 44.T. recurvata seeds. Two of the five chemical compounds correlated positively with the inhibition of seed germination of T. recurvata were hexadecanoic acid  and squalene. Palmitic acid has been reported as a constituent of I. murucoides and as an inhibitor of seed germination of T. recurvata, also it has been confirmed that commercial compound inhibited 65% the seed germination of this epiphyte [Sebastiana adenophora (Euphorbiaceae) [Mesh bag exclusion was done in order to prevent subsequent arrival of xylophages to the experimental branches, but contrary to expectation, the extracts from excluded branches had a greater inhibitory effect on the germination of epiphyte . This coepiphyte . In the biaceae) , and maybiaceae) . It is pOn the other hand, the treatment of presence/absence of epiphytes did not show differences in terms of the inhibitory effect of the extracts obtained from these two treatments. It is reasonable to consider that the branches with epiphytes would be more shaded than those without epiphytes and thus the chemical constitution could change as occurred in the exclusion treatment; however, it is possible that the coverage provided by the four epiphytes that were transplanted on the branches of this treatment was insufficient to bring about a change in the chemical content of the bark of these branches; in fact PC1 separated control branches along with branches with only epiphytes.T. recurvata was the interaction between exclusion and presence of epiphytes. However, as can be seen from the results, this was determined basically by the treatment of exclusion; i.e., regardless of the presence of T. recurvata, the greatest inhibition of germination was produced when exclusion of the branches was included in the interaction. In fact, no interaction presented among the eight treatments of this experimental design exercised an inhibitory effect that was distinctively greater than the others. The fact that no statistical differences were found in the inhibition of the germination of T. recurvata seeds as a function of the treatments does not mean that there may not have been inhibitory effects, but rather that there is no relationship between this effect and the damage while there is a relationship with the exclusion and presence of epiphytes. As stated previously, all of the treatments inhibited germination, a result that corroborates the allelopathic effect of I. murucoides [The only interaction of the treatments in which there were differences in the inhibition of seed germination in rucoides , 27.Ipomoea murucoides, I. pauciflora and Bursera fagaroides) there is a negative association between the xylophages (damage and abundance) and the epiphytes (coverage and abundance) they host. This pattern is most evident in I. murucoides [T. recurvata.At the interspecific level, there is evidence that in host species with a low abundance of epiphytes (rucoides . We foun


Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents . Here, wDOI:http://dx.doi.org/10.7554/eLife.05033.001 Diverse cellular conditions activate an integrated stress response (ISR) that rapidly reduces overall protein synthesis while sustaining or enhancing translation of specific transcripts whose products support adaptive stress responses. The ISR is mediated by diverse stress-sensing kinases that converge on a common target, serine 51 in eukaryotic translation initiation factor alpha (eIF2α) eliciting both global and gene-specific translational effects . Mammalii) to translation initiation complexes and mediates start codon recognition. It binds GTP along with Met-tRNAi to form a ternary complex (eIF2-GTP-Met-tRNAi) that assembles, along with the 40S ribosomal subunit and several other initiation factors, into the 43S pre-initiation complex (PIC). The 43S PIC is recruited to the 5′ methylguanine cap of an mRNA and scans the 5′UTR for an AUG initiation codon  brings initiator methionyl tRNA  and form messenger ribonucleoproteins, which can further assemble into stress granules (SGs). These cytoplasmic, non-membrane bounded organelles contain translationally stalled and silent mRNAs, 40S ribosomal subunits and their associated pre-initiation factors and RBPs; these RBPs facilitate the nucleation and reversible aggregation of SGs through reversible, low-affinity protein–protein interactions mediated by their low complexity domains .Paradoxically, under conditions of reduced ternary complex formation and protein synthesis, a group of mRNAs is translationally up-regulated. These mRNAs contain short upstream open reading frames (uORFs) in their 5′ UTRs, which are required for their ISR-responsive translational control . These tS51A (Eif2s1S51A). Homozygous loss of eIF2α phosphorylation leads to perinatal death but heterozygous eIF2α+/S51A animals, which have reduced levels of eIF2α phosphorylation, grow into healthy adults showing phenotypes that demonstrate the importance of translation initiation in establishment of long-term memories  kinase inhibitors that target PERK or PKR  an K or PKR ; (3) salK or PKR ; and (4)K or PKR . By a yeHere, we show that ISRIB reverses comprehensively and specifically the effects of eIF2α phosphorylation. By profiling the genome-wide translational program downstream of the ISR, we present the application of ribosome profiling to the ISR in mammalian cells, which allowed us to identify and quantify the translational changes that take place upon its induction and ISRIB treatment. Moreover, live cell imaging revealed that ISRIB addition can trigger a remarkably fast dissolution of phospho-eIF2α-dependent SGs in stressed cells, restoring translation.We used ribosome profiling to characterize translational changes induced by ER stress. Deep sequencing of ribosome-protected mRNA fragments provides global, quantitative measurements of translation and reveals the precise location of ribosomes on each mRNA , 2011. WConsistent with the well-established presence of regulatory uORFs in their 5′-UTRs, this genome-wide analysis identified four previously extensively studied mRNAs that displayed significant translational upregulation: ATF4, ATF5, CHOP, and GADD34, . They co< 0.003, chi-squared test with Yates correction).We identified a total of 78 mRNAs whose translation changed significantly and substantially (more than twofold) upon ER stress in HEK293T cells  . ISRIB rSG formation can also be induced in the absence of eIF2α phosphorylation by inhibiting the eIF4A helicase, which is part of the cap-binding eIF4F complex . Pateami50 of 5 nM as previously measured using an uORFs-ATF4-driven luciferase reporter in HEK293T cells  and eIF5 (the 48S PIC-associated GTPase-activating protein for eIF2) have been reported to make cells insensitive to this phosphorylation event , liberating mRNAs back into the translational pool. A pulse of -methionine (Perkin Elmer) and incubated for 15 min. Cells were lysed by addition of SDS-PAGE loading buffer. Lysates were sonicated and equal amounts were loaded on SDS-PAGE gels (BioRad). The gel was dried and radioactive methionine incorporation was detected by exposure to a phosphor-screen and visualized with a Typhoon 9400 Variable Mode Imager .U2OS GFP-G3BP/mRFP-DCP1a cells were seeded on 12-well plates, allowed to recover overnight and treated with 100 nM Tg for 40 min. ISRIB (200 nM) or CHX (50 µg/ml) was added at the same time as 50 µCi of  My major concerns are with the robustness of the data reported. In its present version this is not possible to fully assess.1) For RNA and Ribo-Seq experiments, there is no information given concerning the depth of sequencing. How many million mapped reads and how many replicates were done for each sample?We have included biological replicates for all Ribo-Seq and mRNA-seq data for all conditions. Figure 1–figure supplement 7 contains the reads mapped information and Figure 1–figure supplement 8 shows the correlation coefficient between replicates.2) The significant changes in ribosome occupancy are reported in supplementary tables, but changes in RNA transcript abundance are not reported. This data should also be added to the manuscript.RNA transcript abundance is shown in Figure 1–figure supplement 9.3) For the Ribo-Seq fold changes reported, how are reads mapping to overlapping ORFs handled? Eg, shows reads covering the uORFs (green) and main ORFs (blue) that overlap.We removed Ribo-Seq-fold changes of uORFs from the manuscript.4) Is the read density data inreporting a single representative sample, total summed reads from replicates (if done) or mean reads from multiple replicates? The read density is not very high for any of the genes shown, except for ATF4.The read density represents the total reads of two replicates. We have only included ATF4 and SLC35A4, which have sufficient overall higher read density to draw strong conclusions.5) Can the authors confirm and comment on the presence of RPFs between the annotated uORF and main ORF for CHOP mRNA?The data have been removed.6) One perhaps unexpected finding was an apparent increase in uORF RPFs following stress. The authors have chosen not to elaborate on this point. The usual translational control models depicted often show an all or nothing extreme response, but it is not surprising at all that there are still ribosomes on uORFs that may get skipped more frequently under stress conditions. Perhaps an important measure of control is the proportion of ribosomes that skip uORF2 in ATF4 and 5. This could be quantified as the Ribo-Seq read data for the overlapping ORF regions should be in separate reading frames. It should be possible to assign reads to either the uORF or the main ORF. As a measure of stress mediated translational control, the authors could quantify the number and proportion of RPF within the ORFs key for translational control. For ATF4 and ATF5 ORF/uORF2 reads would give an alternative readout of the relative translational control.The data are no longer presented in the manuscript. The analysis of remaining uORF occupancy in the face of downstream ORF translation will require extensive additional studies.7) Blots showing eIF2 phosphorylation relative to total eIF2 should be in each type of cell used (plus minus) treatment for the time points used in the Ribo-Seq study. This could be added to the ATF4 blot which uses different time points.eLife paper (10.7554/eLife.00498). We do not detect phospho-eIF2α phosphorylation or ATF4 production after only 1 hour of Tm treatment by Western blot analysis, which is the condition used for ribo and mRNA-seq analysis. Ribo-Seq analysis is a more sensitive method than Western blotting to detect the early translational changes upon ER-stress.The ATF4 blot has been removed as it related to the MEFs data. We have reported time courses of eIF2α phosphorylation and ATF4 production in HEK293T cells in 8) For the immunofluorescence experiments and for the GFP experiments inand 4 and Figure 4–figure supplement 1, each experiment requires quantification of a larger number of cells than are shown in each representative image. For example in 100 cells in each of three separate experiments what proportion of cells (plus minus) error contain stress granules or P bodies as appropriate. Such an analysis would enable statistical treatment of the significance of the observations made.The quantitation was added to the stress granule data.9) What is the effect of the ISR and ISRIB on the other arms of the UPR at the times used here. Are sampling times too early to observe XBP1 splicing and changes in ribosome binding? If they are this should be stated, if they are not it would helpful to show XBP1 reads.eLife paper (10.7554/eLife.00498), we looked at XBP1s induction upon Tm treatment in HEK293T cells. XBP1s production lags ATF4 translational upregulation and is not detected at 1 hour of Tm treatment. We also reported in our previous manuscript that ISRIB treatment or blocking the PERK branch of the UPR does not alter activation of IRE1 and XBP1 splicing but it prolongs activation of this branch as measured both by IRE1-GFP foci formation and XBP1 splicing .At the time points analyzed, it is too early to detect changes in XBP1 mRNA splicing. In Reviewer #2[…] While the study is potentially interesting, there are some important issues to address.Major issues:Throughout the manuscript, there is a disconnection between the data and the conclusions. I will only highlight the major ones here:1) One of the central conclusions of the paper is that ISRIB eliminated the effects of eIF2α phosphorylation. The authors wrote: “The translational output of ISRIB-treated cells to ER stress was remarkably similar to that of cells with a genetically ablated ISR”. This contradicts the data. In fact, there are major differences between the two data sets . I recommend providing a complete list of genes that change after ISRIB treatment and to study these changes, as it may shed light on ISRIB's function and/or target.eLife Research Advance and in the revised manuscript, we have provided a list of genes that are significantly and substantially translationally upregulated in all conditions reported . We have carefully analyzed the genes that remain translationally upregulated in the presence of Tm + ISRIB. Unfortunately they are all hypothetical proteins with unknown functions and thus do not shed light on ISRIB function. Some of these uncharacterized genes are also down regulated in the presence of ISRIB alone.We have removed the MEF data and thus we do no longer compare ISR-ablated cells with ISRIB-treated cells. In both the original submitted Supporting the idea that the changes caused by ISRIB  are actually tractable, ISRIB caused more changes  than Tm . The secret of ISRIB's function may be hidden in this dataset. This needs to be documented and exploited.As stated above, ISRIB changes have been documented. Compared to Tm treatment, ISRIB induced changes are small.A related issue: The authors conclude that “ISRIB does not have general off-targets effects on translation, transcription or mRNA stability.” Again, this contradicts the data. Beside, “off-target” is a not suitable here, since we don't know what is the target of ISRIB.We have removed off-target from the text. The ribosome and mRNA genome-wide profiling data demonstrates that ISRIB comprehensively blocks the translational effects driven by UPR activation and eIF2α phosphorylation and does not lead to overall and spurious changes in mRNA levels or translation.2): What are the vertical lines on PERK blots? Between first and second lane in the top blot and between the 9th and 10th lane in the bottom blot.All lanes were contiguous in the blots. The film was rescanned and the lines are no longer present.3): It would be good to see images with better resolution to appreciate the localization of eIF3α, which looks very interesting. I still see some stress-granules on Ars+ISRIB in some cells but in most cells, the signal is too strong to be analyzed. It would be good to see images of better resolution and some quantitative assessment of the effects.eIF3α with all stressors used in this paper has been previously published.Quantitative assessment was included for all stress granule data. Immunofluorescence data of 4) The disappearance of stress-granules is the impressive finding of the manuscript but how does this all happen? It is difficult to follow what might be going on because of the lack of consistency in the conditions used and because adequate controls are not presented. From this paper and the previous one, it would appear that ISRIB acts upstream of ATF4 translation and is dependent upon eIF2α-P (but without affecting the latter)? This needs to be explored further to get some understanding of what is happening.The goal of using different stressors was to distinguish between phospho-eIF2α-dependent (thapsigargin and arsenite) and independent (panteamine A and hippuristanol) stress granule formation. As expected by the ability of ISRIB to make cells resistant to the effects of eIF2α phosphorylation, the small molecule was able to block only phospho-eIF2α-dependent stress granule formation. ATF4 is downstream of eIF2α phosphorylation and thus it is also blocked by ISRIB. The mechanism of action of the drug will await identification of its molecular target.Figure 4C: It would be good to present gels to show the effects, as the authors have the images already. It would be interesting to appreciate the qualitative changes in translation. Does this match, what is seen by ribosome profiling?35S-methionine incorporation and recovery experiments were performed in U2OS cells expressing G3BP-GFP using the potent ER-stressor, thapsigargin. It is not the same cell type or the same ER-stressor (tunicamycin) used in the ribosome footprinting analysis.We have included the gel in 5) The Discussion is very broad and not connected to the current dataset. The first sentence of the Discussion is incorrect, as there are other antagonists of the ISR. I suggest two options for a revised discussion: Either to focus the discussion on the current dataset or to provide additional data inline with the discussion .We have substantially rewritten the Discussion. To the best of our knowledge however, ISRIB is indeed the first and to date only known ISR inhibitor. Although there are inhibitors of specific eIF2α kinases (PERK and PKR inhibitors), ISRIB is the only molecule that can block signaling downstream of all eIF2α kinases by making cells resistant to eIF2α phosphorylation. An ISR agonist exists, salubrinal, which prolongs eIF2α phosphorylation. An HRI activator also exists which can also act as an agonist of the ISR in cells that express HRI.6): I didn't understand this panel. It needs to be clarified.This figure was removed.Reviewer #3[…] The lack of any measureable effect of ISRIB on baseline translation is surprising as there is evidence for basal activity of the ISR: For example PERK_KO, eIF2a_S51A and ATF4_KO cells all share a strong baseline requirement for amino acid supplementation (likely indicating that the ISR contributes to baseline ATF4-mediated gene expression). The authors may wish to comment on this point.-/-), ATF4 (ATF4-/-) or non-phosphorylatable eIF2α (eIF2α S51A/S51A).HEK293T cells were only treated with ISRIB for one hour and thus the basal levels of ISR activation may not be as evident as in cells under prolonged ablation of PERK (PERK


Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated.By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%.We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain. These antibodies were preabsorbed by the GST affinity column, then affinity-purified using a CNBr-activated Sepharose 4B column (GE) conjugated to the recombinant. Hybrid peptide  [cMLCK(42-55/93-102)] was synthesized and coupled with KLH. Theses amino acid sequences in cMLCK were conserved among human, bovine, dog and rodents. This rabbit anti-cMLCK antibody was prepared and affinity-purified using Epoxy-activated Sepharose 6B column (GE).3VO4, 1 mM NaF, 10 mM Na4P2O7 and proteinase cocktail inhibitor ]. After centrifugation, equal volume of SDS sample buffer was added to the supernatant and heated . The samples were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The three kinds of anti-cMLCK, anti-smMLCK (Sigma), anti-ZIPK (Abcam) and anti-myosin phosphatase target subunit 2 (MYPT2)  greatly reduced the basal twitch tension. In the presence of ryanodine, phenylephrine induced an inotropic action in C57BL/6J and C57BL/6N  the relationship was positively correlated between the phenylephrine-induced inotropic response and MLC2v phosphorylation levels (within 15–30%).We found the followings: (i) a new isoform of cMLCK (cMLCK-2) with molecular mass of 61-kDa was identified in heart from various mouse strains. (ii) C57BL/6N expressed only cMLCK-2 while other mouse strains  expressed both cMLCK-2 and 86-kDa cMLCK. (iii) The MLC2v phosphorylation levels in papillary muscle were significantly lower in C57BL/6N than in C57BL/6J. (iv) The αcMLCK was identified as the third MLCK in the MLCK family, 7. The in vitro [The levels of MLC2v phosphorylation in papillary muscles were lower in C57BL/6N (31.8%) than in C57BL/6J (47.4%). It was found that smMLCK and ZIPK each phosphorylate MLC2v in vitro , 17. Thein vitro  and of sin vitro . In addiin vitro . Taken t1-adrenoceptor agonists increase MLC2 phosphorylation in electrically paced human atria [Twitch tension induced by electrical field stimulation in papillary muscle was smaller and the time required to the peak and the half relaxation were both longer in C57BL/6N than in C57BL/6J. Phenylephrine more strongly increased the twitch tension in C57BL/6J muscle than in C57BL/6N but it had no effect on MLC2v phosphorylation level in each mouse strain. Thus, we speculate that the positive inotropic response to phenylephrine depends on the levels of basal MLC2v phosphorylation: i.e., the level of MLC2v phosphorylation under the basal conditions is higher the stronger inotropic action to phenylephrine is provoked. The results are not consistent with the previous findings that αan atria –21. Howean atria –21. Thes2+ channel blocker and activator, respectively) each blocked phenylephrine-induced inotropic response in both C57BL/6J and C57BL/6N, suggesting that the fine tuning of L-type Ca2+ channels may be responsible, in part, for the phenylephrine-induced inotropic response. In the presence of ryanodine (an inhibitor of Ca2+-induced Ca2+ release in SR), phenylephrine still induced an inotropic response in both strains. α1-adrenoceptor activation was reported to be able to increase Ca2+ influx through phosphatidylinositol turnover [2+ release from SR, but to Ca2+ infux at least in part through L-type Ca2+ channel. In a case of vascular strip, noradrenaline was reported to be able to induce arterial contraction by activating voltage-dependent Ca2+ channel [Nifedipine and BAY K 8644 , indicating that cMLCK is dependent on Ca2+/calmodulin. The regulation of cMLCK in vivo remains to be determined.BAY K 8644 and ryanodine did not modify papillary muscle MLC2v phosphorylation in either strains, suggesting that Caoversial , 6. Our 2+ sensitivity of force [Calyculin A (an inhibitor of protein phosphatase type1 and 2A) increased MLC2v phosphorylation in cardiomyocytes . It was of force .To understand the physiological roles of MLC2v phosphorylation in this study, the difference in the levels of MLC2v phosphorylation between the two kinds of sample should be considered. The phosphorylation levels of MLC2v in prepared papillary muscle samples was lower than those in whole heart immediately isolated from sacrificed mice  and 6G. 1-adrenergic stimulation exacerbates cardiac dysfunction and decreases the expression of β1-adrenergic receptors, thus leading to heart failure [1-adrenoceptors remain stable or rather increase in heart failure [1-adrenoceptor agonists [1-adrenoceptors play an adaptive and protective role, depending on the levels of MLC2v phosphorylation. Thus, a new therapeutic strategy for heart failure could find out the way to increase and maintain MLC2v phosphorylation levels (over certain ranges).In heart failure, the level of MLC2 phosphorylation decreases , 30 and  failure , 32. On  failure , 34, thuagonists . The levagonists , 30, and1-adrenoceptor agonist in heart under physiological conditions.In summary, we identified the expression of a new isoform of cMLCK with a molecular mass of 61-kDa in mouse heart. In C57BL/6J strain both 86-kDa and 61-kDa cMLCKs were expressed but only 61-kDa cMLCK was detected in C57BL/6N strain. The inotropic response to phenylephrine was closely correlated with MLC2v phosphorylation levels (within the range of 15–30%). Thus, we believe that MLC2v phosphorylation within such ranges plays an important role to induce the inotropic response by an αHowever, whether the phenomenon observed in our models, namely the MLC2v phosphorylation level-dependent inotropic response, is specific for phenylephrine or common observed in other inotropic agents including β-adrenoceptor stimulation is remained to be investigated.S1 FigPhosphorylation and expression were shown by Western blot analysis.(PDF)Click here for additional data file.S2 FigCardiomyocytes were isolated from ventricles of 1-day-old Sprague—Dawley rat pups with the digestion of collagenase IV and trypsin. Following incubation for 24 h in serum-containing medium, the cardiomyocytes were incubated for 24 h in serum-free medium prior to stimulation with each agonist for 24 h. Phosphorylation levels were determined by glycerol-urea PAGE and Western blot analysis. n = 4–5.(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


N = 4,218) or during  the decision-making process. Consistent with previous results, across both studies inhibition predicted reduced cooperation while positive emotion predicted increased cooperation (even when controlling for negative emotion). Importantly, there was a significant interaction between positive emotion and inhibition, such that the most cooperative individuals had high positive emotion and low inhibition. This suggests that inhibition  may undermine cooperative behavior by suppressing the prosocial effects of positive emotion.Cooperation is central to human existence, forming the bedrock of everyday social relationships and larger societal structures. Thus, understanding the psychological underpinnings of cooperation is of both scientific and practical importance. Recent work using a dual-process framework suggests that intuitive processing can promote cooperation while deliberative processing can undermine it. Here we add to this line of research by more specifically identifying deliberative and intuitive processes that affect cooperation. To do so, we applied automated text analysis using the Linguistic Inquiry and Word Count (LIWC) software to investigate the association between behavior in one-shot anonymous economic cooperation games and the presence inhibition (a deliberative process) and positive emotion (an intuitive process) in free-response narratives written after (Study 1,  Cooperation plays an integral role in our lives, sustaining friendships and business relationships and laying the foundation for successful organizations and nations –18. WhenA growing literature uses economic games to explore the cognitive underpinnings of cooperation from a dual process perspective. In these experiments, participants choose between keeping money for themselves or spending money to benefit others cf. , 35). Fo. Fo35]).A series of recent experiments support the latter possibility; namely, that time pressure –47, cognSocial Heuristics Hypothesis (SHH), which has recently been proposed as a mechanistic explanation of the above findings , a significant main effect of Inhibition , and a significant interaction between Positive Emotion and Inhibition . Examining simple effects, we found a significant effect of Positive Emotion in participants both with  and without  Inhibition, and a significant negative effect of Inhibition in participants both with  and without  Positive Emotion.As seen in F = 39.86, p<0.001], Inhibition , Positive Emotion x Inhibition interaction . The robustness of our results for Positive Emotion when controlling for Negative Emotion suggests that it was truly the presence of positive emotion, rather than the absence of negative emotion, that drove our findings.Importantly, these results were robust to controlling for the presence (or absence) of Negative Emotion, as well as age (continuous), gender, education , and total word count (continuous)  = 0.55, p = 0.460], Comprehension x Inhibition , Comprehension x Positive Emotion x Inhibition ), as well as the fact that our results persist when excluding participants who answered any questions incorrectly  = 40.72, p<0.001], Inhibition , Positive Emotion x Inhibition ; with controls: Positive Emotion , Inhibition , Positive Emotion x Inhibition ).We also note a lack of significant interaction between our variables of interest and comprehension of the game payoff structure  = 0.51, p = .475], but did yield a significant main effect of Inhibition , and a significant interaction of Negative Emotion x Inhibition ; leading to a significant negative simple effect of Negative Emotion without Inhibition , and a non-significant but trending positive effect of Negative Emotion with Inhibition .during a one-shot PGG, rather than afterwards, as participants’ post-decision recollections of their decision-making process may be biased. These studies were approved by the Yale University Human Subjects Committee IRB Protocol #1307012383. All subjects provided written informed consent prior to participating, and this was approved by the Human Subjects Committee. See In Study 2, we further investigated the role positive emotion and inhibition play in cooperation using stream-of-consciousness narratives written Mage 30.7 years [SD = 10.64 yrs], median education level “Attended College”) recruited using Amazon Mechanical Turk in January 2013.Participants were 236 adult US residents , but with comprehension assessed before making their decision rather than afterwards (22.0% of participants answered one or both questions incorrectly). To elicit participants’ motivations Participants’ free-response narratives were analyzed using the same LIWC software as in Study 1.F = 4.67, p<0.05]. To avoid bias introduced by misunderstanding the game structure, we therefore focused our analysis on the 184 participants that answered the comprehension questions correctly. As seen in F = 0.11, p = 0.75] or Inhibition . Similarly to Study 1, however, we found a significant Positive Emotion x Inhibition interaction  such that positive emotion increased cooperation, but this effect was suppressed by Inhibition. Examining simple effects, we found a significant effect of Positive Emotion in participants without Inhibition  but not with Inhibition ; and a significant negative effect of Inhibition in participants with Positive Emotion  but not without Positive Emotion .Preliminary analyses found a significant three-way interaction between Positive Emotion, Inhibition and Comprehension failure , Inhibition , Positive Emotion x Inhibition ), again indicating that it is truly the presence of positive emotion, rather than the absence of negative emotion, that drives the observed results.As in Study 1, these results were robust to controlling for the presence of Negative Emotion, as well as age (continuous), gender, education , total word count of the description (continuous) and minimum writing time   = 0.46, p = .498] or a significant Negative Emotion x Inhibition interaction , but did find a significant main effect of Inhibition .Across two studies, we provided evidence that positive emotion motivates cooperation, that inhibition undermines cooperation, and that these two processes interact: positive emotion without inhibition was associated with the highest level of cooperation. This interaction between positive emotion and inhibition helps to provide a mechanism underlying previous results on the negative consequences of deliberation for cooperation: deliberation may undermine cooperative behavior by dampening the socially beneficial effects of positive emotion.We build on previous work related to positive emotion and prosociality , 71, 73 Our use of automated text analysis allowed us to quantitatively represent the emotional content of our participants’ narratives, and avoids potential coder biases. Our results linking inhibitory language to selfishness are consistent with work analyzing the testimony of Carnegie Heroes, people who risked their lives to save strangers . The samspecific positive emotions involved in cooperation. Exploring this issue is an important direction for future work, given divergent associations between self-focused emotion such as pride and other-focused emotions like compassion on social intuitions [The present results should be interpreted within the confines of several limitations. First, we note that our measures of positive (and negative) emotion were obtained at a general valence level, which precluded us from providing insight into the tuitions . Secondltuitions , their dS1 Dataset(CSV)Click here for additional data file.S2 Dataset(CSV)Click here for additional data file.


Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs), c-jun NH2-terminal kinases (JNKs), MAPK kinase (MEK), and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27). The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK) family members to primate torpor were compared in six organs of control (aroused) versus torpid gray mouse lemurs,  Tb) has huge advantages for mammals, e.g., supporting advanced cognitive capabilities, speed and agility, and ability to live in cold climates Tb to fall, and enter either short-term daily torpor or long-term (days or even weeks) continuous hibernation The ability to generate internal heat and maintain a high body temperature  M. murinus and Microcebus griseorufusMouse lemurs of the Tb values Tb. This has been a significant area of controversy in hibernation research Tb fall to near 0 °C and displaying substantially-enhanced hypoxia/ischemia tolerance Many studies of small mammal torpor and hibernation have discovered compelling commonalities. A prominent theme is a strong global suppression of metabolic rate, involving a regulated and coordinated reduction in all metabolic processes etc.) have shown that global metabolic rate suppression, mediated via reversible protein phosphorylation (RPP), is crucial for survival Studies from a range of animal models with various tolerances to different environmental stresses , and p38, as well as the downstream targets heat shock protein 27 (HSP27) and p53, in different tissues of lemurs by comparing torpid animals with control aroused animals. By utilizing an animal that can enter hypometabolism without the confounding effects of low Tb, the majority of cellular reorganization seen during torpor at warm Tb values can be attributed to active induction of metabolic rate depression.It has been well documented that mitogen-activated protein kinase (MAPK) signaling pathways are crucial in the regulation of the cellular stress response, and several studies have documented their role in hibernation at low body temperatures P < 0.05), 1.9 ± 0.1 (P < 0.01), 1.7 ± 0.2 (P < 0.01), and 1.4 ± 0.1 (P < 0.01) fold of those in controls, respectively (A). In contrast, the relative expression of HSP27 in skeletal muscle decreased significantly, being 72 ± 11% of the control value (P < 0.05). The phosphorylation of three targets in skeletal muscle also changed when comparing control and torpor conditions. During torpor, the ratio  of phosphorylation for HSP27 (Ser78) was significantly increased, being 1.4 ± 0.1 fold of that in control animals (P < 0.05), whereas the relative phosphorylation ratios of ERK1/2  and MEK (Ser217/221) phosphorylation were lower, 36 ± 10% (P < 0.01) and 27 ± 8% (P < 0.05) of those in control animals, respectively (B). In the heart tissue (A), ERK levels in the heart during torpor were 87 ± 2% of the control (aroused) value (P < 0.05), whereas JNK levels during torpor were 1.2 ± 0.1 fold of controls (P < 0.01), with no appreciable changes observed in other targets. Assessment of the relative phosphorylation ratios of the targets in the heart showed no significant changes (B).Using custom multiplex panels, which were validated to cross-react in both primate and rodent species , relative changes in total and phosphoprotein expression of ERK, MEK, JNK, p38, HSP27, and p53 were assessed in the skeletal muscle, heart, liver, kidney, brown adipose tissue (BAT), and white adipose tissue (WAT) by comparing control (aroused) and torpid lemurs. In skeletal muscle, relative protein levels of ERK, JNK, p38, and p53 were significantly increased in torpid animals, which are 1.2 ± 0.1 . However, the ratio of relative phosphorylated p53 (Ser15) was significantly higher  during torpor, whereas relative HSP27 (Ser78) phosphorylation was lower  (B). In kidney, the relative protein expression of HSP27 was elevated (1.4 ± 0.1-fold) during daily torpor (P < 0.05), the only one of the targets to show a significant change (A). However, the relative ratios of phosphorylation for three targets were reduced significantly and substantially in kidney (B) during torpor. The relative phosphorylation of MEK (Ser217/221), JNK (Thr183/Tyr185), and p38 (Thr180/Tyr182) decreased to 65 ± 9%, 71 ± 10%, and 64 ± 2% of control (aroused) values, respectively (P < 0.05).In the liver, total protein levels of all six targets were unchanged in response to torpor A. Howeve < 0.01) B. In kidt change A. Howeven kidney B during A), but phosphorylation of ERK1/2  and p38 (Thr180/Tyr182) were significantly elevated, being 1.3 ± 0.1 and 1.4 ± 0.1 fold of that in control animals, respectively . Data for WAT showed that total protein levels of ERK, p38, and HSP27 were significantly increased during torpor, which are 1.1 ± 0.1 (P < 0.05), 1.4 ± 0.1 (P < 0.05), and 1.1 ± 0.1 (P < 0.01) fold of those in control (aroused) animals, respectively (A). Furthermore, in WAT of torpid lemurs, all six targets exhibited significant elevation in phosphorylation as compared to control animals, with fold changes of 1.8 ± 0.3 , 2.3 ± 0.2 , 1.5 ± 0.2 , 1.4 ± 0.2 , 1.4 ± 0.1 , and 1.8 ± 0.1 , in relative to total protein expression (B).No significant changes were observed for any of the six total protein targets analyzed in BAT during torpor A, but phectively A. Furthepression B.Tb over a wide range of ambient temperatures. However, during periods of food restriction, often associated with cold or dry conditions, the high cost of metabolic heat production can be detrimental to survival. Accordingly, some mammals display significant heterothermy and/or use torpor as means to conserve energy during periods of severe environmental stress Tb, often to near ambient temperature, as well as a suppression of multiple physiological functions  Tb at the nadir of daily torpor in the mouse lemurs used in the present study was only 30–33 °C. Hence, studies of torpor in the gray mouse lemur offer the option to explore the regulation of a relatively pure reduction in metabolic rate, without the confounding influence of low temperature.Mammals have the ability to maintain a constant high core To date, the majority of research on mammalian torpor and hibernation has focused on species from seasonally-cold environments such as bats and a variety of rodents including ground squirrels, hamsters, chipmunks, and marmots, whose seasonal heterothermy is necessitated by the subzero winter temperatures that must be endured The ERK signaling pathway is initiated by the activation of G-proteins, which leads to the sequential phosphorylation and activation a three-tiered cascade involving a MAPK kinase kinase, a MAPK kinase and finally the MAPK. For the ERK cascade the three kinases are RAF kinase (MAPKKK) → MEK (MAPKK) → ERK (MAPK). Upon activation by phosphorylation at Thr202 and Tyr204, ERK1 and ERK2 function as potent Ser/Thr kinases that phosphorylate a wide array of downstream targets. To date, approximately 160 different ERK substrates have been discovered with the majority of downstream proteins involved in regulating cellular proliferation, growth, and survival Interestingly, both BAT and WAT showed increases in relative ERK1/2 phosphorylation during torpor, indicative of increased ERK action. The thermogenic BAT is the main source of heat for rewarming the body during the arousal phase of torpor/hibernation, whereas WAT is the primary metabolic fuel storage organ Like ERK1/2, both JNK and p38 are activated via phosphorylation by a direct upstream MAPK kinase and are primarily involved in stress-activated cellular responses. Although both JNK and p38 cascades can be activated through similar stimuli, each cascade elicits distinct downstream responses The third subfamily of MAP kinases, p38, is also activated through similar extracellular stimuli as JNK and, as such, it was predicted that the regulatory pattern of p38 would be similar to that observed for JNK during torpor. Total p38 protein expression was upregulated in skeletal muscle and WAT; however, active phosphorylated p38 (Thr108/Tyr182) increased in BAT, and WAT, while decreasing in the kidney . As mentWhen comparing the regulation pattern of the three MAP kinase subgroups, no uniform pattern was discerned between tissues during torpor. In the skeletal muscle, decrease in relative phosphorylation of ERK and its upstream kinase MEK was most evident. By contrast, kidney showed no changes in total protein levels of any of the MAPKs (or MEK) but differed in relative phosphorylation of the kinases, displaying a notable decrease in phosphorylated JNK and p38 (as well as MEK). The most interesting findings of this study were the patterns observed in the adipose tissues during torpor. Both BAT and WAT showed significant increases in relative ERK and p38 phosphorylation, with WAT exhibiting an increased phosphorylation of all three MAP kinases (and MEK) during torpor. As mentioned earlier, both adipose tissues possess specialized functions that are critical for torpor, with BAT required for non-shivering thermogenesis and WAT as the primary source of metabolic fuel M. murinus, suggest important cellular roles for MAPK signaling in adapting the animal for torpor. The activation of these selected signal transduction pathways may influence the expression of a panel of torpor-responsive genes, which are critical to the regulation of distinct cellular processes that are necessary for survival in the metabolically-depressed state. Hence, the role of MAPK signaling may involve the execution of unique, tissue-specific survival programs that support torpor in this primate model. Given current efforts to sequence the genome of M. murinus, it is possible that future proteomic analysis of primate torpor will be able to identify torpor-responsive signaling pathways unique to either daily torpor or seasonal hibernation.The relative changes in both total and phosphorylated ERK1/2, JNK, MEK and p38 kinase expression observed in the organs of the hibernating gray mouse lemur, M. murinus) used for this study consisted of 8 adult females, 2–3 years of age (mean body mass 106.3 ± 15.5 g), that were born in the authorized breeding colony at the National Museum of Natural History of France . Standard procedures for animal holding, experimentation and sampling were conducted by Dr. Martine Perret and the Adaptive Mechanisms and Evolution team  as previously described Gray mouse lemurs  and receiver boards  placed in each cage. Body temperature was recorded for 10 s every 5 min whereas locomotor activity was continuously recorded.All animals in the present study were under winter short day conditions for at least 6 weeks before being transferred to individual cages (0.5 × 0.3 × 0.3 m) that were visually separated from each other and contained nest-boxes and branches. Cages were housed in a climate chamber where all mouse lemurs were maintained under short day conditions at a thermoneutral ambient temperature (24–25 °C) with a constant relative humidity (55%). n = 4 each for control and torpid groups). Lemurs in the torpor group were exposed to a calorie-restricted diet for 5 days  to enhance the depth of their torpor bouts. Control animals were also capable of entering torpor but were fed ad libitum. Tb and locomotion were monitored and used to determine the state of torpor, which was assessed as a continuously low Tb . Control animals were euthanized at the end of a daily torpor bout , whereas torpid mouse lemurs were euthanized during a torpor bout (when Tb was at its minimum). For the animals in the present study, the nadir Tb was 30.8 ± 1.6 °C (range 30–33 °C). Animals were euthanized by decapitation following approved protocols used by the MECADEV team. Samples of selected tissues were rapidly excised, immediately frozen in liquid nitrogen and subsequently air freighted to Carleton University on dry ice where they were stored continuously at −80 °C until use. All animal experiments were performed in accordance with the Principles of Laboratory Animal Care  and the European Communities Council Directive (86/609/EEC). All experiments were conducted under personal license (license No. C91-563) and the Internal Review Board of the UMR 7179. In accordance with the recommendations of the Weatherall report, “The use of non-human primates in research”, special attention was paid to the welfare of animals during this work. All efforts were made to minimize nociception. For transport, all tissues were logged as per Convention on International Trade in Endangered Species of Wild Flora and Fauna (CITES) regulations under CITES export permit No. FR1009118231-E and CITES import permit No. 10cA02291/QWH.Mouse lemurs were separated into two experimental groups . Lysis, assay, wash and resuspension buffers were all supplied by Bio-Rad (Cat. No. 171-304011).Two custom multiplex assay panels were prepared by Bio-Rad  and used to measure the total protein levels of ERK1/2, MEK1, JNK, p38, HSP27, and p53 as well as their phosphorylated forms including p-ERK1/2 , p-MEK (Ser 217/221), p-JNK (Thr183/Tyr185), p-p38 (Thr180/Tyr182), p-HSP27 (Ser 78), and p-p53 (Ser15). All antibodies assay panels used were previously demonstrated by the manufacturer to cross-react with both primate and rodent species  added. Samples were then frozen at −80 °C and thawed at room temperature. After another freeze-thaw treatment at −20 °C, homogenates were centrifuged at 4000 × Premixed coupled beads for all the protein targets were diluted by mixing with wash buffer. A 96-well filter microplate was then prepared by adding wash buffer to the desired number of wells and drawing the buffer through the plate by vacuum. A 50 μl aliquot of diluted coupled beads was then added to each well. After washing twice, 50 μl of sample lysate (protein concentration 500 μg/ml) was added to the wells and incubated overnight on a shaker. The detection antibodies (25 μl) were then added to each well and incubated for 30 min. The antibody solution was then drawn through the wells by vacuum pressure. After washing, 50 μl of 1 × streptavidin-PE (diluted in wash buffer) was added to each well and incubated for 10 min. Wells were then vacuumed and washed with 100 μl of resuspension buffer for a total of three washes. After washing, 125 μl of re-suspension buffer was added into each well and then data acquisition was carried out on a Luminex 100 instrument  with Milliplex analyst software .n = 4). Statistical analysis was performed using SigmaPlot (v.11) software using a two-tailed Student’s t-test. Differences were considered significant at P < 0.05 or P < 0.01.Data was collected as median fluorescent intensity (MFI) of the immunoreactive multiplex beads detected by the Luminex 100 instrument. All numerical data are expressed as means ± SEM (All authors contributed to the conception and design of the project and to the editing of the manuscript. MP and FP carried out the animal experiments. KKB, CWW, SNT, and JZ conducted biochemical assays. Data analysis and assembly of the draft manuscript was carried out by KBS, KKB and CWW. All authors read and approved the final manuscript.The authors have declared no competing interests.


Ligand activation of peroxisome proliferator-activated receptors (PPARs) prevents cardiac myocyte hypertrophy, and we previously reported that diacylglycerol kinase zeta (DGKζ) is critically involved. DGKζ is an intracellular lipid kinase that catalyzes phosphorylation of diacylglycerol; by attenuating DAG signaling, DGKζ suppresses protein kinase C (PKC) and G-protein signaling. Here, we investigated how PPAR-DGKζ signaling blocks activation of the hypertrophic gene program. We focused on export of histone deacetylase 5 (HDAC5) from the nucleus, a key event during hypertrophy, since crosstalk occurs between PPARs and other members of the HDAC family. Using cardiac myocytes isolated from Sprague-Dawley rats, we determined that liganded PPARs disrupt endothelin-1 (ET1)-induced nuclear export of HDAC5 in a manner that is dependent on DGKζ. When DGKζ-mediated PKC inhibition was circumvented using a constitutively-active PKCε mutant, PPARs failed to block ET1-induced nuclear retention of HDAC5. Liganded PPARs also prevented (i) activation of protein kinase D (the downstream effector of PKC), (ii) HDAC5 phosphorylation at 14-3-3 protein chaperone binding sites (serines 259 and 498), and (iii) physical interaction between HDAC5 and 14-3-3, all of which are consistent with blockade of nucleo-cytoplasmic shuttling of HDAC5. Finally, the ability of PPARs to prevent neutralization of HDAC5 activity was associated with transcriptional repression of hypertrophic genes. This occurred by first, reduced MEF2 transcriptional activity and second, augmented deacetylation of histone H3 associated with hypertrophic genes expressing brain natriuretic peptide, β-myosin heavy chain, skeletal muscle α-actin, and cardiac muscle α-actin. Our findings identify spatial regulation of HDAC5 as a target for liganded PPARs, and to our knowledge, are the first to describe a mechanistic role for nuclear DGKζ in cardiac myocytes. In conclusion, these results implicate modulation of HDAC5 as a mechanism by which liganded PPARs suppress the hypertrophic gene program. Cardiac hypertrophy is the increase in myocardial mass provoked by hemodynamic stress or myocardial injury, and is a convergence point for many risk factors leading to heart failure. If left unchecked, prolonged hypertrophy is maladaptive and gives rise to cardiac arrest and/or failure At the cardiac myocyte level, hypertrophy is characterized by increases in cell size, protein synthesis, and changes in gene expression 2+/calmodulin-dependent protein kinase Gene expression is regulated by the balance between histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. HATs promote transcription by acetylating nucleosomal histones to relax chromatin structure It is therefore well-established that a key event in the hypertrophic gene response is phosphorylation of HDAC5 Peroxisome proliferator-activated receptors (PPARs) have beneficial effects in the cardiovascular system. Activation of PPARs α The aim of this study was to determine the mechanisms by which PPAR-DGKζ signaling inhibits hypertrophic gene expression. Because PKC/PKD activity opposes the anti-hypertrophic actions of HDAC5 ET1, troglitazone, fenofibrate, GW501516, and antibodies against phospho-HDAC5 and β-actin were from Sigma-Aldrich . DGKζ and 14-3-3 antibodies were from Santa Cruz Biotechnology . PKCε antibody was from Millipore . HDAC5, PKD, and lamin B1 antibodies were from Cell Signaling Technology . Acetyl H3 antibody was from Actif Motif (Japan). R59022 was from EMD Biosciences, Inc. . The PKCε kinase activity assay kit was Enzo Life Sciences .The study is in full compliance with the Canadian Council on Animal Care, and approval was granted by the University of Manitoba Animal Care Committee (protocol 09-064). 1-day-old Sprague-Dawley rats were sacrificed by decapitation, ventricular myocytes were isolated by alternate cycles of 0.05% trypsin plus mechanical disruption as described Constitutively active (ca)PKCε was constructed by first cloning rat PKCε cDNA into a pCDH lentivirus vector . The enzyme was then made constitutively-active by deletion of residues 154–163 of its inhibitory pseudosubstrate domain Lentiviruses expressing small hairpin RNAs (shRNA) against DGKζ using shRNA-expressing plasmids  or caPKCε were prepared as previously described As applicable, myocytes were subjected to lentiviral infection. Myocytes were then rendered quiescent by serum deprivation (0.5% serum) for 24 h and pre-treated for 1 h with vehicle, troglitazone (1 µM), fenofibrate (10 µM), GW501516 (1 µM), or R59022 (10 µM).Following the 1 h pre-treatment, hypertrophy was stimulated by addition of ET1 (0.1 µM).Myocyte size was assessed by immunofluorescent microscopy and computer-assisted planimetry, as previously described Myocyte nuclear isolations were performed from 107 cells using a method adopted and modified from Cayman Chemical’s nuclear extraction kit . After treatments, myocytes were washed twice and suspended in phosphate-buffered saline (PBS) and centrifuged at 500×g for 5 minutes at 4°C. Cell pellets were re-suspended in a phosphatase inhibitor solution in PBS . After centrifugation at 500×g for 5 minutes at 4°C, cell lysis was performed by incubation with hypotonic buffer  for 15 minutes on ice. Supernatants containing the cytosolic fraction were removed after the addition of 10% Igepal and subsequent centrifugation at 1000×g for 10 minutes at 4°C. The remaining pellets were re-suspended by vortex in extraction buffer  and rocked at 4°C for 15 minutes twice with mixing by vortex in between. Supernatants containing the nuclear fraction were collected by pelleting at 14, 000×g for 10 minutes at 4°C. Total protein content was quantified from the resulting nuclear extracts using the bicinchoninic acid (BCA) method.Nuclear DAG levels were quantified from myocyte nuclear isolates by competitive enzyme-linked immunosorbant assay (ELISA) using a Rat DAG ELISA kit . In accordance with manufacturer’s instructions, 50 µg of total protein was incubated with DAG-HRP conjugate in a total volume of 160 µl in plates pre-coated with monoclonal anti-DAG for 1 hour at 37°C. Plates were washed five times and incubated with a proprietary HRP-substrate at 37°C for 15 minutes. After the addition of a stop solution the amount of DAG was measured at 450 nm.Cell lysates were prepared in RIPA buffer and clarified by centrifugation. Conventional western blotting of cytosolic and nuclear extracts was used to assess levels of DGKζ, native and phosphorylated HDAC5, and native and phosphorylated PKD. To assess physical interaction between HDAC5 and 14-3-3 proteins, HDAC5 precipitates were generated from equal amounts of cardiac myocyte nuclear extracts, and 14-3-3 was detected by western blotting.Cells were processed as previously described PKCε was immunoprecipitated from equal amounts of cardiac myocyte nuclear extracts. PKCε activity was assessed using a commercially-available ELISA-based PKC activity assay kit according to the manufacturer’s protocol.Myocytes were infected with lentivirus that expresses a MEF2-luciferase reporter . Myocytes were maintained in DMEM/10% CSC for 24 h, then serum-deprived for 24 h. Following treatments (as described in figure legends), luciferase activity was measured from lysates using a Luciferase Reporter Assay System (Promega).5′-CCGGACACCCAGCCCCAGGATA-3′ and 5′-GTCTCGCCTCCGCAAGCAGCTC-3′), β-myosin heavy chain (5′-TGGTGGAAGGCGGCGTACAGGT-3′ and 5′-CGCCTCCAGCCGCTCCTTCTC-3′), skeletal muscle α-actin (5′-GTCCAGCCCAGCCCTTCAGCAG-3′ and 5′-CCGGACCGGGCCGTATATGGAG-3′) or cardiac muscle α-actin (5′-GTCTGGGAGCCCCCTGGCTGAT-3′ and 5′-TGGACGGGGTCAGTTGGAGCAG-3′). Aliquots of chromatin obtained before immunoprecipitation were also analyzed (input control). Results are expressed as the relative amount of PCR product aliquots immunoprecipitated with acetylated H3, normalized by the input control.Cells were cross-linked with 1% formaldehyde, collected and lysed. Soluble chromatin was fragmented by partial digestion with micrococcal nuclease (NEB), and proteins were immunoprecipitated using an anti-acetylated histone H3 antibody. Following reversal of protein-DNA cross-linking, the DNA was purified by phenol/chloroform extraction and used for quantitative PCR with primers specific for the gene promoters of brain natriuretic peptide .We first verified the effects of liganded PPARs, and the role of DGK, using the DGK inhibitor, R59022. ET1 increased myocyte size, and this was attenuated by troglitazone, fenofibrate, and GW501516. The anti-hypertrophic actions of PPAR ligands were abolished by R59022 .ET-1  reduced HDAC5 content in nuclear extracts , and this was attenuated by troglitazone (1 µM), fenofibrate (10 µM), and GW501516 (1 µM) . RNA silOur previous report of PPAR-DGKζ crosstalk As the ability of PPARs to preserve nuclear HDAC5 levels relies on DGKζ activity and suppression of PKCε activity , we deteET1 induced activation of PKD, the downstream effector of PKC known to directly phosphorylate HDAC5 Binding of 14-3-3 chaperone proteins facilitates phosphorylation and export of HDAC5 from the nucleus and subsequent de-repression of pro-hypertrophic genes Repression of fetal cardiac genes by HDAC5 is achieved, at least in part, through association with MEF2 in vitroin vivoζ.Acetylation of histone H3 is a response to hypertrophic stimuli These findings provide insight into the mechanisms by which liganded PPARs suppress hypertrophic gene activation in cardiac myocytes. As depicted in et al. reported that PKC regulates the nuclear localization of DGKζ via phosphorylation of the MARCKS-homology domain, a motif which is similar to some nuclear localization sequences. However, this regulatory mechanism was restricted to only the PKC α and γ isoforms et al. detected 167 gene promoters with strict PPARγ binding during adipocyte differentiation in 3T3-L1 cells, yet PPRE consensus sequences were present in only 15 of these target genes Our finding that liganded PPARs increase nuclear DGKζ levels may be attributable to either their inhibitory effects on PKCε or their function as nuclear receptor transcription factors DGKζ overexpression suppresses cardiac hypertrophy through inhibition of PKCε et al showed that MC1568, impairs PPARγ signaling mostly in heart and adipose tissues Our notion, that preserved HDAC function is responsible for PPAR effects, is consistent with a previous report in which PPARγ-induced adipogenesis was attenuated by a chemical inhibitor of class II HDACs, MC1568 In addition to MEF2 sequestration, our data suggest a secondary anti-hypertrophic action of HDAC5. In myocytes, induction of fetal genes such as the atrial natriuretic peptide (ANP) and β-myosin heavy chain genes is associated with acetylation of histone H3 Other signaling mechanisms within the nucleus may also contribute to PPAR-induced repression of hypertrophic genes. For example, PPARs might disrupt HAT activity. Work by others suggests that the anti-hypertrophic actions of PPARα are related to suppression of AP-1 DNA-binding activity The present study shows for the first time that the ability of PPAR ligands to block activation of hypertrophic genes in response to ET1 involves PPAR/DGKζ crosstalk in the cardiac myocyte nucleus. The mechanistic contribution of DGKζ is likely due to attenuation of PKCε signaling by depleting its substrate, DAG. Retention of HDAC5 in the nucleus appears to be a consequence of attenuated PKCε and PKD signaling, leading to suppression of pro-hypertrophic events such as histone acetylation of hypertrophic genes and MEF2 activation. Taken in context with other studies on DGKζ, interventions that target PPAR/DGKζ signaling might represent a novel therapeutic approach to address the problem of cardiac hypertrophy.S1 FigureTroglitazone promotes recruitment of PPARγ and Sp1 to the DGKζ promoter.(PDF)Click here for additional data file.


Zizyphus nummularia (Rhamnaceae) is traditionally used as an anti-inflammatory agent. The current study aimed to explore the anti-inflammatory activity (in vivo) of a crude ethanolic extract (EE) and the pure identified octadecahydro-picene-2,3,14,15-tetranone (IC) in the root bark of Z. nummularia. IC was further subjected to suitable in vitro and in silico studies to find out the mechanistic pharmacology.The root bark of 2 (PGE-2) and tumor necrosis factor-α (TNF-α) production in PBS stimulated RAW 264.7 cells. Based on the observation of in vitro studies, finally, ADME prediction and molecular docking studies of IC were performed for better understanding of interaction of IC with TNF-α.EE  and (IC)  were subjected to in vivo anti-inflammatory assays to evaluate the anti-inflammatory activity and predict the probable mechanism(s) of action. Suitable acute  and chronic (cotton pellet granuloma) models were employed to investigate in vivo the anti-inflammatory activity. Based on in vivo observation, IC was further subjected to in vitro assays to estimate the inhibition of nitric oxide (NO), prostaglandin-Ep < 0.05) and arachidonic acid (p < 0.05) induced oedema, and the reduced the granuloma tissue formation (p < 0.05) in experimental mice. IC  exhibited significant (p < 0.01) inhibition of carrageenan, xylene and arachidonic acid-induced edema, and reduced the granuloma tissue formation. In in vitro assays, IC caused a concentration-dependent inhibition of LPS stimulated NO (up to ~ 67.4 % at 50 μM) and TNF-α (~84.5 % at 50 μM) production. However, the PGE-2 inhibition did not follow dose dependent pattern. Based on in vitro observations, the molecular docking has been performed on the basis of interaction with TNF-α. In in silico studies, it was observed that IC showed hydrogen bonding with GLN 47 amino acid residue of TNF-α protein.Oral administration of EE (100 and 200 mg/kg) exhibited significant inhibition of carrageenan (IC possibly produces anti-inflammatory activity through inhibition of TNF-α and NO production.The online version of this article (doi:10.1186/s12906-015-0942-7) contains supplementary material, which is available to authorized users. Therefore, the combination of ethnopharmacological literature and modern scientific tools including molecular docking is now believed to offer a holistic approach of novel drug discovery.Inflammation is the complex biological response to pathophysiological events mediated by various signalling molecules produced by leukocytes, macrophages and mast cells . During Zizyphus nummularia Aubrev. (Rhamnaceae), a thorny small bush or shrub, grows in abundance in the grazing lands of the arid and semi-arid regions of India. The plant is used in traditional medicine as analgesic, anti-inflammatory, antitussive, anthelmintic, and anti-cancer drug [Z. nummularia by experimental models. However, the root barks of different species under the same genus, Zizyphus, have been reported to possess significant anti-inflammatory activity [Zizyphus species, the present study was designed to rationalize the anti-inflammatory activity of the crude extract and of the isolated compound, octadecahydro-picene-2,3,14,15-tetranone, from the root bark of Z. nummularia employing in vivo animal models. The earlier reports regarding anti-inflammatory activity of some small molecules bearing similar pentacyclic structure [in silico study to predict its possible orientation at receptor level.cer drug –12. Leavcer drug –16. On oactivity –19. Constructure –22 encouZ. nummularia was collected in September 2010 from Durgapur, India and authenticated (Ref. CNH/I-I/20/2010/Tech.II/171) by Dr. V. P. Parsad, Taxonomist, Central National Herbarium, Botanical Survey of India, Shibpur, India. A voucher specimen (BCRCP/DP/PT/02/06) was deposited at Dr. B. C. Roy College of Pharmacy & Allied Health Sciences, Durgapur, India for future reference. The detailed methods of extraction, isolation and structure elucidation have been described in our previous publication [Root bark of lication . The strE. coli (serotype 0111:B4), dimethylsulfoxide (DMSO) and sodium nitrite were procured from Sigma . All solvents and reagents used were of analytical grade.Cell line RAW264.7 was obtained from SIGMA-RBI, Switzerland. Dulbecco’s Modified Eagle Medium (DMEM), phosphate buffered saline (PBS) and Griess reagent were procured from Invitrogen, Carlsbad, USA. Lipopolysaccharide (LPS), foetal bovine serum (FBS) from ad libitum. The animal experiments were conducted in accordance with the institutional animal ethical committee of Dr. B. C. Roy College of Pharmacy and AHS (Reg No: BCRCP/IAEC/8/2012).Swiss albino mice  were housed in standard polyprophylene cages (3 mice/cage) under standard laboratory conditions of 12:12 light–dark cycle, temperature (20 ± 2 °C), relative humidity (55 ± 5 %), standard diet  and water n = 6) and the acute inflammation was induced by carrageenan [Mice were divided into six groups  by applying on the inner and outer surfaces of the right ear 30 μl of following irritants: arachidonic acid 0.1 mg/μl in acetone [2 euthanasia and sacrificed by cervical dislocation. Both ears were removed and weighed. The data were represented as percent of oedema weight.Inflammation was induced in mice ( acetone  and xyle acetone . Thirty n = 6/group) of mice were treated with aqueous tween 80 (1 %), EE (100 and 200 mg/kg), IC (400 and 600 μg/kg) and aspirin (10 mg/kg) orally, once daily over 7 consecutive days. On day 8, the mice were subjected to CO2 euthanasia and sacrificed by cervical dislocation. The cotton pellet were removed, dried overnight at 60 °C and weighed.This study was carried out following the protocol of Ismail and co-authors  with lit2. The medium was routinely changed on alternate days. The cells were passaged by trypsinization (Trypsin-EDTA) to disrupt cell monolayer at confluence while splitting RAW264.7 cells for the routine culture and plating the cells for the in vitro assays.The RAW 264.7 cells were maintained in DMEM supplemented with FBS (10 %), glucose (4.5 g/l), sodium pyruvate (1 mM), L-glutamine (2 mM), streptomycin (50 μg/ml) and penicillin (50 U/ml) at 37 °C and 5 % CO5 cells/well) and incubated for 12 h  to allow cell attachment. The cells were then incubated with IC (1–1000 μM) at 37 °C and 5 % CO2 tension. The cell viability was assessed at 2 h and 4 h by MTT assay [The cytotoxic effect of IC was determined by cell viability assay. Briefly, Cell suspension was seeded into a 96-well plate (~4 × 10TT assay . IC did 5 cells/well) and incubated for 12 h  to allow cell attachment. The cells were then stimulated with lipopolysaccharide (1 μg/ml) and different concentrations of IC. Nitrite accumulation, an indicator of NO synthesis, was measured in culture media based on a diazotization reaction using the Griess reagent [Cell suspension was seeded into a 96-well plate  prediction methods. The compound was subjected to evaluation by the QikProp® (Version 3.2) module of the Maestro Schrodinger (MS) software for prediction of pharmacokinetic properties. IC was neutralized before being subjected to QikProp® analysis and significant pharmacokinetic properties consisting of principal descriptors such as mol_MW, SASA, FOSA, FISA, PISA, volume, donarHB, accptHB, QPlogPo/w, human oral absorption, percent human oral absorption, #rtvFG, CNS activity and finally Lipinski’s rule of five. The details of QikProp® properties and descriptors are listed in Table TNF-α inhibitory activity of IC in the in vitro assay was taken in to consideration to study the mode of inhibition of the selected TNF-α protein. The 3D structures of the IC was built using Maestro 9.0 build panel and prepared by LigPrep 2.3 version v23118 . The application uses Optimized Potentials for Liquid Simulations (OPLS) 2005 force field and energy minimized with Macromodel-v97110.A docking study was carried out at the receptor site of TNF-α protein to find out the putative binding mode of the isolated compounds. The crystal structure of recombinant human TNF-α with a resolution of 2.30 Å was retrieved from the protein data bank (PDB ID: 1A8M) . The strt-test using computerised GraphPad InStat version 3.05, Graph pad software, USA. The differences are considered significant when p < 0.05.The experimental data were statistically analyzed by the One-way Analysis of Variance (ANOVA) and expressed as mean ± S.E.M. followed by Dunnett’s p < 0.01) anti-inflammatory activity 3 h after carrageenan injection. On other hand, IC (400 and 600 μg/kg) treatment significantly (p < 0.05–0.01) inhibited carrageenan induced paw edema 2 h after carrageenan administration. The maximum inhibitory values of oedema at 3 h post-carrageenan were 16.2 and 17.9 % with the doses of 100 and 200 mg/kg of EE, respectively. However, IC (400 and 600 μg/kg) ensured maximum inhibition of 32.0 and 35.2 %, respectively, after 4 h of carrageenan injection. The anti-inflammatory effects of EE and IC were compared with the standard drug, aspirin (10 mg/kg), which exhibited significant inhibition of paw edema 1 h onward post-carrageenan treatment. Based on this observation, this activity might be attributed to the inhibition of the release of aforementioned inflammatory mediators.Carrageenan-induced inflammation model is a well-established method used for acute inflammation. The edema expansion follows biphasic responses. The early phase (within 1 h) is mediated by the discharge of autacoids, viz. histamine, kinins and serotonin, while the later phase (after 1 h) involves prostaglandins; the link between the two phases is provided by kinins . Oral adp < 0.05) suppression of arachidonic acid and xylene-induced ear oedema in mice, while the effect of IC (400 and 600 μg/kg) is more pronounced (p < 0.01)  Fig. . The actp < 0.05) inhibition of dry weight of the cotton-pellet granuloma. The inhibitory values for 100, 200 mg/kg of the EE were ~ 30.3 and 33.4 %, respectively. IC exhibited significant (p < 0.01) inhibition of granuloma tissue formation with inhibition values of 40.2 and 42.1 % for the doses of 400 and 600 μg/kg, respectively. The effect of IC (600 μg/kg) was found nearly comparable to that of positive control, the standard anti-inflammatory agent, aspirin (10 mg/kg), which exhibited an inhibitory value of ~ 43.3 %. The inhibitory effect of test materials may be due to inhibition of macrophage activation. Based on the results of in vivo studies, IC was further subjected to in vitro experiment to elucidate its possible mechanism.The inflammatory granuloma tissue formation is a feature of chronic inflammation. It evaluates the effects on macrophage dysfunction and granuloma formation . Macroph, PGE-2 and TNF-α production in RAW 264.7 cells were depicted in Fig. The effects of IC on LPS stimulated NOThe predicted value of Lipinski’s rule of five for IC is within the range of stipulated values. The predicted values of each individual parameters necessary for Lipinski’s rule of five  are also well within the recommended range Table  which in2 group of glutamine. This also correlates with the calculated values of accptHB of IC (5.5 in our case). Molecular docking study also reveals the best possible environment necessary for drug receptor interaction. The hydrophilic-hydrophobic domain of the predicted active site of TNF-α comprises of ARG 131, ASP 45, LYS 90, GLN 47, ASN 46, GLU 135, GLN 27, GLN 25, LEU 26, GLU 23 and GLY 24 amino acid residues (Fig. In the present assessment of ligand-receptor interactions using Glide, IC showed hydrogen bonding with GLN 47 amino acid residue Fig. . The G sues Fig. . The simZ. nummularia exhibited significant anti-inflammatory activity. We also observed that, octadecahydro-picene-2,3,14,15-tetranone isolated from the root bark of Z. nummularia exhibited significant anti-inflammatory activity. To predict the mechanism, in vitro assays were performed to see the effect of IC on LPS stimulated NO, PGE-2 and TNF-α production in RAW 264.7 cells. Among the tested mediators, IC significantly inhibited LPS stimulated TNF-α and NO production. However, the inhibitory effect on TNF-α production has been found more pronounced. Based on quantitative value/inhibition characteristics, molecular docking studies were performed on TNF-α protein. Molecular docking study further helped in supporting the observed TNF-α selectivity. Based on these observation, IC can be regarded as an anti-inflammatory agent with possible inhibitory effect on NO and TNF-α production. Therefore, the compound may have clinical potential for the treatment of inflammation in future.In the present study, we found that the root bark of


Coptis chinensis is commonly used in traditional Chinese medicine. The study investigated metabolic interaction of the active constituents  of Coptis chinensis in human liver microsomes. After incubation of the four constituents of Coptis chinensis in HLMs, the metabolism of the four constituents was observed by HPLC. The in vitro inhibition experiment between the active constituents was conducted, and IC50 value was estimated. Coptisine exhibited inhibitions against the formation of the two metabolites of berberine with IC50 values of 6.5 and 8.3 μM, respectively. Palmatine and jatrorrhizine showed the weaker inhibitory effect on the formation of the metabolites of berberine. Berberine showed a weak inhibitory effect on the production of coptisine metabolite with an IC50 value of 115 μM, and palmatine and jatrorrhizine had little inhibitory effect on the formation of coptisine metabolite. Berberine, coptisine, and jatrorrhizine showed no inhibitory effect on the generation of palmatine metabolite (IC50 > 200 μM). The findings suggested that there are different degrees of metabolic interaction between the four components. Coptisine showed the strongest inhibition toward berberine metabolism. Coptis chinensis), the rhizome of Coptis chinensis Franch from the Ranunculaceae family /(Km + [S]), where Vmax is the maximal velocity of formation, [S] is the concentration of the substrate, and Km is the substrate concentration at half-maximal velocity.Berberine, coptisine, or palmatine as the substrate  was used as a substrate, the other two constituents and jatrorrhizine were used as inhibitors. The final concentration of the constituent ofμL), followed by vortexing for 3 min and centrifugation at 20,000 rpm for 10 min at 4°C to remove the denatured proteins. The supernatant (20 μL) was injected into the HPLC  system. An Agilent series 1200 HPLC system was equipped with degasser, quaternary pump, autosampler, and UV detector. Chromatographic separation was achieved on an Agilent Eclipse XDB-C18  with mobile phase of 20 mM ammonium acetate and 0.1% formic acid in water (A)-methanol (B) at a flow rate of 1.0 mL/min. The gradient program was used as follows: 0–5 min, 20%B; 5–15 min, 20%B–35%B; 15–25 min, 35%B–45%B; and 25.1–30 min, 20%B. The column temperature was maintained at 40°C. The peaks were determined using a UV detector set at a wavelength of 354 nm.The reactions were terminated by the addition of ice-cold acetonitrile (200 50) values and enzyme kinetic parameters Km and Vmax were calculated using GraphPad Prism 5.04 . The intrinsic clearance (Clint) is evaluated according to CLint = Vmax/Km.All results are expressed as the mean ± standard deviation (SD) of the estimates obtained from the three different HLMs experiments performed in triplicate. The relative amounts of berberine, palmatine, and coptisine metabolites were expressed as the peak area of the metabolites formed. The percent inhibition was calculated from the ratio of the amount of metabolites formed with and without the specific inhibitor, and the 50% inhibitory concentration  of berberine was slightly inhibited by palmatine with IC50 values of 185 and 78.5 μM, respectively. The production of metabolites (B2) was inhibited by jatrorrhizine with an IC50 value of 28.5 μM, whereas jatrorrhizine had little inhibitory effect on the formation of B1 (IC50 > 200 μM)  of berberine to a similar extent with IC 200 μM) .50 value of 115 μM. In addition, palmatine and jatrorrhizine had little inhibitory effect on the formation of coptisine metabolite (IC50 > 200 μM) (Berberine showed an inhibitory effect on the production of coptisine metabolite with an IC 200 μM) .50 > 200 μM)  . Coptis chinensis  in human liver microsomes for the first time. In this study, two metabolites, one metabolite, and one metabolite of berberine, coptisine, and palmatine were observed by HPLC but no metabolite of jatrorrhizine was observed after incubation of the four constituents of Coptis chinensis in HLMs with NADPH. LC-MS/MS was used as a guide to identify these metabolites. B1 corresponded to an [M]+ ion at m/z 324, which was 12 Da less than that of berberine, suggesting that B1 was a demethylated ring-opened product of berberine. B2 had an [M]+ ion at m/z 322, which was a loss of 14 Da (CH2) compared with berberine, and the metabolite (C) of coptisine had an [M]+ ion at m/z 308, which was 14 Da (CH2) lower than that for coptisine, and the metabolite (P) of palmatine had an [M]+ ion at m/z 338, which was 14 Da (CH2) lower than that of palmatine. These findings were consistent with the results of some reports [This is investigation of metabolic interaction of the active constituents of reports –17 and sUsing recombinant human CYP enzyme and chemical inhibition analysis in HLMs, we found that berberine, coptisine, and palmatine were metabolized by CYP2D6, CYP3A4, and CYP1A2. CYP2D6 was the predominant enzyme involved in the metabolism of berberine (consistent with Guo's finding ) and cop in vitro intrinsic clearance (CLint) values for the formation of two berberine metabolites in HLMs were approximately 2 to 3-fold higher than those of coptisine and palmatine.The enzymatic kinetic studies revealed that the50 value of 115 μM. Palmatine and jatrorrhizine had little inhibitory effect on the formation of coptisine metabolite. Furthermore, berberine, coptisine, and jatrorrhizine showed no inhibitory effect on the generation of palmatine metabolite (IC50 > 200 μM). However, coptisine showed the strongest inhibition toward berberine metabolism. As described above, berberine was metabolized mainly via CYP2D6 in HLMs and produced two major metabolites (B1 and B2), while coptisine had a strong inhibitory effect on CYP2D6 with IC50 values of 4.4 μM [50 values of 6.5 and 8.3 μM in HLMs, respectively. It indicated that the production of B1 and B2 was decreased in liver due to CYP2D6 being inhibited by coptisine.In this study, we found that there were different degrees of metabolic interaction between the four components. Berberine showed a weak inhibitory effect on the production of coptisine metabolite with an ICf 4.4 μM . Coptisi Coptis chinensis are poorly absorbed when taken orally, resulting in low oral bioavailability [μM. However, this concentration can be reached in liver because of tissue distribution [The alkaloids inlability , 18, 19;ribution . Coptis chinensis inhibited the metabolism of berberine in liver and might increase its bioavailability. The present finding provides novel insight into the understanding of the metabolism-based synergistic mechanism of the coexisting constituents in herb.In conclusion, coexisting constituents (coptisine) in