Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC AQPs belong to a highly conserved group of membrane proteins called the major intrinsic proteins (MIPs) present in all type of organisms and involved in the transport of water and small solutes such as glycerol, nitrate and urea 23 and HAuCl4, that are prone to interact with sulfhydryl groups of proteins as mercurials, have been also shown to inhibit water permeability in plasma membrane from roots, and in particular AgNO3 has been reported to efficiently inhibit water permeability in human red blood cells (EC50 = 3.9 µM) in vivo using PET in vivo experiments There are at present very few reported AQP inhibitors that are suitable candidates for clinical trials and none of them showed specificity for AQP3 so far. Though various AQPs are inhibited by mercurial compounds, such as HgClcis-[PtCl2(NH3)2] (cisplatin) 2]Cl  in vitro, was also selected. For most of these compounds, the mechanisms of pharmacological actions are still poorly understood Within this frame, we decided to reconsider metal-based compounds as possible AQP inhibitors, and we report here the inhibitory effect on the water and glycerol permeability mediated by AQP1 and AQP3 of a series of metal complexes based on different transition metals. The selected compounds are metal-based drugs already known to possess different therapeutic properties as anticancer, antirheumatic and antibacterial agents. Among them were the anticancer drug The effect of the compounds was tested by a stopped-flow method on human red blood cells (hRBC) that specifically express large amount of AQP1 and AQP3 The effect of different metallo-drugs based on Pt(II), Ru(III), Ag(I), Au  was test2, a well-known inhibitor of aquaporin activity, tested in the same conditions. The obtained results demonstrated that the Au(III) complex Auphen is the most effective of the series on glycerol permeability, and far more effective than the mercurial compound . For control hRBCs, the osmotic water (fP) and glycerol (glyP) permeability values were respectively (4.2±0.4)×10−2 cm s−1 (n = 5) at 10°C and (1.8±0.2)×10−5 cm s−1 (n = 5) at 23°C. As shown in Representative traces of stopped-flow experiments with control and hRBCs treated with even lower concentration of Auphen  showing the inhibition of the osmotic water flux or glycerol flux are reported in To assure the compound selectivity, the effect of Auphen was also tested on urea transport. Notably, no significant effect could be observed for both concentrations tested in comparison to the controls  . The abi50 value for Auphen was calculated as 0.8±0.08 µM.Following these promising results we further investigated the inhibition of glycerol transport through AQP3 evaluating the effect of the incubation time of hRBCs with Auphen. aE) for water and glycerol transport, a valuable parameter indicating the contribution of protein channels to permeation, was also estimated from an Arrhenius plot  and Auphen treated hRBCs . However, the aE for glycerol permeation increased ca. 54% when Auphen was present . Since the Auphen concentration used in the assay was higher than the IC50 (corresponding approximately to 80% inhibition), the increase in aE is in accordance with a blockage of the AQP3 channel. Regarding water transport, the observed variation on the aE was not significant (P>0.05); indeed, in hRBC the contribution of the channel pathway to the total water permeability is usually considered to be 90% while the bilayer adds the remaining 10% aE for water transport is predicted not to be significantly affected.The activation energy . As seen in 2. Indeed, mercury inhibition is likely to occur both via covalent modification to Cys189 located immediately after the extracellular entrance of the water pore of hAQP1 The results obtained with EtSH, as well as the known affinity of gold ions for binding to sulfhydryl groups of proteins, suggest that AQP3 inhibition by Auphen might involve direct protein binding of the Au centre to Cys residues as it has already been reported for HgCl50 should not be disregarded. The PC12 cells are much larger than RBCs; hence, the presence of a larger number of these bias reactive groups may eventually contribute to a higher decrease in the effective Auphen concentration available for AQP3 blockage.To further confirm the specific effect of Auphen on AQP3 glycerol transport, the inhibition of glycerol permeation by Auphen was also assessed on PC12 cells stably transfected with rat AQP1 or AQP3  [43]. Ce24)2Cl 2Cl2 and AuN3Cl cores, can undergo substitution of the chlorides with other nucleophiles present in physiological environment. Instead, Aucyclam is a gold(III) complex with a AuN4 chromophore which lacks of chemical activation resulting into poor reactivity and scarce biological (e.g. anticancer) effects 50 is 20-fold higher than observed for Auphen  complexes [Au(dien)Cl]Cl2]+ or [Au(phen)Cl(OH)]+, and [Au(dien)Cl]2+ or [Au(dien)(OH)]2+), were taken into account for the DFT evaluation of the Auphen and Audien binding at putative protein target residues through the calculation of thermodynamics of the ligand exchange process , Met (L = CH3SCH3), and His residues (L = CH3-Im), in different polarity media.The inhibition of AQP3 by Auphen and Audien is believed to be related to the coordination of the Au(III) centre to a protein residue in the pore, presumably cysteine, methionine or histidine side-chains  process  between 3S>CH3Im>CH3SCH3>CH3SH indicating the thiolate form of Cys as the most stable binding site for Au(III) complexes, and the only one showing a favorable reaction free energy with both Cl− and OH− ligands, together with the CH3Im which, however, show a much smaller reaction free energy. It must be noted that, while at physiological pH cysteine residues are predominantly in their neutral form, therefore appearing the least favorable target of Au(III) binding, calculations on the acidic dissociation of thioalcohol complexes gave negative free energy values and very low pKa's (below zero), indicating the facile deprotonation at neutral and even acidic pH with formation of a metal-thiolate complex  complexes is different from that for cisplatin and other Pt(II) complexes where the aqua-species are well known to be more reactive. This could be due to the differences in redox properties of the Au(III) compounds with respect to the Pt(II) complexes. Finally, the free energy for Cl− substitution by thiolate is slightly but significantly, 10–20 kJ mol−1, more favorable for Auphen than for Audien, suggesting a stronger binding of Aupen to Cys, in agreement with its experimentally observed higher AQP3 inhibition.It is worth noting that the reaction free energy for ClEscherichia coli glycerol facilitator (GlpF) Three homology models of human AQP3, namely hmod1, hmod2 and hmod3, were obtained by the use of freely available web-servers SWISS-MODEL, Phyre, and ESyPred, respectively, based on the structurally characterized The family of the aquaporins and the aquaglyceroporin subfamily are known to share a common protein fold. It comprises six membrane-spanning helices plus two half-helices with their positive, N-terminal ends located at the centre of the protein and their C-terminal ends pointing towards either side of the membrane. The helices surround the 20-Å-long and 3–4-Å-wide amphipathic AQP channel. AQPs are identified by two asparagine-proline-alanine (NPA) sequence motifs located at the ends of the two quasi 2-fold related half-spanning helices. The selectivity filter, a constricted region formed by four residues near the periplasmic/extracellular entrance, provides distinguishing features that identify the subfamilies. In water selective AQPs this region is smaller and more polar and contains a conserved histidine residue, while in aquaglyceroporins it is larger and more hydrophobic with two conserved aromatic residues The structure of our homology model of AQP3 was analyzed in detail and compared with the crystal structure of human AQP1 to characterize the different propensity of both proteins for the binding of the considered Au(III) complexes. Thus, the two AQP1 and AQP3 structures were analyzed by the SiteMap module of Maestro for the identification and characterization of protein binding pockets. Two major binding pockets were observed in both AQP1 and AQP3, and, as expected, the mapping retraced the cavities corresponding to the water channels identifying a binding pocket for each side of the protein, namely a periplasmic and a cytoplasmic pocket separated by the selectivity filter (SF) domain  [47].The SiteMap comparison of periplasmic pockets of AQP1 and AQP3 evidenced the main distinctive features of these protein surfaces. In particular, an extended hydrophobic region was detected in proximity of the SF of AQP3, but not in AQP1, the latter showing a markedly higher hydrophilic character. This result is in agreement with several literature evidences suggesting the different hydrophilic/hydrophobic balance on the periplasmic pocket as the major difference between orthodox aquaporin and aquaglycerolporin subfamilies, including AQP1 and AQP3, respectively Afterwards, the position and the chemical environment of the most suitable soft-metal ion protein binding sites, such as cysteine, methionine and histidine side chain residues, was analyzed in detail. First of all, it must be noted that AQP3 is characterized by a higher content of these residues  in comparison to AQP1 . The mapping of the periplasmic surface provided a valuable assessment of these possible gold-binding sites location and exposure on both aquaporin isoforms. In AQP1, Cys189 and His180 resulted to be the only residues proximal to the periplasmic pore, therefore, directly accessible to gold complexes. However, the exposure of these residues is quite scarce and, being part of the SF domain, they are located in the narrowest section of the membrane filter, which can be easily approached only by small molecules. Moreover, being the size filter function a conserved property of SF On the other hand, AQP3 is characterized by the presence of a MxxC motif located on the H1 domain, close enough to the periplasmic pocket to be potentially targeted by metal complexes. In this homology model, while Met37 is projected toward the outer space of the protein (quite far from the pore surface and probably in close contact with the membrane phospholipidic layer), the thiol group of Cys40 is projected toward the periplasmic space approaching the channel pore . The proi.e. Auphen and Audien and their monohydroxo derivatives, and through the calculation of the docking poses and their corresponding scoring we made a preliminary estimation of both the affinity and accessibility of these metal complexes to the targeted pores. In particular, the molecular geometries of the Au(III) complexes obtained at DFT level of theory were rigidly docked into the periplasmic pocket of both AQP1 and AQP3 by using the Glide algorithm. The results of docking studies are summarized in Although the Auphen and Audien inhibition is essentially irreversible, a preliminary docking calculation has been carried out to evaluate the accessibility of the proposed cysteine binding side of AQP1 and AQP3, trying to identify non-covalent binding poses and possible steric crashes. Thus, we considered the same species assumed in the DFT calculations, It is also worth noting that the Cys40 thiol group in AQP3 is in proximity (9–10 Å) and well-oriented with respect to the gold centre of top ranked poses , thus coWhile AQP1 is a selective water channel, AQP3 permeates both water and glycerol on human erythrocytes. In spite of being also a water-transporting channel 4, is stabilized by the presence of nitrogens on the phenantroline and diethylentriamine ligands. Auphen and Audien resulted to inhibit glycerol transport in hRBC with an IC50 = 0.8±0.08 µM and IC50 = 16.6±1.6 µM, respectively, while having only a modest inhibitory effect on water permeability. Moreover, the gold(III) compounds also resulted to be ineffective as inhibitor of urea transport on hRBC.In the present study we report on the screening of different known metallodrugs, with various therapeutic applications, for the inhibition of AQP1 and AQP3. Thus, the potent and selective inhibition of the glycerol permeability through AQP3 in hRBC by water soluble gold(III) complexes, namely Auphen and Audien, was observed, while the other metal complexes were completely ineffective as AQPs inhibitors. Both the effective compounds are tetracoordinated gold(III) complexes with square planar geometry in which the Au(III) oxidation state, at variance with the case of NaAuClaE for glycerol transport in hRBC with no concomitant aE change for water transport upon treatment with Auphen, points to a potent blockage of the AQP3 channel with a much lower effect on AQP1. A similar effect has already been reported in the case of Cu2+ and Ni2+ ions, which were shown to inhibit selectively AQP3 in transfected human cell lines, although at higher concentrations in the mM range 2, and most importantly they are not toxic for the cells in the entire range of investigated concentrations as suggested by the absence of hemolysis and by the recovery of activity after treatment with EtSH.The observed increase in the The specificity of Auphen towards AQP3 was further confirmed by assessing glycerol transport on PC12 cell lines transfected with either AQP1 or AQP3 from rat. The marked inhibitory effect of the compound for the PC12-AQP3 cell clone denotes its higher affinity for AQP3 residues.“prodrugs”2Cl250 value. Conversely, the chemically stable Au(III) complex Aucyclam shows lack of inhibition properties supporting the hypothesis that the gold centre is essential for inhibition and most likely is involved in protein binding.The time-dependent inhibition of AQP3 by Auphen is in accordance with the typical reactivity pattern of this gold(III) complex in aqueous solution. It is worth mentioning that, as commonly found for several other metallodrugs, gold(III) compounds behave as The mechanism of gold inhibition is probably due to the ability of Au(III) to interact with sulphydryl groups of proteins such as the thiolate of cysteine. This hypothesis is partly confirmed by the almost complete recovery of AQP3 activity upon treatment of hRBC with EtSH. However, other modes of Auphen binding might also occur (e.g. with histidine groups). In order to shed light on the putative binding modes of gold complexes to AQPs we used DFT analysis. Thus, DFT calculations on the reactions of Auphen and Audien with model side chain residues indicated the thiolate form of Cys as the most favorable binding site for Au(III) complexes both in the intact or mono-hydroxo species.To further investigate the mechanisms of AQP inhibition by gold compounds at a molecular level, we undertook molecular modeling studies. Initially, a homology model of human AQP3 was built and compared to the structure of human AQP1 via molecular modeling SiteMap analysis, allowing the identification and characterization of protein binding pockets. Our model confirmed the well-established presence of an extended hydrophobic area in the periplasmic region of AQP3, while a marked hydrophilic character was evidenced in the same region of AQP1. This important difference might account for a higher binding affinity of the Auphen complex for AQP3 with respect to Audien, resulting in higher inhibition potency, being the phenantroline ligand likely to establish hydrophobic interactions at the entrance of the glyceroporin channel. Most importantly, the mapping of the periplasmic surface allowed establishing the possible gold binding sites and their exposure on both aquaporin isoforms. In AQP1 none of the cysteine, methionine or histidine residues appear to be accessible for gold binding, including Cys189 previously reported to be the binding site of mercurial AQP inhibitors The investigation of non-covalent binding of Auphen and Audien and their monohydroxo species at AQP1 and AQP3 by docking approaches allowed to ascertain the possibility for the gold complexes to reach the SF domain of AQP3 in closer proximity with respect to AQP1, therefore allowing the compounds to bind at protein sites closer to the constriction pore of the aquaglyceroporin such as the side chain of Cys40. Indeed, it is known that the AQP1 channel cross-section size is the major determinant of selectivity for larger amphipathic molecules such as glycerol in vitroInterestingly, both Auphen and Audien have been previously reported to possess anticancer properties in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.In conclusion, metal compounds, in particular mercurial salts, have already been reported as inhibitors of AQPs, but have major drawbacks such as poor selectivity and extreme toxicity, which hamper their development as therapeutic agents. We described here the selective and potent inhibitory effect (in the nanomolar range) of two Au(III) complexes bearing nitrogen donor ligands on AQP3, which together with their limited toxicity and high water solubility makes them suitable candidates for future Gold compounds and NAMI-A were prepared according to literature procedures (see references throughout the text). The purity of the compounds was confirmed by elemental analysis, and all of them showed purity greater than 98%. Cisplatin, silver sulfadiazine, aurothioglucose and 2-Mercaptoethanol were from Sigma.Venous blood samples were obtained from healthy human volunteers following a protocol approved by the Ethics Committee of the Faculty of Pharmacy of the University of Lisbon. Informed written consent was obtained from all participants. Rat adrenal medulla pheochromocytoma cells (PC12) were kindly provided by Dr. M. Eschevarria, Virgen del Rocio University Hospital, Seville g for 5 min at 4°C and plasma and buffy coat were discarded. Packed erythrocytes were washed three times in PBS , diluted to 0.5% haematocrit and immediately used for experiments Venous blood samples, collected in citrate anticoagulant . Fresh blood was centrifuged at 750×2 (10%) incubator at 37°C. Geneticin at 0.2 mg/ml was added to culture AQP-overexpressing clones. Levels of AQP overexpression were determined by Northern blot analysis and expressed relative to wild type PC12. RNA analysis and functional characterization of these clones have been described in detail previously To obtain stable clones of PC12  that overexpress either rat AQP1 or rat AQP3, twenty micrograms of pcDNA3-AQP1 or pcDNA3-AQP3 were transfected into wild type PC12 by electroporation. After selection with geneticin sulphate (GIBCO) 40 clones were analyzed for levels of expression of either AQP1 or AQP3. Out of 20 positive clones with variable levels of AQPs expression, for each AQP those with higher expression were selected. PC12 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% foetal bovine serum, 10% horse serum, and 1% penicillin/streptomycin (Invitrogen) in a COhRBC mean volume in isotonic solution was determined using a CASY-1 Cell Counter  and was calculated as 82 fL. Equilibrium volumes of PC12 cells were obtained by phase contrast microscopy on an inverted microscope (Axiovert Zeiss 100M) equipped with a digital camera.3 for all the clones measured.Plated cells were dislodged by mechanical aspiration with pipette, washed and re-suspended in PBS. For each measured set of data, an aliquot of cell suspension was placed on a microscope slide and an average of 6 pictures with 4–6 cells each were taken and analyzed using NIH ImageJ software. Cells were assumed to have a spherical shape with a diameter calculated as the average of the maximum and minimum dimensions of each cell. Calculated volumes were 1076±123 µmfP), 100 µL of a suspension of fresh erythrocytes (0.5%) or PC12 cells (1.5×103 to 3.5×103 cells/mm3) was mixed with an equal volume of PBS containing 200 mM sucrose as a non-permeable osmolyte to produce a 100 mM inwardly directed sucrose gradient Stopped-flow experiments were performed on a HI-TECH Scientific PQ/SF-53 apparatus, with 2 ms dead time, temperature controlled and interfaced with a microcomputer. Experiments were performed at 23°C for glycerol permeability and at 10°C for water and urea permeability; for activation energy measurements temperatures were ranged from 10°C to 37°C. For each experimental condition, 5–7 replicates were analyzed. For measuring the osmotic water permeability (Pf was estimated by o/A)(1/Vw(osmout)∞)Pf = k ∞/(ΔI1+ΔI2), where ΔI1 and ΔI2 correspond to the signal changes with either a slow rate constant k1 or a fast one k2, was alternatively used to calculate fPglyP), 100 µL of erythrocyte or PC12 cell suspension was mixed with an equal volume of hyperosmotic PBS containing 200 mM glycerol creating a 100 mM inwardly directed glycerol gradient. After the first fast cell shrinkage due to water outflow, glycerol influx in response to its chemical gradient was followed by water influx with subsequent cell reswelling. glyP was calculated as o/A)Pgly = k . The inhibitor concentration necessary to achieve 50% inhibition (IC50) was calculated by nonlinear regression of dose-response curves  to the equation: y = ymin+(ymax−ymin)/(1+10((LogIC50-[Inh]) H)), where y is the percentage inhibition obtained for each concentration of inhibitor [Inh] and H is the Hill slope. The activation energy (aE) of water and glycerol transport was calculated from the slope of the Arrhenius plot (lnfP or lnglyP as a function of 1/T) multiplied by the gas constant R. All solution osmolarities were determined from freezing point depression on a semi-micro osmometer  using standards of 100 and 400 mOsM.For inhibition experiments cells were incubated with different concentrations of complexes, from freshly prepared stock aqueous solutions, for various times at room temperature before stopped-flow experiments. A time dependent inhibition assay for Auphen over several hours incubation with hRBC and PC12 cells showed no further increase of inhibition after 30 min at r.t. A similar time dependent inhibition assay was performed for all the compounds up to 4 hours incubation at r.t. except for the highly toxic HgClData were presented as mean ± standard error of the mean (SEM) of at least four independent experiments, and were analyzed with either the paired Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's test. A value of P≤0.01 was considered to be statistically significant.The structures of Au(III) complexes, i.e. Auphen and Audien, and their respective hydrolyzed forms were investigated at DFT level of theory with the B3LYP hybrid functional G°I, ΔG°II and ΔG°III, were obtained as reported in G° values obtained by this indirect procedure are expectedly subjected to less approximations than those calculated for the respective direct processes because the inclusion of empirical parameters such as the solvation energy of proton and chloride ion, characterized by large and negative values (and thus affected by larger approximation), is avoided. The corresponding values of apK for the acidic dissociation reaction II and III were calculated by the equation apK = ΔreaG°/2.303 RT, where R is the ideal gas constant and T is 298.15 K.The thermodynamics of Auphen and Audien aquation (I) and acidity of the corresponding aquo (II) and methanethiol (III) complexes was investigated by means of the thermodynamic cycles depicted in The Jaguar 7.7 quantum chemistry package was used for all calculations −1 Å−1, allowing to preserve the main features of the parent crystal conformation in the final structure of AQP1.The crystal structure of AQP1 was downloaded from the PDB archive (entry code 1H6I) The 3D structure of AQP3, not yet resolved, was obtained by homology modeling on accessible web-servers as follow. Initially, possible AQP3 homologues were searched using the HHmod web-server The three homology models were subjected to a Protein Preparation procedure by the same treatment employed for AQP1 (see above). After this step, the structures of AQP1 and hmod1-3 were superimposed in the Maestro workspace by using the protein alignment tool and then processed by the SiteMap module The SiteMap calculation on AQP1 was performed by essentially using the default parameters with the exception of the mapping resolution set to 0.3 to gain the maximum mapping accuracy.Docking calculations were carried out with Glide 3.5 that makes use of a series of hierarchical filters to calculate the ligand binding poses in the active-site region of the target Figure S1aE) values for water transport were not affected by Auphen treatment . The aE for glycerol permeation increased ca. 54% when Auphen was present .Arrhenius plots showing the water and glycerol permeability of hRBS, in the absence (control) or presence of 5 µM Auphen (30 min incubation at r.t. previous to permeability measurements). Activation energy ((TIF)Click here for additional data file.Figure S2Reaction of Au(III) complexes with soft-metal protein sites investigated at DFT level of theory.(TIF)Click here for additional data file.Figure S3Two-step formation of the Au–thiolate adduct between Auphen and Cys side chain.(TIF)Click here for additional data file.Figure S42) and Auphen (X = Cl), II) acidic dissociation of their respective aquo forms and III) acidic dissociation of corresponding thiol adducts, and the corresponding pKa values of the two latter processes. All free energy values were reported in kJ mol−1. Values of hydKPt and a1KPt, referred to the cisplatin first hydrolysis and to the acidity of the corresponding monaquo form, respectively, were taken from B. Lippert, “Cisplatin” 1999, Wiley-VCH, Weinheim, Germany, pp 184, 186.Calculation of reaction free energies in water for the I) aquation of Audien (X = NH(TIF)Click here for additional data file.Table S1−1.Calculated enthalpies and free energies for the reaction of Auphen and Audien with soft-metal protein sites in different polarity media. All values are in kJ mol(DOC)Click here for additional data file.Table S2Docking score of Auphen and Audien at periplasmic pocket of AQP1 and AQP3.(DOC)Click here for additional data file.Dataset S1http://www.ff.ul.pt/fct/dataset_s1.pdf.Calculated homology modelling structures (pdb format) of AQP3. Available at (PDF)Click here for additional data file.


Penicillium verrucosum, P. nordicum and Aspergillus carbonarius are three important ochratoxin A producing species. P. verrucosum is in addition able to produce citrinin. It has been shown earlier that P. nordicum is adapted to NaCl rich environments like salt rich dry cured foods or even salines. In this organism, the biosynthesis of ochratoxin A plays an adaptive role in this habitat. P. verrucosum generally can be found on cereals, but occasionally also on salt rich dry cured foods. In contrast A. carbonarius usually cannot be found in NaCl rich environments, but it occurs in another environment with high concentration of solutes, e.g., in sugar rich substrates like grapes and grape juices. Usually osmotic challenging conditions activate the HOG MAP kinase signal cascade, which in turn activates various osmo-regulated genes. In the current analysis, it could be demonstrated that in case of P. nordicum and P. verrucosum the NaCl induced production of ochratoxin A is correlated to the phosphorylation status of the HOG MAP kinase. Just the opposite was true for A. carbonarius. In this case, also higher amounts of NaCl in the medium lead to an increased phosphorylation status of HOG, but no increase in ochratoxin biosynthesis was observed. In contrast to the Penicillia, higher NaCl concentrations lead to a rapid cessation of growth by A. carbonarius. High glucose concentrations have much less impact on growth and the phosphorylation of HOG.  Aspergillus and Penicillium species. Because of the temperature and humidity preferences ochratoxigenic Aspergilli occur mainly in regions with elevated temperatures and occur on food commodities like coffee   ethan sulfonic acid, pH 5.3, and 0.5% glycerol) in the presence of 200 µmol/L acetosyringone (AS). The cells were further grown for 9 h before mixing with an equal volume of a spore suspension from P. verrucosum BFE808. This solution was plated onto cellophane sheets which were placed on the agar plates containing the co-cultivation medium (IM + AS supplemented with 5 mmol/L instead of 10 mmol/L of glucose). After the co-cultivation procedure at 25 °C for 36 h, the cellophane sheets were transferred to M-100 plates (55 mmol/L glucose and 30 mmol/L KNO3) plus mineral solution  and 1.5% agar supplemented with hygromycin B (100 μg/mL) as selection medium for fungal transformants and cefoxitin (150 μg/mL) to inhibit the growth of A. tumefaciens cells. After incubation for 10 days at 25 °C, the number of hygromycin B-resistant colonies was counted and analyzed further.DNA Fragments covering about 500 bp of the 5' and the 3' end of the tor pPK2 . The resEcoRI restriction enzyme digested DNA was separated on a 0.8% agarose gel and transferred onto a nylon membrane filter according to the method of Southern [hog was labeled in a PCR reaction using the primers HOG_Pdigitatum_kpl_for (5'-ATGGCGGAATTCGTGCGTG-3') and HOG_Pdigitatum_kpl_rev (5'-TTATGCGAATGCTTGTCCAGTAGC-3'). The nucleotide mixture contained 11-DIG-UTP. Hybridization and staining of the filter were performed according to the recommendations of the manufacturer of the DIG-UTP Labelling and Detection kit . For Southern blotting experiments the Southern . HybridiSouthern ). The 16P. nordicum, ensured by a consistent phosphorylation status of HOG, renders P. nordicum competitive under high salt conditions. The shift from citrinin towards ochratoxin A biosynthesis in P. verrucosum under high NaCl conditions, which is also regulated by HOG phosphorylation, make this cereal adapted organism partly competitive on substrates with high NaCl conditions and finally the uncoupling of ochratoxin A biosynthesis and HOG phosphorylation in A. carbonarius is apparently one of the reasons why this species is not adapted to NaCl environments. Therefore, one of the ecological reasons for ochratoxin A biosynthesis in Penicillium is apparently the maintenance of chloride homeostasis [A. carbonarius. The ecological reason for the production of ochratoxin A by A. carbonarius must be different. Glucose (or other sugars) as a typical osmolytic active constituent of its natural habitat only has a negative influence of the biosynthesis of ochratoxin A. Therefore, a reasonable ecological reason for the biosynthesis of ochratoxin A by A. carbonarius cannot yet be deduced. Taken together, the ochratoxin A biosynthesis in Penicillia is NaCl concentration dependent and regulated via the phosphorylation of HOG. The constant high production of ochratoxin A by eostasis . This is


Adherens junctions connect the actin cytoskeleton of neighboring cells through transmembrane cadherin receptors and a network of adaptor proteins. The interactions between these adaptors and cadherin as well as the activity of actin regulators localized to adherens junctions are tightly controlled to facilitate cell junction assembly or disassembly in response to changes in external or internal forces and/or signaling. Phosphorylation of tyrosine, serine, or threonine residues acts as a switch on the majority of adherens junction proteins, turning “on” or “off” their interactions with other proteins and/or their enzymatic activity. Here, we provide an overview of the kinases and phosphatases regulating phosphorylation of adherens junction proteins and bring examples of phosphorylation events leading to the assembly or disassembly of adherens junctions, highlighting the important role of phosphorylation switches in regulating their dynamics.  Adherens junctions (AJs) are cell-cell adhesion sites where calcium-dependent cadherin receptors bind with their extracellular domains to cadherins on opposing cells and with their cytoplasmic tails connect—via adaptors—to filamentous actin , 2. By eDuring the development and lifetime of an organism, cells frequently change shape and position relative to their neighbors. Hence, the ability of cells to regulate their adhesive interactions plays a key role during tissue morphogenesis, repair, and renewal , 7, 8. Din vitro and in vivo )-catenin , 27 and -catenin , 29.While it is most likely every kinase and phosphatase can recognize at least one docking site within the AJ, it is not currently known which of the kinases and phosphatases reside in AJ permanently and which are transient components, homing in to phosphorylate or dephosphorylate AJ proteins only under specific conditions. Even permanent residents may not always be active, as most kinases and phosphatases need themselves to be activated.Receptor tyrosine kinases are commonly activated by an external ligand, such as a growth factor or cytokine, which induces dimerization, cis-phosphorylation or autophosphorylation and activation of the catalytic domain , 125. ReAs will be discussed further below, some of the phosphorylation events serve to activate the kinases or phosphatases and others are inhibitory. One well-understood example of kinase activation is the mechanism of activation of Src. As reviewed in , the famβ-catenin (bind cadherin) and afadin (binds nectin). The following layer is composed of adaptor proteins, such as α-catenin and vinculin, which bind to the second layer adaptors and also bind F-actin. F-actin, along with actin-binding proteins, such as α-actinin, and actin regulators, such as cortactin, would be considered the last layer. Regulatory proteins, such as GAPs, GEFs, and GTPases, can be found throughout the AJ as reviewed in [The AJ can conceptually be divided into four layers . The firiewed in , 142.δ-catenin [There is evidence demonstrating both Y and S/T phosphorylation of proteins in all layers of the AJ . As illu-catenin  and seco-catenin , 147.β-catenin, which plays an important role in the Wnt signaling pathway as a cotranscription factor of TCF/LEF [β-catenin will reach the nucleus or not depends on whether it is phosphorylated by GSK3 and Casein Kinse I in the “destruction complex” [Some AJ proteins have additional functions in the cell, and phosphorylation is also involved in regulating their non-AJ roles . The mos TCF/LEF . Whethercomplex” . HoweverFor the phosphorylation events occurring within AJ an important question is how do they affect the target proteins?A phosphorylated tyrosine, serine, or threonine residue can affect a protein in three major ways: it can increase the affinity for another protein, it can inhibit a protein-protein interaction, or it can activate enzymatic activity. In proteins with an intramolecular interaction, phosphorylation and dephosphorylation can elicit a conformational change in the protein. AJ components provide examples of each type of these outcomes, as detailed henceforth. Tyrosine phosphorylation can create a docking site for an SH2 or PTB domain of a partner protein. For example, tyrosine phosphorylation of cadherin creates docking sites for the SH2 domain of the adaptor SHC1  and the β-catenin [ Examples of interaction inhibition by phosphorylation are also found both between different proteins and intramolecularly: tyrosine phosphorylation of VE-cadherin at certain residues prevents the binding of p120-catenin and -catenin ; phospho-catenin . Activation of the catalytic activity of tyrosine kinases and phosphatases by tyrosine phosphorylation has already been mentioned above . AnotherWe next address the question what are the ramifications of phosphorylation of AJ proteins on AJ structure and dynamics.β-catenin [Numerous experiments have been carried out over the years to address the role of Y and S/T phosphorylation in regulating AJ. Early experiments used broad-spectrum chemical inhibitors of kinases or phosphatases to conclude that phosphorylation negatively impacts cadherin function. For example, inhibition of S/T phosphatases by Okadaic acid or Calyculin-A was reported to lead to complete disassembly of AJ within an hour, and this disruption was attributed to an increase in S/T phosphorylation of -catenin . However-catenin , suggest-catenin . Consist-catenin . These ain vitro to be important for the recruitment of PI3K to AJ and the ability of cells to expand nascent cadherin-adhesive contacts [However, in more recent years, there is accumulating evidence for a positive role of phosphorylation in AJ assembly, mainly coming out of loss-of-function experiments of specific kinases. For example, SRC and FYN were found to be essential for the formation of AJ in mouse keratinocytes . Moreovecontacts . Along tcontacts , and S/Tcontacts .Hence, the emerging view is that it is not possible to generalize the effect of phosphorylation on AJ. With some phosphorylation events leading to the switching “on” of a protein or interaction and other phosphorylation events, even on the same protein, serving as a switch “off”, the effect of phosphorylation on AJ dynamics has to be examined on a residue-by-residue basis. After we delineate the effect of each individual phosphorylation event, we should be able to integrate this information into a single network of interconnected switches and perhaps then we can follow the global effects of a single phosphorylation switch, starting, for example, with hepatocyte growth factor stimulation .We close this paper by giving a few examples of cases in which the consequences of specific phosphorylation events are known. The phosphorylation events presented occur on proteins from each layer of the AJ as well as one cell polarity protein.β-catenin, whereas phosphorylation of S846 is said to inhibit the same interaction [β-catenin to E-cadherin is conducive to a stronger AJ structure. Tyrosine phosphorylation of VE-cadherin at two critical tyrosines, Y658 and Y731, is sufficient to prevent the binding of p120- and β-catenin, respectively [ Serine phosphorylation of residues S840, S851 and S853 in the C-terminus of human E-cadherin (likely by CSNK1E or PRKD1) increases the binding affinity towards eraction . Strongeectively . Phosphoectively . Ubiquitectively , 163. Eight tyrosine residues in the N-terminus of p120-catenin can be phosphorylated by Src . Upon ph Phosphorylation of S142 of zyxin is thought to result in the release of an intramolecular head-tail interaction . OpeningAs its name suggests, VASP is often found phosphorylated in cells. Three phosphorylation sites on residues S157, S239, and T274 are phosphorylated by PKA and PKG, as well as PKC , 170 andIn Drosophila epithelial cells the par-3 ortholog Bazooka is confined to AJ as a result of phosphorylation by either apical or basal polarity complexes [omplexes . At the omplexes , 176. Inomplexes . Recentlomplexes .From the examples presented above, it is clear that phosphorylation switches play a pivotal role in regulating AJ assembly and disassembly dynamics. At the same time it is also clear that our knowledge is only scratching the surface of the phosphorylation network regulating AJ. For the majority of known phosphorylation events in AJ, we know either of a kinase or of a phosphatase involved, but rarely do we know both. Furthermore, while traditional biochemistry techniques have facilitated the characterization of a handful of phosphorylation events on AJ proteins, phosphoproteomic data indicates that the majority of AJ proteins are phosphorylated on multiple serine/threonine and tyrosine residues . PhosphoPhospho-proteomics offers an unbiased and comprehensive snapshot of phosphorylation events, and several different approaches can be taken to elucidate phosphorylation switches in AJ: during normal assembly and maturation, following a signal for disassembly, or when a certain kinase or phosphatase is activated or missing (e.g., , 182–184β-catenin and p-120-catenin [While for the discovery and mapping of phosphorylation events in AJ, one wants to be as comprehensive as possible, when it comes to characterizing a particular switch the more specific the tools, the better. One example of a specific tool is phosphorylation site-specific antibodies, such as those recognizing individual phosphorylation events on -catenin , 188. An-catenin , 189–191Facing an ever-changing landscape of forces and signaling cues, a cell must respond rapidly by adjusting the strength of its AJs according to need. For this it relies on continuous turnover and assembly of core AJ components. Phosphorylation is particularly suitable for regulating the balance between assembly and disassembly as it is rapid and affects the AJ proteins directly. Feedback loops must guarantee a combination of phosphorylated residues at AJ that matches the requirements for a given condition. Experiments have shown that when it comes to phosphorylation both “all on” and “all off” treatments are deleterious to AJ. The challenge now is to elucidate the mechanisms by which the cell maintains a “just right” level of phosphorylation in AJ. While phosphorylation is probably the most prominent regulatory switch controlling cell adhesion, other switches, such as GTPases, lipids and proteases, do exist . A futur


A receptors (GABAAR), containing α4and δ GABAAR subunits, are thought to be activated by GABA spillover outside of the synapse following release resulting in a tonic inhibitory Cl− current which could account for up to 90% of total inhibition in visual and somatosensory thalamus. However, the presence of this unique type of inhibition has not been identified in auditory thalamus.Neural inhibition plays an important role in auditory processing and attentional gating. Extrasynaptic GABAA receptor constructs to elucidate the presence of extrasynaptic GABAARs using both a quantitative receptor binding assay and patch-clamp electrophysiology in thalamic brain slices. Intense [3H]gaboxadol binding was found to be localized to the MGB while whole cell recordings from MGB neurons in the presence of gaboxadol demonstrated the expression of δ-subunit containing GABAARs capable of mediating a tonic inhibitory Cl− current.The present study used gaboxadol, a partially selective potent agonist for δ-subunit containing GABAAR responses mediated by extrasynaptic receptors may be important in understanding how acoustic information is processed by auditory thalamic neurons as it ascends to auditory cortex. In addition to affecting cellular behavior and possibly neurotransmission, functional extrasynaptic δ-subunit containing GABAARs may represent a novel pharmacological target for the treatment of auditory pathologies including temporal processing disorders or tinnitus.Potent tonic inhibitory GABA The medial geniculate body (MGB) is the thalamic nucleus of the central auditory system serving to shape and/or gate information as it is passed on to auditory cortical neurons. Like other sensory thalamic structures, the MGB is considered more than a simple relay nucleus as evidenced by recent data showing important roles for MGB neurons in coding stimulus specific adaptation and processing temporally complex stimuli A receptor (GABAAR) is a heteromeric member of the cys-loop superfamily. It forms a Cl− permeable ion channel pore and serves as the primary inhibitory neurotransmitter receptor in the brain. Nineteen GABAAR subunits  are known, specific combinations of which form functional GABAARs. Extensively reviewed by others, GABAARs lacking the γ subunit and containing the δ-subunit (δ-GABAARs) are benzodiazepine insensitive, located extrasynaptically, show high ligand affinity, exhibit relatively slow desensitization and mediate a tonic inhibitory Cl− current ARs that mediate tonic inhibition and alter neuronal excitability are expressed in visual and somatosensory thalamocortical neurons, the dorsal lateral geniculate nucleus and ventrobasal complex, respectively The GABA4 and δ subunits within the same GABAAR construct appears required for tonic current activation and existing data indicate that these subunits preferentially co-assemble 4δ subunit mRNA in the MGB, suggesting that functional α4δ-GABAAR constructs could be present in rat auditory thalamus 4δ-GABAARs and tonic inhibition in the MGB.In thalamic nuclei, the incorporation of both the α2, δ-subunit containing GABAARs The present study used gaboxadol (formerly THIP), a δ-subunit specific agonist, which, when present at low (µM) concentrations, preferentially binds and activates non-γAll experiments were completed using Fischer Brown Norway (FBN) male rats maintained on an ad libitum diet and reversed light-dark cycle. Procedures were in accordance to protocols approved by the Laboratory Animal Care and Use Committee of Southern Illinois University-School of Medicine .FBN rats (11-months-old) were decapitated and brains were rapidly removed, rinsed in ice-cold phosphate buffer at 4°C (pH 7.4), frozen in powdered dry ice and stored at −80°C. Serial transverse sections were cut at 16 µm using a Leica CM1850 cryostat at −18°C. Selected sections were thaw-mounted onto Superfrost/Plus slides and stored at −20°C. Anatomical locations of the MGB were verified to match neural structures with those previously described 3H]Gaboxadol  was used with modified protocols from Milbrandt and Caspary 3H]gaboxadol: 10–400 nM and post-wash with buffers for 4 quick dips. Buffer solutions used were 50 mM Tris-citrate (pH 7.1). Non-specific binding was determined in adjacent sections by the addition of cold excessive GABA to the ligand buffer.[3H]-hypersensitive phosphor screens for 3–5 days at room temperature. The phosphor screens were scanned using a Cyclone storage phosphor system. The MGB was outlined and analyzed using OptiQuant image analysis software which provided tools for gray-scale quantification in digital light units (DLU). DLUs were then converted to nCi/mg protein using a standard curve generated from co-exposed 3H-embedded plastic standards  Dried slides were apposed to gaboxadol displayed high levels of binding at low ligand concentrations in the rat MGB (n = 4) (max of 151.11±21.47 nCi/mg protein and Kd of 194.64±36.92 nM in the MGB (mean ± SEM). 3H]gaboxadol binding in hippocampus. It is likely that other sensory thalamic areas, cortex, cerebellum, etc. known to express δ-GABAAR s would show similar levels of binding, but are not present in 3H]gaboxadol binding was seen in the MGB. This was in contrast to the relative absence of binding in neighboring structures, but consistent reports providing evidence for α4δ-GABAARs in the hippocampus 3H]gaboxadol binding in both MGB and hippocampus contrasts with the relative absence of [3H]gaboxadol binding at low ligand concentrations in other brain regions, consistent with the low levels of α4 and δ-GABAAR subunit proteingaboxadol binding results. These recordings suggest a qualitative similarity between 22-30-day-old and adult MGB neurons, but find a developmental increase in the amplitude of the tonic current. This increase in current amplitude likely reflects the developmental increase in δ-subunit containing GABAAR expression seen in the cerebellum and thalamus These findings strongly support the presence of functional αAR currents through feedforward or feedback inhibition (4δ-GABAARs have been shown to regulate neuronal excitability different from classical synaptic GABAARs in other sensory systems, they may prove to play a significant role in processing acoustic information. Enhanced inhibitory tone in auditory thalamic neurons may then serve to increase signal fidelity by decreasing “jitter” or noise level through hyperpolarization of the resting membrane potential 4δ-GABAARs in stimulus coding in sensory thalamus is sparse.GABAergic inputs onto MGB neurons from IC and mostly from TRN are likely to activate tonic GABAhibition  [1], [7]4δ-GABAAR activation on cellular excitability has been examined in the studies described above but understanding the role of this receptor subtype in terms of neurotransmission per se has received less attention. For example, hyperpolarization via α4δ-GABAAR mediated tonic inhibition has been shown to be involved in the transition from a tonic to burst response mode in thalamic neurons through the hyperpolarized potential's interaction with T-type Ca2+ channels 4δ-GABAARs is in mediating novelty detection through stimulus specific adaptation AR mediated hyperpolarizing currents. This type of regulation of firing rates in response to repetitive acoustic stimuli in the MGB may be similar to what is observed in stimulus specific adaptation in the IC and AC 4δ-GABAARs in the auditory system include, but are not limited to, the regulation of general cellular excitability, enhancement of temporal coding fidelity and novel stimuli detection. Future studies will focus on examining the role of α4δ-GABAARs in the auditory thalamus, providing insight into how tonic inhibition may contribute to the extraction and/or processing of meaningful sounds. Finally, this GABAA receptor subtype could provide a unique target for new therapeutic agents to treat tinnitus or improve speech processing in age-related hearing loss, two maladies thought to involve the selective down-regulation of inhibition as a function of partial peripheral deafferentation.The effect of α


Cross-reactive carbohydrate determinants (CCD) are carbohydrates chains in glycoproteins. Capable of binding human IgE from allergic they could play a role in the cross-reactivity between allergens from unrelated sources; their function still a matter of debate.Pollen is the most important cause of the production of IgE anti-CCD. Which have been described in trees and herbs. We investigated the relationship between clinical anaphylaxis to hazelnut and the sierological positivity to nuts allergen, grasses and role of interference by anti-CCD IgE.A six year old twins, atopic risk, father suffering from pollen asthma, reported by parents adverse reaction  to hazelnut at first assumption.Skin tests positive for nuts and grasses, no mite high RAST positivity for grasses, over 100 KUI/L, Alternaria 34 KUI/L, Phl-1 83,1KUI/L, Alt a-1- 43KUI/L, Cor a8 negative, Nuts negative, MUFXF3 ( CCD) 2.83 U/ml. Food Challenge after 1 year: negative, Now allergic rhinithis.IgE anti-CCD should not be able to connect mast cells and basophils and release inflammatory mediators. Patients with IgE restricted to CCD have not clinical symptoms. CCD can be considered as a potential interference in atopic diagnosis; especially in polisensitized. It 's important to provide clinical information to laboratory test. IgE -anti CCD create a misdiagnosis of allergies to foods, latex or venom, especially in pollinosis This can lead to unnecessary therapy.


Green tea has various health promotion effects. Although there are numerous tea cultivars, little is known about the differences in their nutraceutical properties. Metabolic profiling techniques can provide information on the relationship between the metabolome and factors such as phenotype or quality. Here, we performed metabolomic analyses to explore the relationship between the metabolome and health-promoting attributes (bioactivity) of diverse Japanese green tea cultivars.We investigated the ability of leaf extracts from 43 Japanese green tea cultivars to inhibit thrombin-induced phosphorylation of myosin regulatory light chain (MRLC) in human umbilical vein endothelial cells (HUVECs). This thrombin-induced phosphorylation is a potential hallmark of vascular endothelial dysfunction. Among the tested cultivars, Cha Chuukanbohon Nou-6 (Nou-6) and Sunrouge (SR) strongly inhibited MRLC phosphorylation. To evaluate the bioactivity of green tea cultivars using a metabolomics approach, the metabolite profiles of all tea extracts were determined by high-performance liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses, principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA), revealed differences among green tea cultivars with respect to their ability to inhibit MRLC phosphorylation. In the SR cultivar, polyphenols were associated with its unique metabolic profile and its bioactivity. In addition, using partial least-squares (PLS) regression analysis, we succeeded in constructing a reliable bioactivity-prediction model to predict the inhibitory effect of tea cultivars based on their metabolome. This model was based on certain identified metabolites that were associated with bioactivity. When added to an extract from the non-bioactive cultivar Yabukita, several metabolites enriched in SR were able to transform the extract into a bioactive extract.Our findings suggest that metabolic profiling is a useful approach for nutraceutical evaluation of the health promotion effects of diverse tea cultivars. This may propose a novel strategy for functional food design. Camellia sinensis L.) is a popular beverage worldwide, and because of its possible health effects, it has received considerable attention as a medicinal herb Natural products derived from medicinal plants are an abundant source of biologically active compounds, many of which have formed the basis for development of nutraceuticals and pharmaceuticals The functional biochemistry of plants is very diverse. The concentrations of many compounds vary widely, and metabolomic analyses are required to determine all metabolites in plant extracts. Among many analytical platforms, mass spectrometry (MS) is the most sensitive and selective technique, and thus it is the method of choice for metabolomic research on plants All the traditional cardiovascular risk factors  are associated with endothelial dysfunction Green tea has various health-promoting activities, and these activities vary from cultivar to cultivar. However, there is little information available for comparing numerous cultivars on the basis of their bioactivity. To properly utilize the nutraceutical properties of green tea, therefore, we need to clarify the relationship between cultivar and bioactivity. For nutraceutical evaluation, it is important to elucidate which cultivars have bioactivity, and which compounds contribute directly or indirectly to this bioactivity. In this study, we applied metabolic profiling techniques to evaluate the bioactivity of 43 representative cultivars of Japanese green tea . The aimO-gallate (EGCG) was obtained from DSM Nutritional Products . Epicatechin-3-O-gallate (ECG), epigallocatechin (EGC), and epicatechin (EC) were obtained from Mitsui Norin Co. Ltd. . Thrombin, myricetin, quercetin-3-β-D-glucoside (Que-glu), anti-β-actin antibody, superoxide dismutase (SOD), and catalase were purchased from Sigma-Aldrich . Caffeine and quercetin (Que) were obtained from Nacalai Tesque Inc. . Theanin was purchased from Taiyo Kagaku . Theobromine was obtained from Wako Pure Chemical Industries, Ltd. . Cyanidin (Cya), cyanidin-3-O-galactoside , cyanidin-3-O-glucoside (Cya-glu), delphinidin (Del), delphinidin-3-O-glucoside (Del-glu), and quercetin-3-O-galactoside  were obtained from ExtraSynthese . Petunidin-3-O-glucoside (Pet-glu) was purchased from Tokiwa Phytochemical Co., Ltd . Rabbit anti-phosphorylated MRLC (Thr-18/Ser-19) antibody was purchased from Cell Signaling Technology, Inc. . Rabbit anti-MLC2 (FL-172) antibody (against whole MRLC) was obtained from Santa Cruz Biotechnology, Inc. . Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody was purchased from Rockland Immunochemicals Inc. . Cyanidin-3-O-(6-O-(E)-p-coumaroyl)-β-galactoside  was kindly provided from Asahi Breweries Ltd. .Epigallocatechin-3-g for 10 min. The supernatant was filtered through a 0.2-µm syringe filter . We used polyvinylpolypyrrolidone  as a polyphenol adsorbent to remove polyphenols from the solution. The PVPP (100 mg) was swollen in 1 mL H2O for 30 min. After centrifugation at 300×g for 5 min, the supernatant was removed and the precipitated gel pellets (300 µL) were mixed with a one-half volume of tea extract (150 µL). After centrifugation at 300×g for 5 min, the recovered supernatant was used as PVPP-treated tea extract.We analyzed 43 major Japanese green tea cultivars , which a2 at 37°C. The level of MRLC phosphorylation was determined by western blot analysis using specific antibodies against phosphorylated MRLC at Thr-18/Ser-19 and antibodies against whole MRLC. For this analysis, the cells were incubated at a density of 2×105 cells/well in 24-well plates for 24 h. Then, the cells were treated with each 1% tea extract or each polyphenol (10 µM) in EGM-2 medium supplemented with 5 U/mL SOD and 200 U/mL catalase for 20 min. These antioxidant enzymes were added to decrease the pro-oxidant effect of green tea polyphenols. Then, thrombin (0.5 U/mL) was added and the cells were incubated for 5 min. This method comprises the modified and optimized conditions based on our previous report Normal human umbilical vein endothelial cells (HUVECs) were obtained from Lonza  and were maintained in EGM-2 (Lonza) supplemented with 5% fetal bovine serum  in a humidified atmosphere with 5% CO4P2O7, 1 mM phenylmethanesulfonyl fluoride, 2 µg/mL aprotinin, and 1 mM pervanadate. Proteins were resolved on 10% SDS-polyacrylamide gels and then transferred onto a nitrocellulose membrane. The membranes were blocked with 2.5% bovine serum albumin and incubated with anti-phosphorylated MRLC (Thr-18/Ser-19) or anti-MLC2 antibody to evaluate phosphorylated or whole MRLC, respectively, followed by incubation with HRP-conjugated anti-rabbit IgG secondary antibodies. Epitopes on proteins specifically recognized by the antibody were visualized using the ECL Advance kit . Band intensities were quantified using NIH Image-J software . Samples were analyzed in triplicate, and representative data are shown in figures. The relative band intensity (phosphorylated MRLC/whole MRLC: pMRLC/MRLC) of each sample was calculated using data from triplicate analyses. The results are shown as a percentage, calculated from the amount of pMRLC/MRLC in treated cells (thrombin + tea extract or single compounds) compared with that in non-treated control cells (+ thrombin only). Data shown are means ± SEM (n = 3). In After stimulation, the cells were lysed in cell lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 30 mM Na2O (0.05% formic acid), and solvent B, methanol (0.05% formic acid). Solvent B was increased from 5% to 60% over 7.5 min, and further increased from 60% to 100% at 10.1 min, at flow rate of 0.1 mL/min. HPLC chromatograms of tea extracts (YB and SR) with or without PVPP treatment were obtained at UV 254 nm. The MS instrument was operated using an ESI source in positive ionization mode with survey scans acquired from m/z 70 to 700. Ionization parameters were as follows: capillary voltage, 4.5 kV; nebulizer gas flow, 1.5 L/min; CDL temperature, 200°C; heat block temperature, 200°C. Tea extracts were diluted 1∶10 with distilled water and 10 µM 4-hydroxybenzophenone (4-HB) was added as an internal standard. Samples were filtered through a 0.2-µm PTFE filter, and 3 µL was injected.All extracts were subjected to high-performance liquid chromatography (HPLC) with time-of-flight MS (LC-MS) analysis using a LCMS-IT-TOF instrument . For the PVPP test, both PVPP-treated and -untreated tea extracts (YB and SR) were analyzed. The instrument was fitted with a Luna 5u C18(2) 100A, 5 µm, 1.0×250 mm column . The oven temperature was 40°C. The conditions of the mobile phase were as follows; linear gradient, consisting of solvent A, Hhttp://masspec.scripps.edu/xcms/xcms.php) to extract and align peaks. Total tea extracts .For all LC-MS datasets, data were processed using the free software XCMS , and OPLS were chosen to create the prediction model. PLS, which can be described as the regression extension of PCA, was calculated using SIMCA-P+. PLS derives latent variables that maximize the covariation between measured metabolite data and the response variable (inhibitory activity) regressed against. This differs from PCA, which utilizes the maximum variation in the metabolite data matrix. OSC is normally used to remove uncorrelated variables or those orthogonal to inhibitory activity from metabolite data using the nonlinear iterative partial least-squares algorithm.http://www.tm4.org/mev/). This summarizes the Z-score values of 541 peaks, which shows differences in metabolite profiles among cultivars.A heat map was generated the statistical package MultiExperiment Viewer (MeV v4.6.1) . In particular, theanin and caffeine strongly contributed to the separation of groups along PC1, and theogallin contributed to the separation of groups along PC2. To further explore the metabolic differences among tea cultivars, we performed another PCA analysis using three representative tea cultivars: the non-bioactive cultivar Yabukita (YB), the bioactive cultivar SR, and the less bioactive cultivar Benifuuki (BF). YB is the most commonly consumed and widely distributed cultivar in Japan, accounting for 70−80% of all green tea consumed. In the bioactivity assay, YB was ranked 32/43 (inhibitory rate <0), SR was ranked 2/43 (inhibitory rate >100), and BF was ranked 18/43 (0<inhibitory rate<50). BF was also selected because it has reported biomedical activities in human models Another unsupervised multivariate analysis method, the PCA model, provides an overview of all observations or samples in a dataset 2 and the predictive ability parameter Q2 were 0.926 and 0.999, respectively (YB vs SR), or 0.921 and 0.999, respectively (BF vs SR). These results indicated that the OPLS-DA models were reliable. The OPLS-DA loading S-plot, a plot of the covariance versus the correlation in conjunction with the variable trend plots, allows easier visualization of the data. The variables that changed most significantly are plotted at the top or bottom of the ‘S’ shape plot, and those that do not vary significantly are plotted in the middle p (corr) > |0.8| were used to select variables that strongly contributed to the difference between the two cultivars. YB > SR was 116 peaks, YB < SR was 200 peaks, BF > SR was 158 peaks, and BF < SR was 145 peaks. As shown on the S-plot, Cya-glu, Cya-gal, and theobromine contributed to the differences between cultivars. These peaks could not be found in the PCA score plot, indicating that OPLS-DA can provide valuable information for clarifying which factors are responsible for differences among cultivars.Although the PCA model provided an overview of all observations or samples, the details of differences in each cluster remained unclear. The supervised method, OPLS-DA, was then used to isolate the variables responsible for differences among the three representative tea cultivars. The OPLS-DA score plots are shown in 2 (0.999) and Q2 (0.999), showed that the OPLS-DA model was statistically significant (p (corr) >0.8 were used to select variables that strongly contributed to differences between the two extracts. In total, 359 peaks were decreased or removed by PVPP treatment, suggesting that some of these peaks are bioactive components.Among green tea constituents, polyphenols are the most abundant and most active components for inhibiting diseases and related reactions. To examine whether polyphenols are involved in the inhibition of thrombin-induced MRLC phosphorylation by tea extracts, we removed polyphenols from samples using the polyphenol adsorbent PVPP. HPLC-UV chromatograms from the non-bioactive tea cultivar YB and highly bioactive tea cultivar SR are shown in nificant . This obnificant . Values p (corr) <|0.8| and w<|0.05| to select metabolites that strongly contributed to the differences between the two cultivars. A Venn diagram illustrating the proportion of unique and overlapping ion features in the three different tea extracts is shown in Taken together with this finding  and the 2 and the cross-validated correlation coefficient Q2, as well as the validation errors of estimation and that between measured and predicted values; these are known as root mean squared error of estimation (RMSEE) and root mean squared error of prediction (RMSEP), respectively. Generally, R2, which describes how well the data of the training set is mathematically reproduced, varies between 0 and 1, where 1 indicates a perfect fit between the model and the data. A prediction model is considered to be good when Q2 >0.5, and excellent if Q2 >0.9. The PLS regression model showed R2 = 0.392 and Q2 = 0.233 with RMSEE = 39.76  was improved by 251% (0.984/0.392), and the predictability was also improved  approach. This decreases the number of variables in the metabolite data matrix by removing those that are linearly unrelated  to the bioactivity matrix 2 and Q2 values of 0.983 and 0.353, respectively. However, the predictive ability of the OPLS regression model did not improve, as the value of RMSEP increased from 33.31 to 38.64.OPLS is a modified version of ordinary PLS. This model may be built with one PLS-factor, and thus is much simpler than the corresponding PLS model 2, Q2, and RMSEP values of the three regression models, we determined that the best bioactive predictive model  was that obtained from the PLS-OSC regression. In this model, variables that were highly relevant for explaining predicted bioactivity were also identified from VIP (variable importance in the projection) values (By comparing the R) values . Large V) values . Theanin) values  showed t) values . These e) values . The culTo further elucidate the potential role of theanin in the bioactivity of tea, we examined its effects on the ability of SR to inhibit MRLC phosphorylation in HUVECs . We addeWhile individual compounds may account for some bioactivity, a combination of compounds might be more effective with respect to the bioactivity of tea. Although successive OPLS-DA  and regrMetabolomic analyses of plants have been used to study genotype, production origin, manufacturing type, sensory evaluation, cultivation method, climatic variables, and postfermentation year Recently, Ku and coworkers used metabolomic approaches  to determine the effect of manufacturing type or cultivation method on chemical composition of a single tea cultivar (green tea or pu-erh tea) In this study on bioactivity of 43 green tea cultivars, Nou-6 showed the highest activity for inhibiting thrombin-induced MRLC phosphorylation in HUVECs . PreviouAt present, human studies suggest that consumption of green tea can reduce the risk of cardiovascular diseases such as atherosclerosis. However, the mechanisms underlying this, including direct involvement in MRLC phosphorylation, and the differences in bioactivity among various green tea cultivars remain unclear In the field of nutraceutical research, there are many reports on screening of plants and foods for positive bioactivity. However, there is little information about negative bioactive regulators. In addition to positive regulators in bioactive plants and foods, elucidation of negative regulators and the unique concomitant factors would be useful for the design of nutraceutical products for use in effective and safe functional foods. In that sense, combining metabolic profiling methods  with bioassays can yield information on both positive and negative bioactive compounds as mentioned above. In fact, several SR-specific biofactors were also found to be able to activate the non-bioactive cultivar YB . This diIn summary, the aim of this study was to investigate the relationship between metabolomic data and bioactivity  of diverse green tea cultivars. Our findings illustrate the usefulness of metabolic profiling with multivariate statistical analysis for evaluation of nutraceutical properties , for identification of bioactive green tea cultivars (Nou-6 and SR), and for identification of bioactive factors (polyphenols). Some bioactive factors that were enriched in SR were able to alter the function of extracts from YB, a major Japanese green tea cultivar, changing it from non-bioactive to active. Thus, the combination of a metabolomic approach and a bioassay, as a simple and effective methodology, may advance nutraceutical research. This has potential applications for the discovery of valuable plant cultivars and bioactive factors.Figure S1Chemical structures of tea constituents.(TIF)Click here for additional data file.Figure S2Chemical structures of theanin showing the highest VIP value.(TIF)Click here for additional data file.Figure S3Chemical structures of 9 biofactors capable of transforming non-bioactive cultivars into bioactive ones.(TIF)Click here for additional data file.Table S1List of MS peaks focused by successive OPLS-DA.(XLS)Click here for additional data file.Table S2List of the representative MS peaks contributing to the separation among three representative cultivars.(XLS)Click here for additional data file.Table S3List of MS peak intensity of all variables in 43 cultivars.(XLS)Click here for additional data file.Table S4List of MS peaks of large VIP values (>1) in bioactivity-predictive regression analysis.(XLS)Click here for additional data file.


Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines  and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC. Molecular targeted therapy, which aims at specific molecular derangements in cancer cells or their microenvironment, is currently standard treatment for patients with advanced hepatocellular carcinoma (HCC) The insulin-like growth factor (IGF) signaling pathway plays important roles in HCC tumorigenesis Activation of the IGF signaling pathway may increase cancer cell proliferation, stimulate aggressive tumor behavior in established cancers The present study sought to clarify whether inhibition of the IGF signaling pathway can enhance the efficacy of molecular targeted therapy in HCC. Effects of IGFR inhibition on the activities of IGF and downstream signaling pathways in HCC cells were determined. Potential synergistic anti-tumor activities between IGFR inhibition and other molecular targeted therapy were explored.The protocol for the xenograft experiments in this study was approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Taiwan University, and conformed to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health..HCC cell lines, including Hep3B, PLC5 and SK-Hep1, were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. Primary human umbilical venous endothelial cells (HUVEC) were cultured as described before 50 at 0.086µM The IGFR inhibitor used in this study was a small-molecule IGFR kinase inhibitor NVP-AEW541 (Novartis), with an in vitro kinase inhibitionIC50 values after drug treatment were calculated using CompuSyn software  based on the changes of absorbance measured by spectrophotometry . All the MTAs used in this study were tested in cell-free condition to confirm the lack of interference with the MTT kit reagents -2, 5- diphenyltetrazolium bromide) assay. The ICents see . The potApoptotic cell death was measured by flow cytometry  and Western blot analysis .Activity of IGFR and downstream signaling pathways, including the PI3K/AKT and MEK/ERK pathways, was measured by Western blot analysis. To explore the effects of IGFR inhibition on other pertinent signaling pathways, a phospho-antibody kinase array , which can simultaneously measure relative levels of phosphorylation of 46 kinase phosphorylation sites, was used  knockdown was used in combination with treatment by MTAs. The candidate si-Chk2 (ID: s22119), si-IGF1R-a (ID: s7211), si-IGF1R-b (ID: s7212) and scrambled nonspecific (negative-control#1) siRNA were purchased from Ambion . Cells were transfected with siRNA using the siPORT NeoFx transfection reagent (Ambion) and treated with the MTAs. The treated cells were then collected for subsequent Western blot or flow cytometry analysis.6cells. The mice were randomized to different treatment groups of MTAs  when the tumor volume reached approximately 100 mm3 (volume [mm3] = [width]2× length ×0.5). MTAs treatment was administered daily by gavage. Tumor volume and body weight were recorded every 4 days. Fresh-frozen tumor samples after MTAs treatment were collected to measure the levels of pertinent proteins by Western blot. Formalin-fixed, paraffin-embedded tumor samples after drug treatment were collected for immunohistochemical analysis of pertinent protein expression and tumor angiogenesis. TUNEL assay was performed to measure the extent of tumor cell apoptosis, as described before Tumor xenograft experiments were performed by using male BALB/c athymic (nu+/nu+) mice and Hep3B cells, which were inoculated subcutaneously at 1×10t test and ANOVA. Significance was defined as p<0.05.All data were representative of at least three independent experiments. Quantitative data are expressed as mean ± SD. Comparisons were analyzed using the Student's 50 of NVP-AEW541 was 2.22±0.55 µM for Hep3B, 2.78±0.67 µM for SK-Hep1, 3.27±0.50 µM for PLC5, and 5.70±0.54 µM for HUVEC. These IC50 levels greatly exceed the IC50 of IGFR inhibition (0.086 µM by in vitro kinase assay) The growth-inhibitory effects of the IGFR inhibitor NVP-AEW541 on HCC cells and HUVEC were determined by MTT assay. The ICTo explore the possible effects of IGF in the tumor microenvironment on cell growth and drug resistance, IGF1 or IGF2 was added to the culture medium of HCC cells and HUVEC. Addition of IGF1 or IGF2 (100 ng/ml) can activate IGFR and AKT signaling in all the HCC cells tested  and HUVEC to a similar extent. This activation can be inhibited by NVP-AEW541 in a dose-dependent manner . HoweverWe hypothesized that IGF stimulation can increase resistance of HCC cells to MTAs. As shown in In the next step we explored the potential synergistic anti-tumor effects between IGFR inhibition and other MTAs. Synergistic growth-inhibitory effects in Hep3B cells were found when the IGFR inhibitor NVP-AEW541 was combined with sunitinib but not with sorafenib. This synergistic effect was similar with or without IGF stimulation in the medium. By contrast, no significant synergistic growth-inhibitory effects were seen in HUVEC .The syneAlthough AKT signaling is considered an important mechanism of drug resistance in cancer cells, our data indicated that modulation of AKT signaling activity did not change the anti-tumor synergy between NVP-AEW541 and sunitinib see . ResultsThe anti-tumor synergy between NVP-AEW541 and sunitinib was also confirmed by xenograft experiments . CombinaThis study sought to explore the roles of IGF signaling pathway in molecular targeted therapy for HCC. Our data suggest that IGF can increase resistance of HCC cells and HUVEC to molecular targeted therapy. The small-molecule IGFR inhibitor NVP-AEW541 can enhance apoptosis of HCC cells induced by selected MTAs, such as sunitinib. The apoptosis-enhancing effects of IGFR inhibition may involve different downstream signaling pathways, including Chk2, when IGFR inhibitors are combined with sunitinib.Although inhibition of IGF signaling pathway has shown promising anti-tumor activity in pre-clinical studies, clinical trials of IGFR inhibitors, including neutralizing antibodies to IGF, receptor-blocking antibodies, and small-molecule IGFR kinase inhibitors, have only limited success An important issue in optimizing treatment targeting the IGFR signaling pathway is identification of pertinent predictive biomarkers for treatment efficacy. Genetic aberrations of IGFR pathway in HCC tumor cells are rare Our data indicate that Chk2, a critical regulator of DNA damage response, may contribute to resistance to selected MTAs. Chk2 activation can lead to cell cycle arrest, senescence, or apoptosis, depending on the cellular genetic context and types and intensity of DNA damage Resistance of cancers to IGFR inhibitors has been extensively studied. An important mechanism of resistance is the compensatory activation of related signaling pathways. The PI3K/AKT and the MEK/ERK pathways are the most extensively studied. AKT signaling has been demonstrated to be a critical mechanism of drug resistance in HCC cells Over-expression of IGF-2 in the tumor microenvironment was hypothesized as another potential mechanism of resistance In conclusion, our data suggest that the IGFR signaling pathway plays important roles in resistance of HCC to MTAs. Multiple downstream signaling pathways may contribute to the resistance phenotypes. Further dissection of the interactions between different signaling pathways is needed to identify the optimal combination strategy for future clinical trials.Figure S1The list of kinases measured by the Human phospho-antibody kinase array kit .(DOCX)Click here for additional data file.Figure S2Modulation of AKT signaling activity did not correlate with the anti-tumor synergy between NVP-AEW541 and sunitinib.(DOCX)Click here for additional data file.Figure S3Screening of phosphor-protein expression in HCC cells after treatment with molecular targeted agents.(DOCX)Click here for additional data file.Figure S4Increased Chk2 phosphorylation by sunitinib plus IGFR inhibition.(DOCX)Click here for additional data file.Figure S5Increased Chk2 phosphorylation contributes to the anti-tumor synergy in SK-hep1 cells between sunitinib and IGFR inhibition.(DOCX)Click here for additional data file.Figure S6The potential anti-angiogenic effects of IGFR inhibition and other MTAs.(DOCX)Click here for additional data file.Table S1Confirmation of the lack of interference with the MTT kit reagents.(DOCX)Click here for additional data file.


Gnetum gnemon L.) seed extract (MSE) containing trans-resveratrol  and other derivatives exerts various beneficial effects. However, its mechanism of action in humans remains unknown. In this study, we aimed to investigate beneficial effects of MSE in healthy adult males. In this double-blind, randomized controlled study, 30 males aged 35–70 years with ≤10% flow-mediated dilatation received placebo or 750 mg MSE powder for 8 weeks, and twenty-nine males (45.1 ± 8.8 years old) completed the trial. There was a significant difference in the melinjo and placebo groups. Compared with the placebo control, MSE significantly reduced serum uric acid at 4 weeks and 8 weeks . HDL cholesterol was significantly increased in the melinjo group. To clarify the mechanism of MSE for reducing uric acid, we investigated xanthine oxidase inhibitory activity, angiotensin II type 1 (AT1) receptor binding inhibition rate, and agonistic activities for PPARα and PPARγ. MSE, trans-resveratrol, and a resveratrol dimer, gnetin C (GC), significantly inhibit AT1 receptor binding and exhibit mild agonistic activities for PPARα and PPARγ. In conclusion, MSE may decrease serum uric acid regardless of insulin resistance and may improve lipid metabolism by increasing HDL cholesterol.Melinjo ( Gnetum gnemon L.) belongs to the family Gnetaceae, native to Indonesia. The tree is small to medium in size, 15–20 m tall, with evergreen leaves. The fruit-like strobilus consists of little skin and a large nut-like seed that is 2–4 cm long inside, with both the fruits and leaves being very popular in Indonesian cuisines.Melinjo , gnetin C , gnetin L (GC derivative), gnemonoside A (GC-diglucoside), gnemonoside C (GC-monoglucoside), and gnemonoside D (GC-monoglucoside) [Sar1-Ile8] angiotensin II, both in the absence or presence of the test compound, in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, and 0.1% BSA. Nonspecific binding was determined in the presence of 10 μM angiotensin II. Following incubation, the samples were rapidly filtered under vacuum through glass fiber filters  presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester . Thereafter, the filters were dried and measured for radioactivity in a scintillation counter  using a scintillation cocktail . The results were expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound was saralasin, which was tested in each experiment at several concentrations to obtain a competition curve from which its IC50 was calculated.Evaluation of the affinity of compounds for the human angiotensin II type 1 (AT1) receptor in the transfected HEK-293 cells was determined using a radioligand binding assay. The cell membrane homogenates  was used to transform the cells. 150 μL Buffer EC, 0.25 μg pPPARα-Gal4 , 1 μg pGal4-Luc, 1 μg pSEAP-control vector, and 18 μL Enhancer were added into a 1.5 mL tube, and the contents in the tube were stirred with the vortex for 10 s. Subsequently, after leaving for 3 min at 25°C, 25 μL Effectene was added to the tube. The contents were stirred with the vortex for 10 s and were left for 7 min at 25°C. The medium of the 60 mm culture dish was removed, and 4 mL fresh medium was introduced there during this time. Subsequently, 7 min later, 1 mL culture medium was added to the 1.5 mL tube and was suspended with a pipette. All the contents were dripped to the 60 mm culture dish, and the contents were incubated for 16 h at 37°C with the presence of 5% CO2.The COS-1 cells were collected by processing of trypsin, centrifuged at 1000 rpm for 3 min at 4°C. After removing the supernatant, the cells were seeded in 60 mm culture dishes at a density of 5 × 10μL medium for each well. The cells were then cultured for 1-2 h at 37°C with the presence of 5% CO2. The test samples (1.25 μL) were added to each well and were cultured with gentle stirring for 24 h at 37°C in the presence of 5% CO2.The transformed cells were collected by processing trypsin. The cells were centrifuged at 1000 rpm for 3 min at 4°C, and the supernatant was removed. The cells were suspended in 10 mL culture medium and were seeded in a 96-well plate with 125 μL) was removed from each well of the 96-well plate and was transferred to each well of a 96-well white plate. Thereafter, a solution for measurement of the luciferase activity was fused at 37°C, and the 100 μL solution was added to the 100 μL medium of the rest in each well. Each luminescence activity was measured after reacting for 35 min in a dark place. 25 μL 1 × dilution buffer was added to each 25 μL medium, which was collected from the 96-well plate. It was stirred gently and left for 30 min at 65°C. Thereafter, they were cooled down to 4°C and then back to 25°C. 90 μL assay buffer was added to each well, was gently stirred, and was left for 5 min at 25°C. 10 μL/MUP solution was added to each medium, and it was gently stirred. After reacting for 60 min at 25°C in a dark place, the fluorescence intensity  based on 4-methylumbelliferone was measured.The medium , flow-mediated dilatation (FMD), and HOMA-IR (a biomarker of insulin sensitivity) .P < 0.05; P < 0.05; Healthy volunteers with administration of 750 mg MSE powder revealed a significant decrease in the uric acid levels at four weeks , suggesting that MSE decreases the serum uric acid levels by a mechanism other than the xanthine oxidase suppression. Next, we explored the possibility whether MSE inhibits the reabsorption of uric acid in the renal tubules. The inhibition of angiotensin decreases the serum uric acid levels by suppressing the reabsorption of uric acid from the renal tubular epithelia [in vitro investigation to evaluate the inhibitory activity of MSE on angiotensin as well as GC, which revealed that MSE and GC have a significant inhibitory activity on AT1 receptor binding, whereas trans-resveratrol revealed no inhibitory activity. These data suggest that MSE inhibits the angiotensin signal, which then downregulates the transporter of uric acid; however, we cannot rule out the possibility that other pathways are involved in the regulation of uric acid because we have not evaluated all the relevant regulatory pathways.In order to clarify the mechanism of MSE in decreasing the serum uric acid levels, we investigated the potential inhibition of uric acid synthesis and uric acid reabsorption in the renal tubular epithelia. With regard to the synthesis of uric acid, we first investigated the inhibitory activity of MSE on xanthine oxidase. Allopurinol, a well-known chemical compound used for the treatment of gout, effectively inhibited the xanthine oxidase activity with IC23 μg/mL . Howeverpithelia . In thisP < 0.05). It is well known that HDL cholesterol transports the deposited cholesterol from atherosclerotic lesions of blood vessels to the liver [Although we failed to detect any beneficial effects of MSE on LDL cholesterol, we found a significant increase in the HDL cholesterol levels in healthy volunteers who consumed 750 mg MSE powder for eight weeks as indicated in he liver , and it α and PPARγ because these receptors can increase the HDL cholesterol levels [α and PPARγ  [trans-resveratrol doses was between 10 mg/day and 1,500 mg/day in the RCTs [trans-resveratrol would not have contributed to the increase of HDL cholesterol level in MSE group. However, it might be possible that trans-resveratrol was one of the ingredients in MSE that affected the parameters in our study since the effects of trans-resveratrol on serum uric acid and HDL-cholesterol still remain unclear.About the effects of ls (RCT) . In addithe RCTs , while ttrans-resveratrol.Since the relation between hyperuricemia and metabolic syndrome has been pointed out these days , our stu


GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary  AMP-activated protein kinase (AMPK) is an evolutionary conserved heterotrimeric serine/threonine kinase that plays a central role in sensing and regulating energy homeostasis at the cellular, organ and whole-body level , although the involved mechanisms are not entirely clear. The known inhibitory effect on mitochondrial ATP generation may simply increase cytosolic ADP/ATP and AMP/ATP ratios 2O2 was reported to activate AMPK in vitro in combination with high glutathione levels 2O22O2, e.g. in human fibroblasts, may at best reach the low nanomolar range in vivo would occur rather slowly and at low levels In vivo, protein glutathionylation is rather facilitated by specific enzymes that may constitute a dynamically regulated S-glutathionylation cycle More recently, S-glutathionylation of Cys299 and Cys304 in the AMPK α-subunit via exposure to the strong oxidant HSchistosoma japonicum belonging to the Mu-family is well known as the GST-tag used in fusion proteins to favor solubility and purification of proteins GSTs occur as a large superfamily of mitochondrial and cytosolic proteins. In mammals, there are seven classes of cytosolic GSTs, including the Alpha-, Mu-, and Pi-families −) While this kind of redox regulation relies exclusively on GST-protein interactions, the catalytic activity of GST is required for its role in protein glutathionylation. By hydrogen bonding of glutathione to their active site tyrosine, GST-Alpha, -Mu and -Pi enzymes decrease the pKa of the glutathione thiol group (R-SH) and thus favor thiol deprotonation to form the highly nucleophilic thiolate anion  and AMPK α2T172Dβ2γ1 mutant (221TD) is described in Cytosolic yeast two-hybrid (Y2H) systems, Cyto-Y2H Rat liver was obtained from animals anesthetized with sodium pentobarbital  according to the protocol approved by the Grenoble Ethics Committee for Animal Experimentation (no. 36_LBFA-LK-01). Liver tissue was immediately extracted in 10 mM HEPES pH 7.4 , 0.2 mM EDTA) and centrifuged twice (1000 g and 12 000 g for 10 min each) to obtain soluble proteins in the supernatant for pull-down and immunoprecipitation. Protein concentrations were determined according to Bradford 2, 10% glycerol, 6 g/L BSA, 0,5% Tween 20, 0,02% NaN3) before addition of Glutathione Sepharose beads and incubation for an additional hour at 4°C. Where indicated, 1 mM glutathione was included. Sepharose beads were washed eight times and resuspended in SDS sample buffer. For immuno precipitation, 1 mg protein from liver extracts were reacted with anti-GSTM  or GSTP1  antibody (1∶240) in PD-buffer overnight at 4°C. Protein A Sepharose was added, incubated for another hour at 4°C, and washed 8 times before being resuspended in SDS-PAGE sample buffer. Solubilized, denaturated proteins were subjected to SDS-PAGE and immunoblotting using anti-AMPKα primary antibody  and anti-rabbit secondary antibody  for detection with a chemiluminescence kit  and a CCD camera . Bands were quantified densitometrically by Image J (imagej.nih.gov/ij) and normalized. Statistical analysis was done by students T-test. Where indicated, proteins stained in PAGE gels with colloidal Coomassie Blue were identified by MALDI-TOF/TOF mass spectrometry.For pull-down assays, either 30 µg of purified recombinant protein  at a flow rate of 20 µl/min. AMPK diluted to different concentrations just prior to measurements was injected onto the GST surfaces for 180 to 300 s at 20 or 30 µl/min which excludes mass transfer limitations (not shown). Experimental curves were corrected for bulk refractive index changes. Fitting of association and dissociation curves for kinetic analysis was done with BIAevaluation software. GSTs were identified by MALDI-TOF/TOF and peptide mass fingerprinting, potential phosphosites by LC-MS/MS as described in Methods S1.For SPR with BIAcore , GSTs were covalently immobilized by standard amine coupling  on the carboxylic functions of two different chips. Gold chips functionalized by mixed self-assembled monolayers as described r shifts due to glutathionylation.AMPK 221WT  in 0.1 M phosphate buffer pH 6.5 was incubated with or without 10 mM glutathione alone or together with GSTM1 or -P1 (0.5 μM) for 10 min at 30°C. Alternatively, AMPK  was incubated with EDTA (1 mM) at 30°C alone or together with GSTM1 or -P1 . After 15 min, 0,1 mM glutathione was added for 2 or 4 min. The reaction was stopped by heating in SDS sample buffer and samples separated by non-reducing SDS-PAGE and immunoblotted using primary anti-glutathione antibody  and anti-mouse secondary antibody  for luminescent detection and quantification as described above. Blotted proteins were also visualized by Ponceau staining to reveal M32P]ATP (specific activity 400 mCi/mmol ATP), 50 μM AMP, 5 mM MgCl2, 1 mM DTT, and 10 mM HEPES (pH 7.4). Recombinant CamKKβ (1.25 nM) was added and samples were incubated for 3 min at 30°C. The reaction was stopped by heating in SDS sample buffer and AMPK phosphorylation at Thr-172 as an indicator of AMPK activity was probed by SDS-PAGE and immunoblotting with anti-phospho-T172 AMPKα primary antibody  and anti-rabbit secondary antibody for luminescent detection and quantification as described above.AMPK 221WT (25 nM) was incubated for 10 min at 30°C with or without glutathione (10 mM) and in presence or absence of GSTM1 or -P1 (125 nM) in kinase buffer containing 200 μM ATP were added and the mixture incubated for 2 min at 37°C. Kinase reactions were stopped as above, separated on SDS-PAGE and analyzed by Typhoon phosphoimager .To analyze GST phosphorylation S. japonicum GST (GST-Sj). In fact, GST-Sj alone can pull down AMPK 221WT  did not interact with AMPK α -subunits in the sensitive Cyto-Y2H assay . HoweverTo test whether also endogenous rat GST isoforms bind to AMPK, we used crude rat liver extracts for co-immunoprecipitation and GST pull-down assays. Liver contains mainly GST-Alpha and -Mu isoforms and few GST-Pi. Endogenous rat AMPK indeed co-immunoprecipitated with antibodies specific for GSTM1/2 and GSTP1  and pullin vitro experiments with surface plasmon resonance spectroscopy (SPR). The GST interaction partner was chosen for covalent immobilization since it appeared more stable in this setup. We first used a sensor chip where the gold surface had been functionalized with a self assembled monolayer that reduces non-specific adsorption to the surface and allows immobilization of low ligand densities for analyzing GST-Sj. Sensorgrams with AMPK 221WT, 221TD or BSA (negative control) injected onto this surface confirmed a direct GST/AMPK interaction  and the very slow dissociation  resulting in a KD of about 5 nM.To obtain quantitative data on the GST/AMPK interaction in respect to kinetics and affinity, we performed a series of eraction  that waseraction . Conventeraction  and to eeraction . The simeraction  as seen In vitro phosphorylation assays with CamKKβ-activated AMPK and a 50-fold excess of GSTs revealed no phosphorylation of GST-Sj and slow, very low level phosphorylation of GSTM1 and GSTP1 as compared to ACC  of GST/AMPK complexes, we first examined whether they lead to GST phosphorylation and/or affect GST activity. d to ACC , reachind to ACC . Presencarent Km , Fig. S3arent Km . Thus, GIt has previously been shown that GSTM1 and -P1 were able to modulate signal transduction through an interaction with JNK and/or other stress activated kinases . Hence, In vitro phosphorylation of AMPK 221WT at αT172 by its upstream kinase CamKKβ was identical in presence or absence of glutathione, and also not further modified by GSTM1 or -P1 . It does not involve the AMPK α-subunit, as we have recently reported for fumarate hydratase in vivo regulation of AMPK activity.GST-mediated glutathionylation and activation of AMPK may be considered a possible additional layer of AMPK regulation linking the energy-stress sensor to redox regulation and anti-oxidative defense. Our present data are novel in that they provide a mechanism for glutathionylation-dependent AMPK activation at low oxidative capacity, as compared to the highly oxidative conditions used in an earlier study Figure S1The Strep-tag in Strep-GST constructs is phosphorylated by AMPK. Phosphorylation of GSTP1(200 pmol) and GSTM1 (40 pmol) in Strep-tagged  and Strep-tag-free forms  by AMPK221 (4 pmol) activated by CamKKβ (1 pmol). In vitro phosphorylation for 10 min at 37°C was analyzed by SDS-PAGE and Typhoon phosphoimager (top panel) and control Coomassie stain for protein loading (bottom panel). Control lanes lack AMPK221 but contain CamKKβ.(PDF)Click here for additional data file.Figure S2Substoichiometric phosphorylation of GSTP1 by AMPK in vitro. (A) Phosphorylation time course of GSTP1 or ACC (200 pmol each) by AMPK221 (4 pmol) activated by CamKKβ (1 pmol). In vitro phosphorylation for 5 to 60 min at 37°C was analyzed by SDS-PAGE and Typhoon phosphoimager. Control lanes lack AMPK221 but contain CamKKβ. (B) Quantification of (A) using Image Quant TL, using normalization to maximal ACC phosphorylation and fitting to phosphorylation enzyme kinetics.(PDF)Click here for additional data file.Figure S3GSTM1 and -P1 are activated in complexes with AMPK in vitro. Enzyme activity of or 20 μg GSTM1 (A) or 30 μg GSTP1 (B) in absence or presence of 5 or 15 µg AMPK221WT at different concentrations of the model substrate CDNB and saturating glutathione concentrations.(PDF)Click here for additional data file.Figure S4Increased phosphorylation of AMPK downstream substrate depends on the presence of AMPK-activating upstream kinase CamKKβ. AMPK 221WT preactivated with CamKKβ in kinase buffer with cold ATP and glutathionylated with 0,1 mM glutathione in presence or absence of GSTM1 or -P1, both as described in 32P]ATP. In vitro phosphorylation assays were analyzed by SDS-PAGE, Ponceau protein staining (lower panel) and Typhoon phosphoimager (upper panel) are shown. Note: AMPK autophosphorylation in particular of the α-subunit.(PDF)Click here for additional data file.