Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.Expression of the catabolic network in  Escherichia coli is a widely studied model organism, not only because of fundamental interests, but also due to increasing societal demand for a number of (relatively reduced) products of its catabolism, such as organic acids and alcohols which can be obtained in high yield through metabolic engineering. E. coli can operate in three basic modes of metabolism: aerobic respiration, anaerobic respiration and fermentation. Depending on the availability of oxygen, or of alternative electron acceptors like nitrate and fumarate, E. coli switches between these modes and therefore requires a well-orchestrated gene expression repertoire to accomplish this switch E. coli’s preferred final electron acceptor, in its absence, alternative electron acceptors such as fumarate, nitrate, DMSO and TMAO can be used instead 2, which transfer the electrons to the quinone pool(s) in the respiratory chain via specific dehydrogenases. Quinols then transfer the electrons to the final electron acceptor oxygen, or to alternate acceptors, via quinol oxidase complexes or dedicated reductases The enterobacterium E. coli contains three different types of quinone: ubiquinone (UQ), demethylmenaquinone (DMK) and menaquinone (MK), where DMK is the biosynthetic precursor of MK. The redox midpoint potential of these three quinones is 110 mV, 40 mV and −80 mV, respectively Quinones are lipid-soluble electron, or better said ‘hydrogen’ carriers that reside in the cytoplasmic membrane and function as mobile redox carriers in the respiratory chain. Depending on the midpoint potential of a specific dehydrogenase/oxidase combination, we expect that the quinone that will preferably function as their mobile electron carrier, will differ anoxic redox control) is an indirect oxygen sensor and functions as a transcriptional regulator of the oxidative- and fermentative catabolism in E. coli54 His717 Asp576 Pi phosphorelay 717 of ArcB to the aspartate residue (Asp54) in the active site of ArcA The ArcB/ArcA two-component signal transduction system (in vitro experiments that oxidized ubiquinone (250 µM of UQ0) inhibits the kinase activity of ArcB, which led them to the prediction that ArcB activation will decrease in parallel with the lowering of the reduction level of the ubiquinone pool et al., in a detailed reporter-enzyme study of the Arc system in which the rate of oxygen supply to chemostat-grown cells was dosed with high precision, showed that the level of Arc activation is highest under specific micro-aerobic conditions with a second, but lower, maximum under anaerobic conditions et al. speculated that not only ubiquinone but also oxidized menaquinone may de-activate ArcB, which then might explain the decrease in ArcB activity at low oxygen-supply rates Georgellis and coworkers observed in E. coli. To this end our wild type E. coli (MG1655), which contains all three types of quinone, has been compared with mutants containing UQ only, DMK only, or a combination of DMK and MK only  present in the respective strains, using HPLC .The utations . A ubiCACells were grown in batch culture at 37°C using Evans salt medium with nitrilo-acetic acid (2 mM) and sodium phosphate buffer  to increase buffering capacity The extraction and analysis of quinones was accomplished essentially as described in Sharma et al. 2012 8H2 and UQ8, respectively, whereas peaks eluting at around 20.9 and 24.4 minutes revealed spectra of DMK8 and MK8, respectively. In the case of UQ8, both the reduced- and the oxidized form can be detected. For DMK8 and MK8 however, only the amount of the oxidized form can be quantified reliably, probably due to rapid auto-oxidation of the napthaquinones. The amount of quinones was calculated from the area under the peak by using UQ10 and MK4 as calibration standards.Before fractionating the samples with high-performance liquid chromatography  using a reversed-phase Lichrosorb  RP10 C18 column , the extracted quinones were re-suspended using a glass rod, in 80 µl ethanol. The column was equilibrated with pure methanol as the mobile phase at the flow rate of 2 ml/min. Detection of the quinones was performed at 290 nm for ubiquinone (UQ) and at 248 nm for napthaquinones (DMK and MK). The amount of each quinone species was calculated from the relevant area under the peak. Peaks were identified by UV/visible spectroscopy and tandem mass spectral analysis. The peaks around 8.6 and 13.7 minutes elution time showed spectra for UQet al.ArcA phosphorylation levels were measured with Phos-tag™-acrylamide gel electrophoresis and Western immunoblotting as described by Rolfe 2+-Phos-tag SDS gels. For gel preparation a 10% (w/v) solution of acrylamide/bis-acrylamide was used for the resolving gel, to which 5 mM Phos-tag-acrylamide  and MnCl2  was added. In the gel-electrophoresis device, the resolving gel was cast in the chamber until 75% of the gel chamber size. For the upper part of the chamber a stacking gel of 3% (w/v) acrylamide was used. The gel was placed in the running device with SDS running buffer Protein sample processing for the detection of the mobility shift of phosphorylated ArcA was carried out with polyacrylamide-bound Mnet al.Before loading the gel, samples were diluted to an optical density of approximately 4, at 600 nm, in a v/v mixture of 61.5% 1 M formic acid, 33% loading buffer and 5.5% 10 M NaOH, as described by Barbieri After blotting, the membrane was blocked with milk powder to prepare it for Western analysis. The membrane was first hybridized with a specific rabbit anti-ArcA antibody at a dilution of 1∶10,000. Secondly, a 1∶5,000 dilution of secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad), was added and visualized with a chemi-luminescence assay based on peroxidase activity. In this gel system the phosphorylated- and non-phosphorylated form of ArcA run at an apparent size of approximately 34 and 28 kDa, respectively. The relative level of ArcA phosphorylation was calculated by analysis of the relevant band intensities using a Phosphor-Imager and software for the analysis of Western blots from Image studio .E. coli, and the corresponding wild type strain, were physiologically characterized with respect to growth rate via optical density measurements at 600 nm. ΔubiE) and AV34 (ΔmenA) is similar under aerobic conditions, again showing the functionality of DMK in aerobic respiration In order to get more insight into the regulation of ArcB activity through the relationship between ArcA phosphorylation and the redox state of the three different quinone pools, three different mutant strains of ΔmenA), while anaerobically menaquinones are most abundant  (ΔubiE and ΔubiCA), indicating that menaquinone is particularly abundant anaerobically. These results are in agreement with the results of ΔubiCA) both aerobically and anaerobically, but not when fumarate is added as an external electron acceptor  . Both DMacceptor .E. coli MC4100 derivative ASA12 via binding of ArcA-P to a modified cydAB promoter, which controls expression of the reading frame of lacZ, and was engineered to be ArcA-P selective i.e. via Phos-tag SDS-PAGE, however, is a more direct method to measure this parameter (ΔubiE) under aerobic conditions, we conclude that demethylmenaquinone is also able to inhibit kinase activity of ArcB, just as ubiquinone does (In previous reports from our group arameter  [1]. We one does .0 in ArcB kinase activity inhibition ΔubiCA) and AV36 (ΔubiE) clearly show a role for napthaquinones in the regulation of ArcAB phosphorylation, such that the (oxidized) quinones (i.e. DMK and MK) decrease the level of ArcA phosphorylation. Furthermore, under anaerobic conditions the level of ArcA phosphorylation is consistently higher in AV36 (ΔubiE) than in AV33 (ΔubiCA), which indicates (D)MK to be sufficient for ArcB kinase inactivation under oxidizing conditions  clearly increases from aerobic to anaerobic conditions. Furthermore, a small increase in ArcA phosphorylation was observed in anaerobic conditions when the external electron accepter is omitted, irrespective of the type of quinone present . Therefonditions . Based o0 inhibits ArcB activity in vitro, the data on the relative level of ArcA-P under aerobic conditions suggests that ArcA phosphorylation is inhibited by increasing concentrations of (oxidized) ubiquinone. Furthermore, the data from anaerobic conditions supports this, as the oxidized/reduced ratio of the ubiquinones in AV34 (ΔmenA) is likely much lower than that of the menaquinones in AV33 (ΔubiCA). This assumption is based on the more negative redox midpoint potential of the demethylmenaquinone and menaquinone. Strikingly, under anaerobic conditions, the total concentration of ubiquinone is higher in AV34 (ΔmenA) than in the wild type. This is possibly caused by up-regulation of ubiquinone biosynthetic enzyme(s), or their activity, in AV34 (ΔmenA), so as to compensate for the absence of menaquinones. Additionally, the fact that the level of ArcA phosphorylation is slightly higher in AV34 (ΔmenA) than in the wild type (MG1655), may be due to the absence of (oxidized) menaquinones in this strain.Considering the way UQIn order to investigate the role of the napthaquinones in more detail and to further verify the above observations, it is essential to be able to differentiate between the reduced and oxidised forms of MK and DMK. So far, however, this latter issue has remained unsolved, due to a very high auto-oxidation rate of napthaquinones during their isolation and subsequent HPLC analysis. The total DMK and MK content are shown in 8H2 (or: the UQ8H2/UQ8 ratio), however, increased rapidly during exponential growth, due to the limited availability of dissolved oxygen at increasing cell densities 8H2 content started to decrease as a response to restoration of complete oxygen saturation (2), and therefore the level of ArcA-P is high  remains rather constant during all phases of growth in these batch cultures, which is according to expectations. The relative level of UQturation  due to aturation . This co is high . Under aThis difference between the aerobic- and anaerobic batch experiment is fully consistent with a role of both the ubiquinones and the napthaquinones in the regulation of ArcB activity. The napthaquinones presumably stay longer in an oxidized state when, due to limited oxygen inflow, the ubiquinones are converted to their ubiquinol form  2 cannot activate ArcB, which is in contrast to the results presented here and their own previous work  Science 292, 2314) While this manuscript was in preparation, Alvarez Figure S1Representative picture of a Phos-tag gel. The upper band represents the phosphorylated form of ArcA (corresponding to 35 kD) and the lower band represents the un-phosphorylated form of ArcA (corresponding to 28 kD). This gel shows samples from exponential-phase cultures grown under anaerobic batch conditions in Evan’s medium supplemented with 50 mM glucose and 1% (v/v) LB at 37°C. The lanes from left to right are loaded with A: ΔubiE, B: ΔmenA, C: ΔubiCA, D: MG1655 (wild type) and E: molecular weight marker.(TIF)Click here for additional data file.Table S1Calculations and comparison of total intensities of ArcA and ArcA-P per sample.(DOC)Click here for additional data file.Table S2Total DMK and MK content for MG1655 (wild type) under aerobic and anaerobic conditions.(DOCX)Click here for additional data file.


Although anterior cervical arthrodesis is an effective procedure for the treatment of cervical disorders, the method has some complications. Here, we describe this rare complication of cervical instrumentation with a literature review. A 23-year-old male patient was operated for a C6-C7 dislocation. At postoperative month 10, he presented with hemoptysis and dysphagia. Cervical roentgenograms showed anterior migrations of one broken screw and a plate-locking screw at the C6 corpus. One screw was missing. We concluded that the missing screw had perforated the esophagus and had been eliminated spontaneously through the gastrointestinal tract. No screw should migrate. Even loose screws should be noted in follow-up X-ray studies. If such findings are detected, a second operation for revision should be considered as soon as possible to prevent potentially fatal complications. Anterior cervical spine surgery has been used in the treatment of cervical disc herniation, trauma, neoplasm, spondylosis, osteomyelitis, and ossification of the posterior longitudinal ligament , 2. AlthA 23-year-old male patient was previously operated on for C6-C7 dislocation 10 months earlier in another institution. Surgical treatment included anterior discectomy and iliac crest graft placement at the C6-C7 level, untouched C5-6 level, and plate-screw fixation performed between the C5–C7 levels. Postoperative early roentgenogram after the previous operation showed error in operative design . Late roAt 10 months after the operation, he presented with hemoptysis and dysphagia. Cervical roentgenograms showed anterior migration of one broken screw and plate-locking screw at the C6 corpus. In Figures Chest, abdominal, and pelvic roentgenograms failed to reveal the missing screw. During a second operation, the missing screw was confirmed, and we found the head portion of the broken screw and the plate-locking screw. We also noted the empty screw hole in the cervical plate. We did not see esophageal perforation. Cultures were negative. Because we determined that fusion at the C6-C7 level was complete, all instruments were removed. Esophagography was normal, and a fistula test with methylene blue was negative. As a result, we concluded that the missing screw had perforated the esophagus and had been eliminated spontaneously through the gastrointestinal tract.Anterior cervical arthrodesis and instrumentation is an effective procedure for the treatment of cervical disorders, including cervical disc herniation, trauma, neoplasm, spondylosis, osteomyelitis, and ossification of the posterior longitudinal ligament , 2. AnteThere are some reports on the displacement of grafts and screws resulting in esophageal perforation. Pompili et al. reported a missing screw 2 years after anterior decompression from C4 to C7, and esophagoscopy was normal. They concluded that the screw had likely perforated the esophagus and was eliminated spontaneously through the gastrointestinal tract . Wong etEsophageal injuries can be divided into intraoperative and delayed postoperative complications. Intraoperative complications can occur at the time of surgery or during the early postoperative phase, such as a decubitus wound . BecauseManagement of esophageal perforation depends on the time of the laceration. Conservative treatment consisting of intravenous antibiotics, elimination of oral intake, and parenteral nutrition can be preferred for minimal defects . If the Although loosening of anterior cervical instruments is not rare, the number of patients reported with a missing screw following loosening is small , 6, 10. Although anterior instrumentation is safe for use during surgery to treat cervical spine disorders, this approach has some complications because of the complex anatomy of the anterior neck region.Failure of an anterior instrumentation system may cause rare serious complications, with possible damage to the esophagus, trachea, and vessels. For this reason, patients who undergo anterior cervical instrumentation should be followedup for a period of time to detect whether there may be a failure of the anterior plate screw system.No screw should migrate. Even loose screws should be noted in follow-up X-ray studies. If such findings are detected, a second operation for revision should be considered as soon as possible to prevent potentially fatal complications.


The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion. The Cas family protein NEDD9/HEF1/Cas-L has emerged as a critical regulator of cancer invasion and metastasis in a variety of different cancers The Cas family proteins include NEDD9, p130Cas, Efs/Sin and the most recently described member HEPL/CASS4 The substrate domains of both NEDD9 and p130Cas are highly tyrosine phosphorylated in response to adhesion to integrin ligands and as a result of constitutive activation of regulatory kinases including FAK and Src We recently reported that mouse embryo fibroblasts (MEFs) from NEDD9−/− mice have faster rates of focal adhesion disassembly and correspondingly migrate more rapidly on 2D surfaces Given the role of mouse p130CasY253 in the regulation of p130Cas-mediated cell migration, we questioned whether there may be a functional equivalent of this residue in NEDD9. To assess potential conserved tyrosines between the Cas protein family members we performed a global sequence alignment of multiple vertebrate isoforms  of the 4 Cas family members . A portiin vivo we analysed publicly available data at the PhosphositePlus website. This revealed frequent detection of phosphorylated NEDD9Y189 in high throughput mass-spectrometry based screens in a variety of different cancer cell types  to prevent phosphorylation . The effNext, we analysed the role of NEDD9 Y189 and NEDD9 Y214 in the promotion of cell migration. NEDD9−/− mouse embryo fibroblasts were transfected with pGFP.NEDD9, pGFP.NEDD9 Y189F and pGFP.NEDD9 Y214F and individual migrating cells were imaged by time-lapse microscopy. Analysis of the migration paths traced by the cells indicated that cells expressing NEDD9 Y189F elaborated extensive migration paths . QuantifHaving previously demonstrated that NEDD9 expression regulates focal adhesion dynamics To next confirm whether phosphorylation of Y189 is sufficient to regulate cell migration speed, we tested the effect of the phospho-mimetic, Y189D . ImportaPrevious studies have suggested an important role for phosphorylation of Y253 mouse/Y249 human p130Cas in the regulation of cell migration. Using a global sequence alignment strategy we identified that Y189 in the human NEDD9 substrate domain appears to be conserved with Y249 of human p130Cas. We further show that NEDD9 Y189 appears to contribute to focal adhesion dynamics and cell migration. Thus our study has identified a unique role for a single amino acid residue in the substrate domain of NEDD9. Collectively, our data and previously published investigations of p130Cas tyrosine residues The global alignment of the Cas family sequences revealed a striking consensus between NEDD9 and p130Cas tyrosine phosphorylation motifs (-YXXP-) in the substrate domains. NEDD9 Y189 is one of 10 tyrosines that align with p130Cas tyrosines and only 3 NEDD9 tyrosines (including Y214) show no concordance. Interestingly, two tyrosines – NEDD9 Y166 and NEDD9 Y317 - showed concordance in all 4 Cas proteins. The p130Cas mouse tyrosine Y391 that aligns with NEDD9 Y317 has previously been shown to be a substrate for Src kinase We have previously shown that mouse embryo fibroblasts (MEFs) from NEDD9−/− mice have faster rates of focal adhesion disassembly and correspondingly migrate more rapidly on 2D surfaces At present the protein(s) that associate with phosphorylated Y189 NEDD9 are not known. Our data suggests that mutation of this residue does not affect global tyrosine phosphorylation of NEDD9. Therefore, it appears likely that phosphorylated Y189 may mediate interaction with a specific protein partner that is required for NEDD9 stabilization of focal adhesions. Conceptually, the abundance of phosphorylation sites in both the p130Cas/BCAR1 and NEDD9 substrate binding domains suggests the possibility of redundancy among these sites. However, together with our study, evidence is now mounting to suggest that at least some individual sites may be necessary for discrete functions of each molecule. Given the critical role of NEDD9 in the promotion of cancer invasion http://www.ibi.vu.nl/programs/pralinewww/) Homo sapiens (AAH20686), Mus musculus (NM_017464), Rattus norvegicus (NM_001011922), Gallus gallus (XM_418946); p130Cas Homo sapiens (P56945), Mus musculus (NM_009954), Rattus norvegicus (NM_012931), Gallus gallus (XM_414057); Efs Homo sapiens (NP_005855.1), Mus musculus (AAC52340.1), Rattus norvegicus (AAI61942); and HEPL Homo sapiens (Q9NQ75), Mus musculus (AAI29977.1), Rattus norvegicus (NP_001178673.1), Gallus gallus (XP_417499.2). Curated, publicly available, high-throughput tandem mass spectrometry-derived phosphorylation modification data from Phophositeplus (www.phosphosite.org) Global sequence alignment was performed using PRALINE  from homozygous null NEDD9 (NEDD9−/−) mice have been previously described 5′-GGGGTATTTGACATCCCTCCCTCATCAGCAAAAGGC-3′ and to generate Y214F was 5′-GATAAAACCTCAAGGGGTGTTTGAGCATCCCGCCTACAAAAGGG-3′. Forward primers and the matched reverse complementary primers were purchased from Sigma (USA). Generation of the Y189D mutant construct was performed by Genscript . All cDNA constructs were sequenced in both directions to confirm sequence fidelity. Transient transfection of MEF cells was achieved using a nucleofector (Amaxa) and MEF2 nucleofector kit, as per the manufacturer’s protocol (Integrated Sciences). MCF-7 cells were transfected using Lipofectamine 2000 as per the manufacturer’s instructions .The GFP.NEDD9 fusion protein expression construct has been previously described  and was used as the template to create single amino acid substitutions of tyrosine (Y) to phenylalanine (F) by site-directed mutagenesis using the Quikchange mutagenesis kit from Stratagene . The forward primer sequence to generate Y189F was 3VO4, 1 mM PMSF, 10 µg/ml Leupeptin, 10 µg/ml Aprotinin immediately prior to extraction). Extracted NEDD9 proteins were immunoprecipitated with anti-NEDD9 antibodies coupled to protein A Sepharose .Conditions of protein extraction and immunoblotting were carried out as previously described 2-independent media (Invitrogen), supplemented with 15% FBS and pen-strep). Immediately prior to imaging, recording media was supplemented with Oxyrase  (60 µl per 2 mL media). Time-lapse images were captured using an ORCA ERG cooled CCD camera  and Olmypus IX81 inverted microscope equipped with an environmental chamber heated to 37°C. For assay of focal adhesion dynamics and random cell motility glass bottom dishes were pre-treated with poly-L-lysine (50 µg/mL) and then coated with a solution of fibronectin (20 µg/mL) for 2 hours at 37°C. For fixed imaging, cells were fixed in 4% PFA in PBS for 10 minutes, permeabilised  and then immunostained. Imaging of fixed cells was performed with the Olympus IX81 inverted microscope.Cells were grown in 35 mm glass bottom dishes (MatTek) in recording media . Cells undergoing division or apoptosis were excluded from analyses and random migration analyses were performed on sparsely plated cultures. Post image capture, nuclear translocation was tracked in time-lapse stacks using Metamorph V6.3 software (Molecular Devices). Generation of migration traces and the calculation Mean Squared Displacement and cell speeds were performed as previously described GFP-NEDD9-positive focal adhesions were imaged with an Olympus IX81 inverted microscope, with a 60X (NA 1.35) oil objective, using fluorescence filters of BP 460–495/BP510–550 (GFP). Images were captured every 2 minutes for 90 minutes total, 250 ms exposure times. Focal adhesions at the protruding edge of the membrane were analysed by measuring the temporal changes in integrated pixel intensity of an individual focal adhesion. Quantification of adhesion dynamics was performed as previously described Final micrograph images and grey level adjustments were prepared in Adobe Photoshop.t test and for greater than two means using one-way ANOVA with Tukey's Multiple Comparison Test.All error bars on histograms show the standard error of the mean (SEM). Statistical comparison of two means was performed in Graph Pad Prism using a Student’s Figure S1Cas family proteins sequence alignment. Sequences aligned at the PRALINE website.(PDF)Click here for additional data file.


The present study focused on Daiokanzoto (Da-Huang-Gan-Cao-Tang), which shows the most potent inhibitory effects on P-gp among the 50 Kampo medicines studied, and investigated the P-gp inhibitory effects of Daiokanzoto herbal ingredients (rhubarb and licorice root) and their components by an ATPase assay using human P-gp membrane. Both rhubarb and licorice root significantly inhibited ATPase activity, and the effects of rhubarb were more potent than those of licorice root. The content of rhubarb in Daiokanzoto is double that in licorice root, and the inhibition patterns of Daiokanzoto and rhubarb involve both competitive and noncompetitive inhibition, suggesting that the inhibitory effects of Daiokanzoto are mainly due to rhubarb. Concerning the components of rhubarb, concentration-dependent inhibitory effects were observed for (−)-catechin gallate, (−)-epicatechin gallate, and (−)-epigallocatechin gallate. In conclusion, rhubarb may cause changes in the drug dispositions of P-gp substrates through the inhibition of P-gp. It appears that attention should be given to the interactions between these drugs and Kampo medicines containing rhubarb as an herbal ingredient.We have studied the effects of various Kampo medicines on P-glycoprotein (P-gp), a drug transporter,  P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) transporter family, has a molecular weight of 170 kDa and is a glycoprotein consisting of 1,280 amino acids. P-gp was discovered in 1986 as a product of the MDRI gene that causes multidrug resistance in cancer cells . When P-in vitro ) as the abscissa; linear regression was performed for each concentration of Daiokanzoto. A Dixon plot was prepared with the reciprocal of ATPase activity (1/V) as the ordinate and the reciprocal of the Daiokanzoto concentration as the abscissa. Linear regressions were performed for each concentration of verapamil, and Ki values were calculated from the values of the x coordinates at the intersection points on the graphs.Four concentrations of Daiokanzoto  and seven concentrations of verapamil -catechin, (−)-catechin, (−)-catechin gallate, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, and (−)-epigallocatechin gallate were 8, 40, and 200 μM.The inhibitory effects of the 13 components of rhubarb -catechin, (−)-catechin, (−)-catechin gallate, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, and (−)-epigallocatechin gallate) on ATPase activity were studied in the presence of 50 t-test.The results are shown as the means ± SD, and statistical significance was evaluated via Student's μM each; verapamil, digoxin, vinblastine, and ketoconazole) [This study investigates the effects of Daiokanzoto on ATPase activity in the presence of four P-gp substrates (50 onazole) . ControlAmong the P-gp substrates, verapamil resulted in the strongest inhibition of ATPase activity by Daiokanzoto. Therefore, the inhibitory effects of Daiokanzoto on ATPase activity in the presence of various concentrations of verapamil were studied. Daiokanzoto resulted in a concentration-dependent inhibition of ATPase activity at all concentrations of verapamil .x coordinate of the intersection point, was 0.057 mg/mL.The resulting inhibition patterns were investigated using the Lineweaver-Burk and Dixon plots. When linear regression was performed at each concentration of Daiokanzoto, the regression curves for both plots showed intersection points in the second quadrant . These rμM verapamil  significantly inhibited ATPase activity in the presence of 50 erapamil .The inhibitory effects of rhubarb on ATPase activity in the presence of verapamil at various concentrations were studied. Rhubarb demonstrated a concentration-dependent inhibition of ATPase activity at all concentrations of verapamil studied .x coordinate of the intersection point) was 0.063 mg/mL.The resulting inhibition patterns were investigated using Lineweaver-Burk and Dixon plots. Linear regressions were performed at each concentration of rhubarb, and the regression curves of both plots contained intersections in the second quadrant. These results suggest that the P-gp inhibition pattern of rhubarb and verapamil is a mix of competitive inhibition and noncompetitive inhibition similar to that observed for Daiokanzoto . The Ki μM verapamil. Sennoside A, sennoside B, rhein, aloe emodin, emodin, gallic acid, (+)-catechin, (−)-catechin, (−)-epicatechin, and (−)-epigallocatechin did not show significant effects on ATPase activity in the presence of verapamil  [The present study investigates the inhibitory effects of Daiokanzoto and its herbal ingredients on P-gp via an ATPase assay using human P-gp membranes. The reaction solution in ATPase assay contained EGTA, ouabain, and sodium azide, which inhibits Cat assay) .Daiokanzoto showed significant inhibitory effects on ATPase activity in the presence of all P-gp substrates used . A kinetThe present study regarding the inhibitory effects of rhubarb and licorice root  on ATPase activity in the presence of verapamil reveals that both components exert inhibitory effects, with rhubarb being the stronger of the two . CompariRhubarb showed concentration-dependent inhibition of ATPase activity in the presence of verapamil . When kiin vivo inhibition.Thirteen components of rhubarb were studied to clarify which are responsible for the inhibitory effects of rhubarb on ATPase. Of these, sennoside A, sennoside B, rhein, aloe emodin, emodin, gallic acid, (+)-catechin, (−)-catechin, (−)-epicatechin, and (−)-epigallocatechin were found to exhibit no inhibitory effects on ATPase activity even at sufficiently high concentrations, whereas (−)-catechin gallate, (−)-epicatechin gallate, and (−)-epigallocatechin gallate demonstrated concentration-dependent inhibitory effects . A studyThe present results indicate that rhubarb may cause changes in the disposition of P-gp substrates via the inhibition of P-gp. It is known that P-pg-expressing cancer cells are resistant to anticancer drugs. Therefore, rhubarb may potentiate the anticancer effect by the inhibition of P-gp. In the future, it is worthwhile to study interactions with Kampo medicines containing rhubarb as a herbal ingredient.


Neocortical inhibitory interneurons play a critical role in shaping the network activity patterns by directly controlling the firing rates of pyramidal cells (PC) . Evidenc


Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.0/Gi receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A4 attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G0/Gi receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G Chronic mast cell activation is a characteristic feature of asthma The outcome of mast cells interacting with the airway epithelium is poorly understood. Airway epithelial cells (AEC) are capable of suppressing mast cell chymase expression We have previously demonstrated that HLMC in contact with BEAS-2B AEC exhibit a marked reduction in both constitutive and IgE-dependent HLMC histamine release To further our understanding of the mechanisms regulating HLMC function by AEC, we have now studied the effects of primary human AEC including air liquid interface (ALI) cultures, derived from both healthy and asthmatic subject cultures, on HLMC degranulation.The BEAS-2B epithelial cell line was purchased from the European Collection of Animal Cell Cultures . Cells (passages 8–12) were grown on human plasma fibronectin-coated T75 culture flasks in BEBM media (Clonetics Cat. No. CC4175), with an added enhancement bullet kit (Clonetics Cat. No. CC4175), Pen/Strep (5 ml) and fungizone (5 ml) to create basal epithelial growth media (BEGM). BEAS-2B were then passaged on to human plasma fibronectin-coated 16-well 0.40 µm Transwell plates and then grown to confluence prior to use in assays.All subjects donating lung tissue gave written informed consent, and the study was approved by the Leicestershire Research Ethics Committee. HLMC were dispersed from macroscopically normal lung obtained within 1 h of resection for lung cancer using immunoaffinity magnetic selection as described previously Asthmatic subjects (n = 6) and healthy controls (n = 6) were recruited from Glenfield Hospital, Leicester, UK. Asthmatic subjects had a consistent history and objective evidence of asthma, as described previously 4) in direct contact with confluent BEAS-2B cells cultured in 24 well plates, ii) BEAS-2B grown to confluence initially on an inverted 0.4 µm Transwell membrane which was then inserted into the plate in the correct orientation, with HLMC (5×104) then added to the top chamber, and iii) HLMC (5×104) cultured alone on plastic in the bottom of a 24-well plate. The cells were cultured for 16 h, the media then removed gently, any non adherent cells in the removed media recovered by centrifugation and the cells returned to their respective wells in fresh media. They were then activated with anti-human IgE for 30 minutes, supernatants removed and the cells recovered by centrifugation. The remaining cells were then lysed with sterile water to allow for measurement of cell histamine content.Three conditions were compared initially: i) HLMC  was used at a final concentration of 10 µM based on our previous experience with this Mast cells were sensitised using 2.5 µg/ml human IgE  for at least 1 h, washed and re-suspended in fresh media. They were stimulated with anti-IgE  at a final concentration of 1/1000 for 30 minutes. Pertussis toxin (PTX),  was used at a final concentration of 500 ng/ml, the EP4 was measured by ELISA  according to the manufacturer’s instructions.Histamine was measured by sensitive radioenzymatic assay as described previously 18 solid phase extraction (SPE)  to remove small lipid mediators We fractionated AEC supernatants from primary AEC monolayers using reversed phase C18 solid phase extraction (see above), which purifies lipid mediators and removes interfering substances. The extracted samples were kept as solutions in methanol at −20°C; these were then dried by rotary evaporation and reconstituted in LC solvent prior to analysis. Samples were analysed using a Waters  Acquity ultra performance (UP)LC system coupled to a Waters Xevo TQ triple quadrupole mass spectrometer operated in negative ion mode with selected reaction monitoring. Chromatography: Solvents were 0.02% acetic acid (solvent A) and 45% acetonitrile (solvent B), used in a gradient system varying between 50∶50 A:B and 20∶80 A:B. The column was a Waters Acquity BEH C18 1.7 µm, 2.1×150 mm. Injection volume was 10 µl, total run time 17 minutes. Mass spectrometry: Capillary voltage was 2.6 kV, cone voltage 25 V; desolvation temperature was 550°C with gas flow 800 l/hr. Collision gas flow was 0.2 l/min. Ion transitions monitored for selected reaction monitoring were: resolvin D1 and D2 375.2 → 141.2; lipoxin A4 351.2 → 115.2; deuterated-lipoxin A4 356.2 → 115.2; resolvin E1 349 → 195; resolvin E2 333.2 → 199.2; protectin D1 359.2 → 206.2; maresin 1 359.2 → 250.2 4) was added to the media, enabling the accurate quantitative determination of each mediator.We used liquid chromatography (LC)-mass spectrometry (MS) to look for the presence of resolving mediators of inflammation  in both primary AEC monoculture supernatants and HLMC-co-culture supernatants. For targeted determination, cell culture media were subject to CAll statistical analysis was carried out using SPSS Version 14.1 (IBM). All experiments were performed with at least 4 replicates per condition. The mean of the replicates was used for statistical analysis. Comparisons across groups were made using a repeated measures ANOVA with post test Bonferroni correction, a p value of <0.05 was considered statistically significant.Initially we extended our original work examining the suppression of HLMC degranulation in direct contact with confluent BEAS-2B cells, by comparing the co-culture of HLMC and BEAS-2B cells separated by a 0.40 µm Transwell membrane . Analysis of cell supernatants revealed a 55%  suppression of constitutive histamine release over 16 h in the presence of BEAS-2B in either direct contact  or separated by a Transwell membrane  when compared to HLMC cultured alone . There wBEAS-2B airway epithelial cells therefore consistently inhibit HLMC constitutive and IgE-dependent histamine release, but cell-cell contact is not required indicating that a soluble mediator(s) is involved.We then repeated the latter Transwell experiments described above using ALI AEC cultures that had been grown from well characterised donors either with or without a diagnosis of asthma. Constitutive (16 h) histamine release was significantly lower from HLMC co-cultured with healthy ALI AEC compared to HLMC co-cultured with asthmatic ALI AEC (p = 0.03) or HLMC cultured alone (p = 0.001) . AsthmatTo assess further, whether the AEC-inhibitory activity is transferable, we measured constitutive histamine release from HLMC cultured in primary AEC monolayer supernatants for 16 h, compared to control AEC media. There was a significant reduction in constitutive release , but thiTo investigate further the mechanism(s) surrounding the inhibitory effect of AEC on HLMC, firstly we wounded a BEAS-2B Transwell monolayer subtly by scraping the monolayer twice with pipette tip to create an X of damaged cells. Interestingly, this manoeuvre markedly reduced the inhibition of the 16 h constitutive histamine release from HLMC (p = 0.03), but had a much lesser effect on IgE-dependent histamine release (p = 0.29)  . Removal of lipid mediators ablated the inhibitory activity present in these AEC supernatants  for resolvin D1, and 0.5 pg/µl (1.3 nM/L) for resolvin D2 and lipoxin A4 . We could also not detect other resolving mediators including resolvin E1 and E2, protectin D1 and maresin 1 which were screened for using published SRM characteristics as listed in Methods.We could accurately and reproducibly measure lipoxin Apoxin A4 . This coWe demonstrated previously that BEAS-2B AEC exert a strong suppressive effect on both constitutive and IgE-dependent HLMC degranulation 2, but signals through a G0/Gi protein coupled receptor mechanism as evidenced by the reversal of this inhibition in the presence of pertussis toxin. We suggest that good candidate molecules for this AE-derived suppressive effect are the family of resolving mediators of inflammation which include the lipoxins, resolvins and protectins 0/Gi protein coupled receptors 1 and resolvin D2, are potent inhibitors of HLMC degranulation and v) biochemical fractionation indicates that the activity in AEC supernatant is a <10 kDa lipid which would fit the profile of a resolving mediator.This mediator is not PGEResolving mediators are generated from arachidonic acid (Lipoxin A4 and B4), docosahexaenoic acid , and eicosapentaenoic acid (resolvin E1) through biochemical synthesis involving the enzymes 5- and 15-lipoxygenase  −10 M) or high performance mass spectrometry techniques  by LC-MS which were screened for using published SRM characteristics. However, such molecules could well be present and exert significant biological activity well below the currently available levels of detection. As well as the characterised resolving mediators mentioned above, there are also potentially hundreds of other arachidonic acid, eicosapentanoic acid and docosahexanoic acid metabolites which might exert similar effects The resolving mediators we tested exhibited a marked inhibition of HLMC degranulation at concentrations well below the current detection limits of current lipoxin ELISAs  is involved, and the family of lipid-derived resolving mediators are candidates, supported by the observation that lipoxin A4, resolvin D1 and resolvin D2 are potent inhibitors of HLMC IgE-dependent activation. These mediators may therefore have potential use as mast cell-targeted therapies. Identifying the inhibitory mediator(s) derived from AEC and understanding the AEC signalling pathways behind its production offers exciting potential for the development of novel approaches to treating asthma.In summary we have demonstrated that primary asthmatic AEC are less effective at suppressing the constitutive release of histamine from HLMC than healthy AEC, while both attenuate IgE-dependent degranulation. These suppressive effects are attenuated by AEC wounding, and by disabling G


Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S-induced angiogenesis.Hydrogen sulfide (H Our present study showed that the pro-angiogenic effect of H2S appears to be regulated by both a NO-dependent and an independent mechanism, whereas NO effect on angiogenesis is partially dependent on H2S. We demonstrated that H2S stimulated NO release by increasing eNOS phosphorylation via a p38 MAPK and Akt-dependent mechanism, which contributes to the stimulatory effect of H2S on EC proliferation and angiogenesis.The angiogenic crosstalk between HHuman umbilical vein endothelial cells-derived EA.hy 926 cells were kindly provided by Dr. Cora-Jean S. Edgell Total nitrate/nitrite concentrations were measured by conversion of nitrate to nitrite after incubating supernatants with nitrate reductase (10 U/ml) and NADPH (5 mM) for 1 hr at 37°C. The total nitrite was measured with a Griess assay kit  using a reference sodium nitrate standard curve Endothelial cells were seeded in 6-well plates and cultured until they reached 70–80% confluence. The cells were then transfected with specific siRNA to knockdown CSE or eNOS gene (50 nM). Negative siRNA was used as transfection control (50 nM), using Lipofectamine™ RNAi-MAX transfection reagent according to the manufacturer's instruction (Invitrogen). Overexpression experiments were carried out with plasmid DNA containing CSE cDNA  or eNOS cDNA . Mock empty vector was used as transfection control  g for 15 min. at 4°C. Supernatants were collected, and the same amounts of proteins were separated on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes . All primary antibody incubations were performed at 4°C overnight. The antibody dilution for phospho-eNOS (Ser1177), eNOS, phospho-ERK, ERK, phospho-p38 MAPK, p38 MAPK, phospho-Akt (S473) and Akt was at 1:1000 . Anti-CSE antibody was used at 1:5000 , and anti-β-actin antibody was at 1:10000 (Sigma-Aldrich). The membranes were stripped using a buffer containing 100 mM β-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl (pH 6.8) at 50°C for 30 min. Membranes were visualized using enhanced chemiluminescence Western blotting system . Densitometric quantification was performed using Alpha Digi Doctor Software . The protein bands were quantified and normalized against either β-actin or total form levels of the target protein, and expressed as a percentage relative to the controls . The phosphorylation level is defined as the ratio between the phosphorylated target proteins and their total forms and expressed in the summarized bar graphs as the percentage of the untreated controls.Cultured cells were collected and incubated in a lysis buffer containing 0.5 M EDTA, 1 M Tris-Cl (pH 7.4), 0.3 M sucrose and a protease inhibitors mixture (Sigma-Aldrich). The cell extracts were sonicated three times (5–10 sec./each) on ice using a cell sonicator  4 cells) were incubated with different agents in 500 μl DMEM and then seeded onto the surface of Matrigel (BD Biosciences). After 12 hrs, the formation of capillary-like structure was imaged by light microscope. The total lengths of tube-like structures per field were measured using image analysis software (NIH Image software- Image J).The Matrigel matrix gel was thawed overnight at 4°C on ice and then added to pre-chilled culture dishes and allowed to polymerize at 37°C for 1 hr. Endothelial cells (2 × 104 cells/well). After 24 hrs of initial seeding, cells were incubated with DMEM serum-free medium for overnight Cells were counted using automated cell counter TC10™ from BioRad  and seeded into 96-well plates (1 × 10Cystathionine gamma-lyase knockout (KO) mice were generated as described previously t-test. Values of P < 0.05 were considered statistically significant.All data were expressed as mean ± SEM. Each data point represented at least three to four independent experiments. Statistical comparisons were evaluated using Student's 2S precursor) on NO production. L-cysteine pre-treatment stimulated NO production in ECs (Stimulation of ECs with NaHS for 30 min. increased NO production over a concentration range from 10 to 100 μM A. The efn in ECs B. Howeven in ECs B. Cystatn in ECs C and D. n in ECs E and F.NaHS (50 and 100 μM) treatment markedly increased the phosphorylation of eNOS in ECs A. The st2S-induced eNOS phosphorylation and the NO production, we examined the roles of Akt, ERK and p38 MAPK in H2S-stimulated NO production. Treatment with NaHS at 100 μM enhanced the phosphorylation of p38 MAPK, Akt and ERK to different levels  treatment stimulated NO production in bovine arterial ECs. While these authors illustrated the H2S-dependent Akt mechanism that stimulates NO production, the involvement of other kinases, like p38 MAPK, or the synergistic partnership between H2S and NO in angiogenesis were not addressed. Conversely, it has been reported that a high concentration of NaHS (300–3000 μM) significantly inhibited the activity of recombinant bovine eNOS Phosphorylation activates eNOS 2S and NO in angiogenesis regulation. Ex vivo aortic explants isolated from CSE-KO mice showed a remarkable decrease in vascular neogenesis when compared to WT mice. L-arginine treatment stimulated angiogenesis in the WT mice and to a lesser extent, in the CSE-KO mice. On the other hand, L-NAME treatment reduced new vessel formation in WT mice, and this inhibitory effect was not significant in CSE-KO mice, suggesting that the angiogenic effect of NO might be mediated through H2S biosynthesis. Cystathionine gamma-lyase overexpression stimulated EC proliferation, whereas CSE knockdown reversed this effect. Interestingly, we found that the pro-angiogenic effect of H2S was partially attenuated in the presence of eNOS inhibitor L-NAME, or after eNOS knockdown using siRNA. Taken together, our results suggest that both gasotransmitters are required for optimal angiogenic activity, yet angiogenesis still proceeds in the presence of either H2S or NO alone albeit to a reduced degree. Previous studies had reported that H2S and NO can mediate angiogenesis without much knowledge about the H2S–NO interaction on angiogenesis 2S and NO is important for physiological control of different vascular function versus rat) and experimental conditions, and we found that H2S and NO, alone or combined, can cause angiogenesis. Hydrogen sulfide-stimulated angiogenesis was partially but not completely inhibited by NO blockage, whereas in CSE-KO mice, NO treatment stimulated angiogenesis but to a reduced level. The exact molecular mechanism underlying H2S-mediated NO pro-angiogenic response is not clear.We explored the possible interaction between H2S promotes NO production in ECs via the activation of a cascade of phosphorylation events, starting from p38 MAPK, Akt to eNOS. Hydrogen sulfide promotes EC tube formation, proliferation and angiogenesis by NO-dependent and independent mechanisms as outlined in 2S may be a key regulator for angiogenic signalling pathways, whether they required NO or not. The elucidation of the H2S–NO relationship in the vascular biology would improve our understanding of the pathogenic mechanisms for cardiovascular disease in general and angiogenic-related diseases in particular.In summary, our studies demonstrate that H


Lanius collurio. The effect appeared to be strong enough to enhance or counteract the influence of climatic factors and breeding density on the inter-annual variation in mean hatching dates. Moreover, the seasonal distribution of reproductive output was shifted to larger, or smaller, broods early in the season when the nesting mortality increased, or decreased, respectively, during the season. We suggest that population level changes in timing of breeding caused by a general advancement of spring and of the food supplies might be altered by the seasonality in nesting mortality. Hence, we argue that consideration of nesting mortality is of major importance for understanding long-term trends in avian phenology, particularly in species capable of renesting.The seasonal timing of avian reproduction is supposed primarily to coincide with favourable feeding conditions. Long-term changes in avian breeding phenology are thus mostly scrutinized in relation to climatic factors and matching of the food supplies, while the role of nesting mortality is largely unexplored. Here we show that higher seasonal mean daily mortality rate leads to a shift in the distribution of breeding times of the successful nests to later dates in an an open-nesting passerine bird, the red-backed shrike  Optimal timing of reproduction is important from both the life-time and the seasonal perspective. In seasonal environments, animals may often benefit from an early time of breeding Recently, many organisms, and particularly birds, seem to start reproduction earlier in the season as a result of higher spring temperatures Parus major and coal tits P. ater. Seasonal increase in post-fledging predation by European sparrowhawks Accipiter nisusTurdus grayi to breed early, while Pienkowski Charadrius hiaticula may start egg laying relatively late apparently to avoid high nest predation early in the season. Seasonal increase, or decrease, in nesting mortality may thus select for earlier, or later, breeding, respectively. More recently, some studies have provided evidence that birds may delay egg-laying when the perceived predation risk is high Lack In many species, inter-annual trends exist in mean seasonal nesting mortality Lanius collurio, L., recently tends to arrive earlier to the breeding grounds in Poland and Hungary In Central Europe, an open-nesting passerine bird, the red-backed shrike Breeding data were extracted from four sites across the Czech Republic  from a c2) for the respective study sites were available only for the period 1994–2006 from reports by red-backed shrike ringers and observers of the Czech Shrike Working Group 2 from our data . Therefore we used the ln-transformed number of ringed broods per km2 available for all years as a proxy for annual conspecific density (“ln(density)” hereafter).Estimates of breeding density , Coleoptera (up to ca. 23%), and Hymenoptera (2.5–4%) being the most important food items HD) for each nest in each year and at each site was calculated as a sum of the differences in mean daily temperatures and minimal developmental threshold temperature of 0°C from the start of the calculation period (1 January) to hatching date. We considered minimum threshold temperatures of 0, 2, 4, 10°C in analysed models but only present the best fitting model with threshold of 0°C. In central Europe, 1 January is relevant as a starting date for tree and even some Hymenoptera prey development and it is also used when there is no clear phenological event to start calculation from MAY hereafter), i.e. temperatures during the time when the onset of breeding takes place, were also obtained. All climatic data were provided by the Czech Hydrometeorological Institute and the Research and Breeding Institute of Pomology Holovousy Ltd.Following Schwartz j(Xj) is replaced by a sum of smooth functions with estimated degrees of freedom (“edf” hereafter) of explanatory variables ∑ sj(Xj) We were interested in general biological patterns. Therefore, we analyzed the relationships between explanatory and response variables across all sites, i.e. using all combined data and considering site as a random effect. We employed linear mixed models (LMM hereafter) and generalized additive mixed models (GAMMs). Generalized additive model (GAM) is a generalized linear model where the linear predictor of explanatory variables of the form ∑ βUsing data from the Czech nest record scheme, Hušek and Adamík HD). Standardized differentials were calculated by subtracting annual average hatching dates from the annual average hatching dates weighted for the brood size, and dividing this difference by the standard deviation of the hatching dates , trendDMR  at either site A, C, or D (site B was not tested because of short time series). There only was indication of autocorrelation in the mean CDDHD at site C . Trends in DMR were analyzed elsewhere. Hušek et al. First, we analyzed inter-annual trends in variables. There was no need for formal time series modelling as there was no temporal autocorrelation in either first or mean hatching date , TMAY and DMR as explanatory variables. Then, in following two LMMs, we included year as a continuous explanatory variable to test whether the effects of TMAY and DMR were confounded by the effect of inter-annual trend in mean hatching dates. Finally, we included TMAY, DMR and ln(density) in a single complete LMM, and corroborated the results by fitting GAMM. There were no strong correlations between the explanatory variables  as explanatory variables and standardized differentials for hatching dates (SDHD) as response variable in a single GAMM. The precision of estimates of DMR and trendDMR varied across years. When, however, the reciprocal variance of nesting mortality estimates was used as a weighting factor, the results did not change qualitatively and are therefore not presented.Last, to test prediction #2, we analyzed the relationship between the mean CDDAt each study site there was a large inter-annual variation in timing of breeding with a maximum range of 21 days for mean hatching date, and 22 days for first hatching date, respectively, particularly so at site C . Estimatyear = −0.16±0.055, t = −2.95, p = 0.0052; MAY  and mean CDDHD . There was no trend in either the standardized differentials for hatching dates /later  in the season when the spring progress was faster/slower at hatching times  also depends on the seasonality in nesting mortality. Similarly, based on life history modelling, Varpe et al.  density . When th density . In many density . Variabi density .The effect caused by the seasonality in nesting mortality on the distribution of reproductive output  is compaNon-mutually exclusive explanations exist to account for this observation because the distribution of the reproductive output on the population level may either mirror partial offspring mortality, seasonally plastic reproductive investment of individuals, or seasonal variability in timing of breeding of higher quality individuals rearing larger broods. The former might result from partial predation, selective predation on larger broods or partial mortality caused by deteriorated climate, food shortage or stronger competition. Whether or not birds adjust their seasonal reproductive effort in terms of timing of breeding, or clutch size, to seasonal patterns in nest predation, as determined by e.g. perceived predator density and activity, remains a major question Cardellina rubrifrons). In our study system, the association of larger broods occurring early in the season with seasonally increasing nesting mortality might simply be caused by decreased proportion of smaller replacement clutches in the population. This can not, however, explain the association of larger broods occurring later in the season with seasonally decreasing nesting mortality as larger replacement clutches are unlikely in red-backed shrikes Recently, Decker et al. 2012 In the Czech subpopulation of the red-backed shrike, there is a trend over the years for spring to be more progressed at the time of hatching . Yet, thWe did only consider mean annual estimates of breeding density in our study, though the fine tuning seasonality aspect might also be present, e.g. when caused by skewed distribution or arrival dates. Breeding density determines seasonal fitness primarily through competition for resources and altering of social interactions In the studies of nesting mortality on timing of breeding, the main limititation is a demand for large annual sample sizes of nest failure data over many years because lower sample sizes cause less precise estimates of the seasonality trends. However, despite such limitations, we are confident in the main finding of our study, because the results obtained by including reciprocal variance of mortality estimates as weights in the model did not change qualitatively, though appeared a little weaker (or stronger) in some cases. Even if the effects were weak or formally not significant in the red-backed shrike population, they might be of major importance in other species suffering increased (or decreased) nesting mortality over the years in a rapidly changing world. Also, estimates of linear seasonal trend in DMR should rather be handled with caution because generally, data from the beginning of the season  were scarce in our dataset see . We are In summary, we provide the first correlative study to demonstrate the relationship between variation in nesting mortality and the inter-annual seasonal distribution of timing of breeding on the population level in birds with implication for climate change studies. Moreover, we show that the seasonal patterns in nesting mortality affect the seasonal distribution of the reproductive output of the whole population.Figure S1Site-specific temporal variability in selected breeding parameters of the red-backed shrike. Shown are fits of linear regressions with their 95% confidence intervals. Full line for site C. a) Mean hatching date , b) standardized differential between the mean hatching date and mean hatching date weighted by brood size (SDHD), c) mean cumulated total of degree days at hatching date with temperature threshold 0°C , and d) standardized seasonal linear trend in daily mortality rate (trendDMR). Site A: full circles, site B: full triangles, site C: empty circles and site D: empty triangles. 0 = 1 January.(TIF)Click here for additional data file.Figure S2Site-specific effects of selected covariates on mean hatching date of the red-backed shrike. Shown are fits of linear regressions, dot-dashed line for site B, full line for site C, long-dashed line for site D. a) Effect of TMAY; site B , site C  and site D . b) Effect of DMR; site C . c) Effect of ln(density); site B (bln(density) = 12.40±3.08, t = 4.02, F1,2 = 16.18, p = 0.057, r2 = 0.89), site C (bln(density) = −5.53±1.64, t = −3.38, F1,29 = 11.40, p = 0.0021, r2 = 0.28). Site A: full circles, site B: full triangles, site C: empty circles and site D: empty triangles. 0 = 1 January.(TIF)Click here for additional data file.Figure S3Site-specific effects of selected covariates on the seasonal distribution of reproductive output of the red-backed shrike. Shown are fits of linear regressions with its 95% confidence intervals. See title of HD;. site B . b) The effect of trendDMR. Only significant estimates of trendDMR, i.e. confidence intervals of the estimates exluded zero, were considered here and linear regression was fitted to data from all sites (n = 9) . c) The effect of ln-density; site C (bln(density) = −0.06±0.03, t = −1.99, F1,29 = 3.96, p = 0.056, r2 = 0.12).(TIF)Click here for additional data file.Table S1Characteristics of the study sites in the Czech Republic.(DOC)Click here for additional data file.Table S2Summary of the breeding data in the red-backed shrike.(DOC)Click here for additional data file.Table S3Results of multiple linear regression models on mean hatching date at site C.(DOC)Click here for additional data file.Results S1Site-specific analyses.(DOC)Click here for additional data file.Results S2Associations between variables.(DOC)Click here for additional data file.


Interfaces between individual ice crystals, usually referred to as grain boundaries, play an important part in many processes in nature. Grain boundary properties are, for example, governing the sintering processes in snow and ice which transform a snowpack into a glacier. In the case of snow sintering, it has been assumed that there are no variations in surface roughness and surface melting, when considering the ice-air interface of an individual crystal. In contrast to that assumption, the present work suggests that there is an increased probability of molecular surface disorder in the vicinity of a grain boundary. The conclusion is based on the first detailed visualization of the formation of an ice grain boundary. The visualization is enabled by studying ice crystals growing into contact, at temperatures between −20°C and −15°C and pressures of 1–2 Torr, using Environmental Scanning Electron Microscopy. It is observed that the formation of a grain boundary induces a surface transition on the facets in contact. The transition does not propagate across facet edges. The surface transition is interpreted as the spreading of crystal dislocations away from the grain boundary. The observation constitutes a qualitatively new finding, and can potentially increase the understanding of specific processes in nature where ice grain boundaries are involved. The properties of ice grain boundaries are unknown to a large extent, much due to the difficulty in investigating a grain boundary at thermodynamic equilibrium The importance of water on earth, and in the atmosphere, has made it one of the most investigated molecules. Ice crystals are involved in the production of thunderstorms The thermodynamically stable form of ice at ambient temperature and pressure is ice Ih, which has a hexagonal crystal structure This paper explores the possibility of visualizing two ice prisms growing into contact, using Environmental Scanning Electron Microscopy (ESEM). ESEM is a technique that operates at higher pressure than does regular SEM, and its principal of detection takes advantage of a signal amplification from secondary electrons produced by electron-gas interactions On the stainless steel surface, the average distance between nucleation sites was relatively short, which prohibited studying the growth of individual crystals; every crystal was in contact with another crystal already as the first image was recorded, as shown in Supporting Information S1. The facet transition exemplified in Supporting Information S1). For the largest facets observed (facet edges within the range 200–300 µm), the transition from smooth to wavy of the entire facet occurred faster than the time interval between image acquisitions (i.e. 30–40 s), as shown in When ice crystal growth was investigated on the surface of a polyvinyl alcohol cryogel, nucleation occurred at much fewer sites as compared to the stainless steel surface, which enabled monitoring the growth of individual ice crystals. The crystals generally grew with hexagonal prism morphology, with sharp facet edges and smooth facets, until they came into contact with another crystal. It was observed that the transition of a facet structure from smooth to wavy could be catalysed by a contact with an adjacent crystal, as shown in contact between crystals and not, e.g., a certain temperature which initiated the transition, since the transition only was observed on facets directly after they had come into contact with another crystal , and we do not know if similar transitions can occur at ambient pressure. Unfortunately, those conditions can not be investigated with the present experimental setup. Nevertheless, the observed transitions are caused by the contact between two crystals, i.e. two crystal lattices which “mismatch” at the grain boundary, and the mismatch is the same regardless of surrounding pressure. It should also be kept in mind that prism growth is the basic growth morphology of ice Ih, and all understanding of more complex growth morphologies  is based on the understanding of simple prism growth. To summarize the interpretation of the results, they suggest the possibility that there is an increased probability of molecular surface disorder in the vicinity of a grain boundary.To the best of our knowledge, no theoretical model has been presented which describe the spreading of crystal dislocations away from a grain boundary. It is also beyond the scope of the present work to create such a model; the purpose of this report is the rapid communication of a qualitatively new finding. The finding will potentially improve the understanding of processes in nature where ice grain boundaries are involved; one example is the sintering of snow. In the case of snow sintering, it is unknown whether or not the surface structure varies over the ice-air surface of an individual grain When exemplifying the impact the observations might have on the understanding of phenomena in nature, it should be recalled that the grain boundary-induced transition is connected to an increased crystal growth rate . One of In summary, this work shows that the formation of an ice grain boundary can be visualized in detail using ESEM. Experiments are shown where the formation of a grain boundary induces a surface transition on facets which grow into contact, and the transition does not propagate across facet edges. The surface transition is interpreted as the spreading of crystal dislocations away from the grain boundary. The fact that an ice grain boundary can induce a surface transition which does not propagate across facet edges constitutes a novel qualitative finding. The results suggest the possibility that there is an increased probability of molecular surface disorder in the vicinity of a grain boundary. Previous studies of ice grain boundaries have mostly been attempts to investigate grain boundaries at thermodynamic equilibrium; this work shows that information on ice grain boundary properties also can be gained by studying the formation of a grain boundary during ice growth.Cryogels of polyvinyl alcohol, PVA, were produced by dissolving 6 wt% PVA  in water, and the solution was subjected to 7 freeze-thaw cycles (each cycle contained approximately 20 hours at −20°C followed by 4 hours at room temperature). Images of ice crystals were recorded using a Philips ESEM-FEG XL30, operating in wet mode. The environmental gas used was pure water vapor. PVA cryogels, 1–2 mm thick and containing water, were placed on a sample stage (a Peltier cooler stage with a stainless steel surface), and the pressure was first lowered and then raised to replace air with water vapor. The pressure was thereafter lowered to 0.2–0.5 Torr while simultaneously lowering the temperature of the sample stage to between −15°C and −20°C. The lowering of temperature and pressure caused ablation of water from the surface layer of the cryogel. The pressure was subsequently re-raised stepwise by letting in water vapor, 0.1 Torr/step, until the growth of ice crystals was observed. Ice crystal growth was studied within the pressure range 0.9–1.6 Torr, and images of individual ice crystals could be recorded with roughly 30 s intervals. The process of two crystals growing into contact with each other was investigated more than 100 times, at sample stage temperatures between −15°C and −20°C.After investigating crystal growth, the pressure was lowered stepwise, 0.1 Torr/step, in order to study at which pressure crystal growth converted to ablation. The result was then used to estimate the crystal surface temperature Supporting Information S1Supporting material text.(DOC)Click here for additional data file.Figure S1Crystals of hexagonal ice (ice Ih) growing on the surface of a polyvinyl alcohol cryogel. The temperature of the sample stage is −20°C and the pressure of the sample chamber is given in the pictures. Crystal growth converts to ablation as the pressure is lowered from 1.1 to 1.0 Torr, and the temperature of the crystal surface can therefore be estimated to between −16.2°C and −17.4°C. The scale bar is 50 µm.(TIF)Click here for additional data file.Figure S2An example of grain boundary-induced surface transition, observed at a sample stage temperature of −15°C. The crystal surface temperature is estimated to between −13.6°C and −14.5°C. The water vapor density is 1.6 Torr. The scale bar in (d) is 100 µm.(TIF)Click here for additional data file.Figure S3An example of a facet which grew to have edges longer than 200 µm before it came into contact with another crystal. In (TIF)Click here for additional data file.Figure S4Increase in the linear growth rate of the facets which undergo grain boundary-induced surface transition. The figure shows two crystals that grow into contact, referred to as the left and the right crystal. For the left crystal, facets are named F1 and F2 while facet edges are named x and y, as shown in (a). Contact between the crystals is first observed in (l), and the contact results in a surface transition of facet F2. The x/y ratio of facet F1 is constant before the two crystals come into contact, but changes after contact is reached. The change in x/y ratio can be attributed to an increasing linear growth rate of facet F2. The water vapor density was 0.9 Torr. The temperature of the sample stage was −20°C, and the temperature of the crystal surface is estimated to between −18.4°C and −19.7°C. The scale bar in (p) is 50 µm.(TIF)Click here for additional data file.Figure S5An example of a facet which develops an irregularity without contact with another crystal. Such irregularities generally disappeared relatively fast, as exemplified in this figure: the right facet appears perfectly smooth in (a–b), exhibits an irregularity in (c–d) and appears perfectly smooth in (e–f). The water vapor density was 0.9 Torr. The temperature of the sample stage was −20°C, and the temperature of the crystal surface is estimated to between −18.4°C and −19.7°C. The scale bar in (f) is 50 µm.(TIF)Click here for additional data file.


Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity. In our previous studies we indicated that extracellularly applied ASN increases the secretion of Aβ and potentiates its toxicity, thus leading to mitochondrial dysfunction and caspase-dependent PC12 cell death The major function of Tau, like other MAPs, is stabilization and regulation of microtubule (MT) dynamics necessary for neurite outgrowth, morphogenesis, axonal transport and normal neuronal functions The significance of extracellular ASN in Tau phosphorylation and MT instability, which could be involved in the mechanisms of dopaminergic cell death, is unknown. Therefore, this study was performed to investigate the effect of exogenous ASN on phosphorylation state of Tau, microtubule stability and death in PC12 cells. Moreover, the involvement of two major Tau-kinases: glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase (CDK5) in ASN-evoked Tau modification, MT destabilization and death was evaluated. Taken together, our data indicated that extracellular ASN induces GSK-3β-dependent Tau modulation and we suggest that proapoptotic effect of ASN might be mediated at least in part by the GSK-3β-catalyzed Tau hyperphosphorylation, which leads to the tubulin depolymerization and in further consequence to impairment of cytoskeleton stability. Finally, the effects of ASN can be largely prevented by inhibition of GSK-3β kinase. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity. Thus, one of the mechanisms by which ASN could mediate PC12 cell death is the disruption of tubulin polymerization into microtubules (microtubules destabilization), the cell's structural support system.The following antibodies were used in the current study: anti-phospho Tau (Ser396), anti-phospho GSK-3β (Ser9), anti-GSK-3β, anti-α/β-tubulin , anti-phospho-GSK-3β (Ty216) , anti-GAPDH, anti-rabbit IgG , anti-mouse IgG , anti-mouse IgG conjugated with gold particles . ASN was obtained from rPeptide . Reagents for reverse transcription (High Capacity RNA-to-cDNA Master Mix) and PCR (Gene Expression Master Mix) were obtained from Applied Biosystems . Cell culture reagents: Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), Horse Serum (HS), penicillin, streptomycin, G418, L-glutamine, and other reagents such as: deoxyribonuclease I, 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), TRI-reagent, polyethylenoimine (PEI), dimethyl sulfoxide (DMSO), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1Hbenzimidazoletrihydrochloridehydrate (Hoechst 33258), MES (2-N-(morpholino)ethanesulfonic acid), Colchicine and Paclitaxel (Taxol) were obtained from Sigma-Aldrich . Specific GSK-3 inhibitor: 3--4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB-216763) was from Tocris Bioscience , potent inhibitor of CDK5: N-(5-Isopropylthiazol-2-yl)phenylacetamide) (BML-259) was from Enzo Life Sciences . Cell Lysis Buffer (10×) was obtained from Cell Signalling . Reagents for TUNEL assay: FITC-labelled dUTP and PI/RNase Staining Solution were obtained from BD Biosciences, Heidelberg, Germany.Human alpha-synuclein  was dissolved in phosphatebuffered saline (PBS) (pH 7.4) at a concentration of 100 μM and immediately used for experiments as soluble ASN in form of mixture of monomers and oligomers 2 atmosphere.The studies were carried out using rat pheochromocytoma (PC12) cells Equal PC12 cell numbers were seeded into 96-well 0.1% PEI-coated plates or dishes and the growth medium was changed to a low-serum medium . Then PC12 cells were treated with exogenous ASN (10 μM), GSK-3 kinase inhibitor: SB-216763 (10 μM), CDK5 kinase inhibitor: BML-259 (10 μM), Colchicine (0.1 μM) and Taxol (0.1 μM) for 48 h.Cellular viability was evaluated by reduction of 2--2,5-diphenyltetrazolium bromide (MTT) to formazan. After 48 h treatment with the tested compounds, MTT (2.5 mg/ml) was added to all of the wells. Cells were incubated at 37°C for 2 h. Then medium was removed, the cells were dissolved in DMSO and absorbance at 595 nm was measured.The typical apoptotic nuclear morphology (nuclear condensation and shrinkage) was determined by microscopic analysis of the cells stained by Hoechst 33258. After incubation in the presence of ASN, cells were fixed by addition of 50 μl fresh mixture of methanol: glacial acetic acid (3∶1) for 3 min at RT to the culture medium. Cells were gently re-rinsed in methanol: glacial acetic acid (3∶1) for 5 min and left to dry for 30 min at RT. Next the cells were washed with PBS and stained with 1 μg/ml Hoechst 33342  for 30 min. To quantify the apoptotic process, cells with typical apoptotic nuclear morphology were examined under a fluorescence microscope . The results were expressed as the percentages of apoptotic cells.The presence of DNA fragmentation in apoptotic cells was determined by microscopic analysis of the cells stained by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP- fluorescein-isothiocyanate (FITC)-nick end-labelling (TUNEL) method. After incubation in the presence of ASN, cells were fixed as described above (Hoechst staining). The TUNEL assay was performed according to Gavrieli et al. 4, 2 mM EGTA, 0.1 mM EDTA, and 4 M glicerol at 37°C. After incubating the cells at 37°C for 10 min in 400 μl of free tubulin extraction buffer (buffer A plus 0.1% Triton X-100 and protease inhibitors), the extracts were centrifuged at 37°C for 2 min at 16.000×g. The supernatant fractions contained free tubulin that had been extracted from the cytosol. The cell pellet and lysed cells on the bottom of tube were dissolved in 400 μl of 25 mM Tris (pH 6.8) with 0.5% SDS, which contained tubulin in its original polymerized state . Equal amounts of total proteins from the free and polymerized tubulin fractions were analyzed by Western blot analysis using an anti-α/β-tubulin antibody.The microtubules stability was analyzed according to method described by Hongo et al. Cells were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PBS (pH 7.4) for 2 h at 4°C. After dehydration the cells were embedded in Epon 812, and ultrathin sections were processed according to the post-embedding procedure. Monoclonal mouse anti-phospho Tau (Ser396) was diluted 1∶30 in PBS and applied to the slices for 24 h at 4°C. Then the grids were exposed to secondary anti-mouse IgG conjugated with gold particles diluted 1∶50 in PBS. The sections were examined and photographed with a JEOL JEM-1011 electron microscope. Mean phospho-Tau expression in the control and ASN-treated cells was calculated as the number of gold particles in 10 randomly selected visual fields ± S.E.M.Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit according to the manufacturer's protocol . The level of mRNA for selected genes was analyzed by using TaqMan Gene Expression Assays  according to the manufacturer's instructions. Plates were analyzed on an ABI PRISM 7500 apparatus . The relative levels of mRNA were calculated using the ΔΔCt method.The cells were washed three times with ice-cold PBS and lyzed in Cell Lysis Buffer (1×). Protein levels were determined using the Lowry method, and then the samples were mixed with Laemmli buffer and denatured at 95°C for 5 min. After standard 10% SDS-PAGE separation, proteins were transferred onto PVDF membranes at 100 V. Next, the membranes were washed for 5 min in TBS-Tween buffer (TBST)  and the non-specific bindings were blocked for 60 min at RT with 2% or 0.5% BSA in TBST or with 5% non-fat milk solution in TBST. Further, membranes were washed three times for 5 min in TBST and incubated with the following primary antibody: mouse monoclonal anti-phospho-Tau (Ser396) (1∶500) in TBST, overnight at RT; rabbit monoclonal anti-phospho-GSK-3β (Ser9) (1∶500) in 5% non-fat milk solution in TBST, 1 h at RT and next overnight at 4°C; rabbit monoclonal anti-GSK-3β (1∶500) in a 5% non-fat milk solution in TBST, 1 h at RT and next overnight at 4°C; mouse monoclonal anti-phospho-GSK-3β (Tyr216) (1∶250) in 0.1% BSA in TBST, 2 h at RT and next overnight at 4°C; rabbit polyclonal anti-α/β-tubulin (1∶1000) in 5% BSA in TBST, overnight at 4°C. The membranes were then washed three times (5 min) in TBST and incubated for 60 min at RT with secondary antibody (anti-rabbit or anti-mouse IgG) (1∶4000) in a 5% non-fat milk/TBST. Antibodies were detected using chemiluminescent reaction and ECL reagent  under standard conditions. After stripping, the immunolabeling of GAPDH was performed as a loading control.t-test between two groups or one-way analysis of variance ANOVA with Dunnett's Multiple Comparison post-hoc test among multiple groups. Statistical significance was accepted at p<0.05. The statistical analyses were performed using GraphPad Prism version 4.0 .The results were expressed as mean values ± S.E.M. Differences between the means were analyzed using a Student's In the present study we investigated the effect of exogenous ASN on MAP-Tau protein in PC12 cells. The Tau phosphorylation status was analyzed by the TEM immunogold method. Ultrathin sections were analyzed and the mean number of gold particles representing the p-Tau protein was calculated. As presented in To study the influence of ASN on GSK-3β expression, we analyzed the level of mRNA using a real-time PCR (RT-PCR) and the immunoreactivity of this enzyme by Western blot analysis. ASN treatment significantly increased GSK-3β immunoreactivity by about 24%  without To evaluate the effect of ASN on microtubule stability, we determined the amounts of free and polymerized tubulin. Our studies showed that exogenously added ASN (10 μM) significantly increased the amount of free tubulin and reduced the amount of polymerized tubulin as compared to the control group . SB-2167Cell viability determined by MTT assay in the present study showed that 10 μM ASN reduced PC12 cell viability by about 27% and that SB-216763 (10 μM) prevented cell death evoked by ASN .ca. 1.5% of the total)  was observed when the cells were treated with exogenous ASN together with the GSK-3β inhibitor SB-216763 (10 μM)    1 and 6B2 (10 μM) 1 and 6B2e cells) 1 and 6C2n cells) 1 and 6C2A broad range of neurodegenerative disorders, including PD and AD, is characterized by neuronal damage that may be caused by toxic proteins in oligomeric form via affecting its phosphorylation status. Finally, ASN-induced apoptotic processes led to PC12 cell death, and the GSK-3β inhibitor attenuated ASN-evoked cytotoxicity and enhanced PC12 cells viability.In this study we have shown, for the first time that extracellularly added ASN in monomeric/oligomeric form increases Tau phosphorylation on Ser396, leads to the tubulin depolymerization and that the GSK-3β inhibitor protects cells against ASN-evoked both Tau hyperphosphorylation and MT destabilization. Moreover, we showed that ASN activated GSK-3β in vitro incubation of Tau and ASN synergistically promoted the fibrillization of both proteins Hyperphosphorylation of Tau has been indicated in various models of Parkinsonism and synucleinopathies and it was suggested that ASN could initiate Tau changes Since the two major protein kinases, GSK-3β and CDK5, are involved in Tau phosphorylation via other kinases in vitro and in vivo experimental models of PD, GSK-3β was activated by phosphorylation at Tyr216 in the presence of ASN, which then led to Tau hyperphosphorylation at Ser262 and Ser396/404 GSK-3β activity is regulated by serine (inhibitory) and tyrosine (stimulatory) phosphorylation, by its subcellular localization, by the formation of protein complexes containing GSK-3β and by the phosphorylation state of GSK-3β substrates via GSK-3β activation. This suggests that GSK-3β has an important role in ASN-evoked MT destabilization. Disruption of tubulin polymerization into microtubules was found by Chen et al., who demonstrated the inhibition of tubulin polymerization in a dopaminergic cell line inducted by oligomeric ASN It is well documented that Tau is cytoskeleton protein and its binding to MT is dependent on its phosphorylation state. Highly phosphorylated Tau is less able to bind and assemble microtubules Since microtubules are involved in cytoskeletal formation, in the regulation of mitotic apparatus, in the transport of intracellular organelles such as mitochondria, in axonal transport and normal neuronal functions; disruption of microtubules (MT destabilization) can induce cell cycle arrest in M phase, formation of abnormal mitotic spindles, inhibition intracellular transport and final triggering of signals for apoptosis Taken together, these studies provide evidence that extracellular ASN is involved in GSK-3β-dependent Tau modulation and that its proapoptotic effect might be mediated at least in part by GSK-3β-catalysed Tau hyperphosphorylation, which leads to the tubulin depolymerization and in further consequence may impair cytoskeleton stability. GSK-3β inhibitors may be used as a potential therapeutic strategy to counteract the effects of ASN toxicity in synucleinopathies. In our study we identified the potential mechanism by which extracellular ASN may disrupt microtubule integrity in PC12 cells, which can provide insights into the early pathogenic mechanism of PD and other synucleinopathies.