These results collectively suggest that MS-induced ERK1/2 activation through AT1-R might be independent of G-protein coupling. Moreover, either knockdown of β-arrestin2 or overexpression of a dominant negative mutant of β-arrestin2 prevents MS-induced activation of ERK1/2. We further identifies a relationship between Src, a non-receptor tyrosine kinase and β-arrestin2 using analyses of co-immunoprecipitation and immunofluorescence after MS stimulation. Furthermore, MS-, but not AngII-induced ERK1/2 phosphorylation is attenuated by Src inhibition, which also significantly improves pressure overload-induced cardiac hypertrophy and dysfunction in mice lacking AngII. Finally, MS-induced Src activation and hypertrophic response are abolished by candesartan but not by valsartan whereas AngII-induced responses can be abrogated by both blockers. Our results suggest that Src plays a critical role in MS-induced cardiomyocyte hypertrophy through β-arrestin2-associated angiotensin II type 1 receptor signaling.Angiotensin II (AngII) type 1 receptor (AT1-R) can be activated by mechanical stress (MS) without the involvement of AngII during the development of cardiomyocyte hypertrophy, in which G protein-independent pathways are critically involved. Although β-arrestin2-biased signaling has been speculated, little is known about how AT1-R/β-arrestin2 leads to ERK1/2 activation. Here, we present a novel mechanism by which Src kinase mediates AT1-R/β-arrestin2-dependent ERK1/2 phosphorylation in response to MS. Differing from stimulation by AngII, MS-triggered ERK1/2 phosphorylation is neither suppressed by overexpression of RGS4  nor by inhibition of Gαq downstream protein kinase C (PKC) with GF109203X. The release of inositol 1,4,5-triphosphate (IP Angiotensin II type 1 (AT1) receptor, belonging to the G-protein-coupled receptor family (GPCRs), shares a common structure of 7-transmembrane receptor (7TMRs), and mediates the signal response of Angiotensin II (AngII), thereby regulates blood pressure, cardiac hypertrophy and heart failure A growing body of evidences indicated that biased agonism might selectively induce the conformational switch of GPCRs, which preferentially activated or inhibited a subset of downstream signaling Src is a stress-sensitive kinase which plays an important role in the pathophysiological mechanisms for pressure overload-induced myocardial hypertrophy and pulmonary arterial hypertension, Src inhibition can effectively reverse the hypertensive response and hypertrophic signaling 202/Tyr204 (#9101) and anti-phospho-Src at Tyr416 (#2101) were purchased from Cell-signaling Technology; anti-Angiotensin II type 1 receptor (ab9391) were purchased from Abcam, plc; anti-FLAG-probe (#F7425), GF109203X (#G2911) were purchased from Sigma-Aldrich; anti-HA-probe (sc-805), anti-β-arrestin2 (sc-13140) were purchased from Santa Cruz Biotechnology; and SU6656 (#572635) was purchased from Calbiochem, Merck KGaA. Mechanic stretch-model culture plates were provided as kind gifts from Chiba University Graduate School of Medicine.Anti-ERK1/2 (#9102), anti-phospho-ERK1/2 at Thr5′-GACCGGAAAGACTTTGTGACC-3′ (forward) and 5′-GGTCACAAAGTCTTTCCGGTC-3′ (reverse). The mutant β-arrestin2-V54D was amplified and then cloned into plasmid pcDNA3. The SRC gene was amplified by PCR using primers 5′-CGGGATCCACTAGTAACGGCCGCCAG-3′ (forward) with a BamHI restriction site and 5′-CGCTCGAGCGAGGTTCTCTCCAGGCTG-3′ with a XhoI restriction site, and subcloned into the pcDNA3 plasmid containing a HA tag-encoding sequence.HA-tagged ERK2, FLAG-tagged β-arrestin1 and 2 were kindly provided by professor Issei Komuro . Wild-type FLAG-tagged β-arrestin2 in vector pcDNA3 was used as the template. PCR based site-directed mutagenesis approach was performed to make a valine(54)-to-aspartic substitution in the wild-type β-arrestin2 using Pfu polymerase (Takara), the mutagenic primers were as follows: 2PO4, 137 mmol/L NaCl, 4.2 mmol/L NaHCO3 and 5 mmol/L glucose at pH 7.4. Cells were then dissociated at 37°C by a combination of mechanical agitation and enzymatic digestion with 0.1 mg/mL DNase II (Sigma) and 0.125% pancreatin trypsin . Cells were pre-plated for 2 h in 100 mm dishes with Dulbecco's modified Eagle's medium (DMEM) with 10% defined bovine calf serum , penicillin (100 units/mL) and streptomycin (100 mg/mL) (Sigma), then the unattached cardiomyocytes were collected and plated at a field density of 1×105 cells/cm2 on silicone rubber culture dishes. Stretching of cardiacmyocytes by 10% was conducted as described previously In vitro cardiacmyocytes were cultured as previously reported Angiotensinogen gene knockout (AGT KO) mice were provided as kind gifts from professor Issei Komuro. Aged 8∼10 weeks of AGT KO mice were used in the present study and wide-type (WT) C57BL/6 mice were used as control littermates. All the procedures involving animals were carried out in accordance with the recommendations of the guidelines for the Care and Use of Laboratory Animals from China Council on Animal Care. The experiment was approved by the Experimental Animal Ethics Committee, Fudan University Shanghai Medical College with the permit number of 20110307-092. Pressure overload model was established by transverse aorta constriction (TAC) for 2 weeks as previously described 32P] ATP, and incubated with 25 mg of MBP as the substrate at 25°C for 10 min. After incubation, the reaction was terminated by adding Laemmli sample buffer  to the samples, and the samples were boiled for 5 min. The supernatants were subjected to SDS-polyacrylamide gel electrophoresis, and the gel was washed with 7% acetic acid for 30 min and with 3% glycerol for 30 min, dried, and then subjected to autoradiography.HA-tagged ERK2 plasmid was transient transfected into cardiomyocytes 24 h after plating the cells on stretch-model culture plates. ERK2 kinase activity was determined as we described previously 5′-CTGGATGTCTTGGGTCTGA-3′ and anti-sense primer: 3′-CGGATGGGGAAGTGGAAAC-5′; β-arrestin2, the sense primer: 5′-CCCTATGCTCAGAAACGAA-3′ and the anti-sense primer: 3′-TAGACAAGTAGCGGTGGAT-5′. The amplification of target genes were carried out on MJ Mini™ Gradient Thermal Cycler with a program of 95°C for 3 min followed by 35 cycles of 95°C for 30 s, 54°C for 30 s and 72°C for 30 s, and by a single incubation at 72°C for 5 min. PCR products were separated by electrophoresis on 1.2% agarose gels.Total RNA were extracted from cardiomyocytes using Trizol (Invitrogen) after mechanical stretch for 10 min. The cDNA was synthesized and optimized using a cDNA reverse transcription Kit (Takara), and the PCR primers used as follows:β-arrestin1, the sense primer:Cardiomyocytes were digested and separated to mini tubes. After incubating with cell lysates for 30 min on ice, cells supernatant was obtained by centrifugating for 30 min.Total protein extracts were size-fractionated by SDS-PAGE and were transferred to PVDF membranes. The membranes were blocked with 5% FBS in TBS-T buffer  for 2 h, then were incubated with antibody at 4°C overnight. After washing them for 3 times, the second antibody was adding, finally the membranes were treated with supersignal West Pico Chemiluminescent Substrate (Thermo), and the immunoreactive bands were detected by chemiluminescence system (Bio-Rad).Cultured cells were transfected with various plasmids as indicated. After transfection for 24 h, cells were harvested by lysis buffer . Cell extracts were sonicated and then centrifuged at 15000 g for 30 min at 4°C. Supernatants were subjected to immunoprecipitaion with indicated antibodies and co-incubated with protein A/G Plus-agarose beads (Santa Cruz Biotechnology) at 4°C for 3 h. Then the beads were spun down at 800 g for 2 min, washed with 700 µL lysis buffer for 3 times. The beads were eluted with SDS/PAGE loading buffer after extensive washed with 0.1 mol/L Tris-buffered saline. Finally the immunoprecipitates and total cell lysates were subjected to SDS/PAGE and determined by Western blotting with indicated antibodies.In vitro cultured cardiomyocytes were seeded in photic-bottomed stretch plate, and were transfected with β-arrestin2-GFP-WT or β-arrestin2-GFP-V54D mutant. Cells were stretched for 10 min, and washed with 1×PBS and fixed in 4% formaldehyde for 15 minutes at room temperature. After permeabililized with 0.1% triton X-100 for 10 minutes at room temperature, cells were blocked in 5% BSA for 1.5 hours. Immunostaining was performed by using anti-phospho-Src antibody overnight at 4°C. Oil immersion lens in a multitrack mode using a dual excitation (Alexa Fluor488 and Alexa Fluor555) filter sets. All Cells were counterstained with DAPI staining. Immunostaining was visualized by using laser scanning microscope .3 was according to the methods as described previously 5 cells per well and labeled for 24 h at 37°C with myo-[3H] inositol (1.0 µCi/mL). After terminated labeling by aspirating the medium, cells were rinsing twice and the pellet was resuspended and harvested with phosphate-buffered saline (0.02% EDTA). Cells were transferred to culture template for re-adherence, and after subjecting to mechanical stretch or incubated with AngII (10−7 mol/L), cell lysates were prepared in 0.4M perchloric acid and neutralized in 0.72M KOH and 0.6M KHCO3 in the presence of 5 mM LiCl. The lysates were then applied to Dowex columns , washed, and eluted. The eluates were counted in a liquid scintillation counter. IP3 releasing was estimated by determining the ratio of inositol phosphate radioactivity to the sum of total inositol phosphate.Determination of IPTransthoracic echocardiography was performed by animal specific instrument . Mice were anesthetized and M-mode images of the left ventricle were record when mice partially recovered from anesthesia. The anterior and posterior wall thickness of left ventricular  at end-diastole and papillary muscle level was measured by two-dimensional short-axis views of the left ventricle and M-mode tracings. Left ventricle ejection fraction (LVEF) was obtained using Simpson approach. Left ventricular hemodynamics was also evaluated at 2 weeks after TAC. Briefly, a micronanometer catheter  was inserted through the right common carotid artery into the aorta and carefully introduced into left ventricular. Left ventricular end systolic pressure (LVSBP) was measured as previously described P values smaller than 0.05 were considered statistical significance.All data were expressed as means ± s.e.m. The between-group comparisons of means were done by one-way ANOVA followed by Tukey-Kramer test. −7 mol/L)-induced cardiomyocytes after overexpressing RGS4. In contrast, RGS4 induced suppression of ERK1/2 was largely prevented by mechanical stretch.Increasing evidence implicated that AT1-R could function through a heterotrimeric G-protein coupling or a G-protein-independent mode −7 mol/L) and mechanical stretch, respectively. Immunoblotting analyses showed that ERK2 phosphorylation (indicated by MBP activity) was markedly attenuated in RGS4 overexpressed cells followed by AngII induction but not by mechanical stretch (−6 mol/L)  , indicat3) in stretch-induced cardiomyocytes, since CAMP-dependent IP3 bioactivity was the major event of GPCRs activation through Gα protein coupling. As shown in 3 release was significantly attenuated in stretching-induced cardiomyocytes compared with that in AngII (10−7 mol/L)-induced cardiomyocytes. Both AngII and mechanical stretch mediated IP3 release were blocked by candesartan (10−6 mol/L), suggesting that AT1 receptor was essential for Gα protein coupling. In addition, inhibition of PKC with GF109203X, a specific PKC inhibitor, effectively attenuated AngII (10−7M) induced ERK1/2 phosphorylation, but the phosphorylation level of ERK1/2 stimulated by stretching was not affected   . Collect2+-ATPase (SERCA)  and posterior wall at end-diastolic (LVPWd) thickerness in both C57BL/6 mice and AGT KO mice, accompanied by higher left ventricular ejection fraction (LVEF) and left ventricular end-systolic pressure (LVESP). However, Efficacy of attenuating myocardial hypertrophy by pretreatment with SU6656 was more pronounced in AGT KO mice than in C57BL/6 mice . Cardiac (SERCA) .We further compared the effects of candesartan and valsartan on myocardial hypertrophic response induced by TAC and AngII stimulation in AGT KO mice. Results showed that both candesartan and valsartan exerted inhibitory impacts on AngII-induced cardiac hypertrophy, but only candesartan effectively reversed TAC-induced hypertrophic response . FurtherCardiac hypertrophy is one of the independent risk factors responsible for myocardial injury and remodeling, and such compensatory response may transit to heart failure. Plenty of previous studies have demonstrated that AT1-R is one of the most important receptors mediating cardiac hypertrophic response through multiple signaling pathways Immerging evidences indicated that activation model of GPCRs might undergo different phases due to different stimuli. It was generally believed the first phase of AT1-R activation was G-protein coupling dependent, and this process was hypersensitive to ligand binding. G-protein signaling subtype 4 (RGS4), an inhibitory regulator of GPCR signaling through accelerating GTPase activity, was mainly expressed on cardiomyocytes 3, two important molecules were required for AngII-mediated modulation of myofilament calcium sensitivity and cardiac excitation-contraction coupling 3 was not fully activated in cardiomyocytes by short time effect of mechanical stretch. Interestingly, MS-induced ERK1/2 phosphorylation was not, suppressed by blocking PKC, suggesting a G-protein coupling independent signaling pathway was involved in stretching-induced cardiac hypertrophy. Of note, SU6656, a Src kinase inhibitor, effectively suppressed stretching-induced ERK1/2 activation, indicating a crucial role of Src but not PKC was required for mechanical stretch-mediated AT1 activation and intracellular signaling.Previous studies illustrated that PKC and IPRakesh K. previously reported that mechanical stretch evoked an abundant increase of ERK1/2 phosphorylation in hearts from WT mice, but not from AT1-R KO or arrestin2 KO mice The activation of cellular oncogenes such as c-myc and c-Src were critically involved in the reprogram progress of gene expression during pressure overload-induced myocardial hypertrophy In summary, our study presented the divergent mechanisms for mechanical stretch- and AngII-mediated β-arrestin2-dependent and G-protein coupling-dependent signaling pathway in cardiac hypertrophy. Mechanical stretch-induced conformational switch of AT1-R might be different from that stimulated by AngII, thus resulted in different signal transduction . The unc


Arabidopsis alone there are thousands of experimentally determined phosphorylation sites. To archive these events in a user-intuitive format we have developed P3DB, the Plant Protein Phosphorylation Database (p3db.org). This database is a repository for plant protein phosphorylation site data, currently hosting information on 32,963 non-redundant sites collated from 23 experimental studies from six plant species. These data can be queried for a protein-of-interest using an integrated BLAST module to query similar sequences with known phosphorylation sites among the multiple plants currently investigated. The paper demonstrates how this resource can help identify functionally conserved phosphorylation sites in plants using a multi-system approach.Protein phosphorylation is widely recognized as the most widespread, enzyme-catalyzed post-translational modification in eukaryotes. In particular, plants have appropriated this signaling mechanism as evidenced by the twofold higher frequency of protein kinases within the genome compared to other eukaryotes. While all aspects of plant protein phosphorylation research have grown in the past 10 years; phosphorylation site mapping using high-resolution mass spectrometry has grown exponentially. In  Arabidopsis thaliana contains slightly over 1000 protein kinases, at least twice as many on a gene frequency basis compared to man or fungi , controlling signaling pathways, metabolic, and cellular processes. It is evident that plants are particularly adept at this form of protein modulation as the genome of the reference plant 3DB. PhosPhAt , peptide sequence, phosphorylation site, mass spectra, etc. As a result there is a clear need to hierarchically store and organize the increasingly large phosphoproteomics data, motivating the creation of phosphorylation databases in the past few years. HPRD, the Human Protein Reference Database , with different experimental designs including mass spectrometry instrumentation  as well as data mining algorithms (and associated output). Consequently, the data were of different quality. To maintain the high-quality data within P3DB, while also expanding this database in accordance with the growing body of global phosphoproteomic studies, simple quality control standards were implemented. To be deposited into P3DB, datasets must meet the following criteria: FDR <1%; precursor mass accuracy <15 ppm; and PTM-score >84, in which case identification of the phosphorylation site is statistically significant compared to background. Additionally, all database searching must be performed with homologous proteome databases.In P3DB 2.0 has a new user-friendly web interface with better visualization for phosphoproteomics data and for data searching. Hierarchical design was applied to the front-end display as well as the database schema which not only facilitates user access to the interested portion of the database but also provides a comprehensive viewer to explore the data from all aspects. The data were first classified by organisms as the highest level, then proteins, followed by peptides, finishing with phosphorylation sites. Annotated mass spectra were mapped to the peptides if provided from experimental side. The data sources were listed in a drop-down list, which can be used as a filter by user selection at any scale level. Phosphoproteins in P3DB are also seamlessly linked to prediction results from the phosphorylation site prediction tool Musite . Original citations were also linked to all of the archived data.MySQL was used for storage, maintenance, and operation of Phttp://p3db.org, the new home page for the P3DB 2.0 website is shown  in the search module and does the mapping internally. Other than querying by accession number, data can also be searched using protein description, annotations, and protein names within the search module. The search module also allows the user to filter by organisms or studies to narrow down the search space. On the protein page, all accession numbers in the system can be obtained so that we can easily know or verify the TAIR numbers (AT4G00810 and AT2G27710) for our target proteins O23095 and AAC73029. The protein page shows the phosphorylation sites on the whole sequence with the sites highlighted. By looking at the protein pages for our P1 and P2 subunits . Although the best hit is a P3 subunit, it does contain the conserved phosphorylation site too. We can also try to search for the other homologous proteins within Arabidopsis, i.e., paralogs. With the best hit of AT3G44590, two sites are observed to be phosphorylated other than the one that is strongly conserved. Sequence conservation of phosphorylation sites can be a useful indicator for conservation of regulatory function.To determine if this phospho-serine residue is conserved, we can perform a BLAST search through P3DB. Since the same site might be observed in different peptides, each non-redundant peptide link leads to the specific peptide page. The peptide page contains the details of the experimental condition and parameters, each of which is mapped to the spectrum. If the annotated mass spectrum is available , the visualization tool will bring the mass spectrum and annotated peptide fragmentation series in the new screen.Continuing with protein AT3G44590, we can explore for further information in a hierarchical manner  of the comprehensive protein database UniProt. This is because the phosphorylation data within UniProt is almost exclusively from Arabidopsis. P3DB 2.0 has a wide range of different plant species, reflecting the expansion of phosphoproteomics research in Viridiplantae. The overall variability in phospho-amino acid frequency among the various plants likely reflects the preliminary nature of cataloging the plant phosphoproteome, particularly for crop species.Table The plots in Figure Although the plots are more descriptive Figure , they ar3DB 2.0 displays the data in a relational, hierarchical manner that integrates proteins, peptides, phosphosites, and spectra for each phosphorylation event. Various search and query tools  are embedded within the P3DB website framework allowing for seamless interrogation of the data without leaving the site. For example, the search module provides the user with multiple ways to access to proteins of interest and the BLAST search tool allows for comparative studies at the primary sequence level.P3DB version 2.0 now has become an integrated data bank and portal driven by the rapidly growing field of high-throughput phosphorylation site mapping. For bioinformaticist or experimental biologist, P3DB provides both the tools and experimental resources for querying plant protein phosphorylation data.In summary, PThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, Mn exposure significantly increased extracellular signal-regulated kinase (ERK) 1/2, as well as c-Jun N-terminal kinase (JNK) 1/2/3 phosphorylation at both 3 and 6 hours incubations, in both brain structures. Furthermore, Mn exposure did not change the total content or phosphorylation of TH at the serine 40 site in striatal slices. Thus, Mn at concentrations that do not disrupt cell viability causes activation of MAPKs (ERK1/2 and JNK1/2/3) in immature hippocampal and striatal slices. These findings suggest that altered intracellular MAPKs signaling pathways may represent an early event concerning the effects of Mn in the immature brain.The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the immature central nervous system, have yet to be completely understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs) and tyrosine hydroxylase (TH) could represent potential targets of Mn in striatal and hippocampal slices obtained from immature rats (14 days old). The aim of this study was to evaluate if the MAPK pathways are modulated after subtoxic Mn exposure, which do not significantly affect cell viability. The concentrations of manganese chloride (MnCl Manganese (Mn) is an essential metal for humans and animals. Its essentiality is due to its requirement as a cofactor in chemical reactions catalyzed by several enzymes involved in cellular homeostasis, such as mitochondrial superoxide dismutase and glutamine synthetase. However, exposure to high Mn levels may cause a neurological syndrome referred to as manganism, which shares multiple analogous symptoms with Parkinson's disease (PD) . Within the central nervous system (CNS), Mn accumulates predominantly in structures of the basal ganglia, such as the striatum , globus pallidus, and substantia nigra, consistent with dystonia, bradykinesia and rigidity secondary to damage to dopaminergic neurons and gliosis , 3. NonmNeonatal and developing brains are more susceptible to Mn toxicity. The uptake of metal in the intestine is usually elevated in the first week of life, followed by a steady decrease with age. These factors contribute to the increased risk for neurotoxicity in newborns when exposed to excess Mn . In addiMAPK  reduction assay . At the β-mercaptoethanol, pH 6.8) for 5 minutes at 100°C. Aliquots for protein concentration determination were collected before β-mercaptoethanol addition and protein concentration was later determined by the Peterson method [μg of total protein/track were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels , followed by transfer to nitrocellulose membranes using a semidry blotting apparatus   as described by Bjerrum and Heegaard (1988). After blocking with 5% skim milk in Tris buffered saline (TBS)  for 1 hour, membranes were incubated overnight (4°C) with primary antibodies to detect the phosphorylated forms of ERK1/2, JNK1/2/3, p38MAPK, and TH, in the dilutions recommended by the manufacturer. Subsequently, membranes were incubated for 1 h at RT with anti-Rabbit IgG HRP conjugate secondary antibodies. All steps were followed by three 5 minutes washing with TBS-T (TBS with the addition of Tween-20 0.1% and pH 7.5). The blots were developed by chemiluminescent reaction. Subsequently, membranes were stripped of the antibodies using a solution of NaOH 0.2 N and then reprobed to detect the total forms (phosphorylated plus nonphosphorylated form) of the target proteins. The bands were quantified using the Scion Image software . The ratio of the optic density (OD) of the phosphorylated protein band over the OD of the total protein band was calculated and the phosphorylation level of each protein was determined as a percentage of the control (considered 100%).To access MAPK activation, western blot analysis of slices samples was performed as previously described , 30, 31.n method . Next, 2MAPK detected a single band of approximately 38 kDa; anti-JNK 1/2/3 detected two bands, one at approximately 54 kDa and the second at approximately 46 kDa, corresponding, respectively, to the three JNK isoforms, JNK2/3 and JNK1.The antibody against ERK1/2 detected two bands, one at approximately 44 kDa and the second at approximately 42 kDa, corresponding, respectively, to the two ERK isoforms, ERK1 and ERK2. Anti-p38post hoc test when appropriate using STATISTICA 5.1 software . Differences were considered to be significant when P < 0.05. The values were expressed as mean ± S.E.M.Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Duncan's in vitro Mn exposure  for 3 or 6 hours did not significantly affect cell viability in either striatal , globus pallidus, and substantia nigra . In thisstriatal  or hippostriatal  assessedMAPK were assessed in striatal or hippocampal slices isolated from immature rats (PND14) in response to in vitro Mn exposure. Slices were exposed for 3 or 6 h to Mn at concentrations ranging from 10–1,000 μM and then analyzed by western blotting.The phosphorylation levels of ERK1/2, JNK1/2/3, and p38μM, 3 h) significantly increased ERK1 and ERK2 phosphorylation (49.81 ± 7.66%)  = 3.53, P = 0.048) and 54.96 ± 6.43%  = 3.64, P = 0.045), respectively, in striatal slices. JNK1/2/3   = 19.79, P = 0.000465), ERK2 (22.65 ± 2.84%)  = 23.25, P = 0.000264)  = 3.88, P = 0.025), and JNK2/3 (30.65 ± 7.29%)  = 5.11, P = 0.00922)  = 3.88  P = 0.025) and JNK2/3 (20.65 ± 4.59%)  = 5.11, P = 0.00922) phosphorylation   phosphor Figures  were sig Figures .F = 3.23, P = 0.046) and ERK2 phosphorylation (29.02 ± 5.45%)  = 3.48, P = 0.042) at 100 μM Mn  = 3.48, P = 0.042) at the highest Mn concentration tested . JNK1/2/3   = 3.21, P = 0.046) and ERK2 (13.34 ± 4.46%)  = 3.02, P = 0.049)  = 5.22, P = 0.008), and JNK2/3 (67.97 ± 24.94%)  = 3.11, P = 0.048) . No significant changes were observed in p38MAPK phosphorylation under the same experimental conditions   exposure were observed (TH is the rate-limiting enzyme for dopamine (DA) synthesis. Medium- and long-term modulation of TH activity occurs by regulation of gene expression and in the short-term by regulation of enzyme activity. Phosphorylation on serine residues, by several enzymes, is the primary mechanism of short-term TH activity regulation , 34. Prese to Mn . Howeverobserved .in vivo developmental exposure [in vitro with particular emphasis on striatal and hippocampal tissues. Employing experimental conditions (Mn concentration and exposure time) that do not cause significant cell death  altered the activity of key cell signaling elements, causing increased phosphorylation of DARPP-32-Thr-34, ERK1/2, and AKT in the striatum of rats on PND14 , PC12 ceexposure . However2 did not induce significant changes in cell viability  for 4 hours. However, a decrease in cell viability in vitro was demonstrated in DAergic PC12 cells [MAPK were increased. These results indicate that even though cell viability is not compromised, toxic metals may still cause changes in cells signaling pathways in vitro that may disturb cellular function [Our results showed that slice incubation with MnCliability . This fi12 cells , 23 afte12 cells . Further12 cells . Lead  for 3 hours , fail to take into account toxic events that are dependent upon neuronal and glial interactions. In contrast, tissue slices from rat brain maintain the natural extracellular matrix, neuronal connectivity, and neuroglial interactions, providing an appropriate experimental model for studies on acute neurotoxic events [μm thick brain slices is well established [in vitro studies on the neurotoxicity of metals, such as cadmium (Cd) [The majority of e models , 39. Howe models , 41. Thuc events , 43. Theablished , 44 and ium (Cd)  and Pb [ium (Cd) .Caenorhabditis elegans showed that extracellular and not intracellular DA is responsible for Mn-induced DAergic neurodegeneration [Striatal slices are rich in DAergic neurons, in contrast to hippocampal slices. Of particular importance, Mn shows selective neurotoxicity to DAergic neurons by mechanisms that have yet to be fully defined but possibly involve interaction with DA, leading to its oxidation forming toxic quinones and consequent oxidative stress –47. A reneration . Moreoveneration . TherefoμM Mn for 3 hours , diminished total thiol content, and increased superoxide dismutase and catalase activity, indicating oxidative stress within this structure [With respect to the susceptibility of hippocampal slices to Mn, we noted increased ERK1/2 phosphorylation following exposure of hippocampal slices to 100 and 1,000  3 hours . ERK and Figures , resp.. tructure . Therefo40 phosphorylation by Mn [40 phosphorylation in striatal slices from immature rats. This site is phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), increasing TH activity. Ser40dephosphorylation by protein phosphatase 2A (PP2A) results in decreased TH activity [40 (μM). In the latter study, a sustained TH-Ser40 phosphorylation (6–24 h) was observed, independent from H2O2 production and in the absence of significant changes in cell viability [Given the (1) importance of TH in controlling DA levels in neurons, (2) the involvement of DA metabolism in Mn neurotoxicity, as well as (3) a plethora of studies showing TH activity modulation and Seron by Mn , 39, we activity . Our resvity [40 . This reiability . in vitro to Mn reproduce (at least in part) the changes observed in vivo.Thus, based on our and previous studies, it can be inferred that Mn requires longer exposure periods to induce cell death in slices. However, important molecular changes were observed within short periods after Mn exposure, such as the activation of ERK1/2 and JNK1/2/3 signaling pathway Figures . These fin vivo conditions, one would posit that Mn is able to modulate the activation of MAPKs in tissues that maintain their metabolic competence and viability. The physiological significance of such activation represents a research topic that needs further attention.A key finding in this study is the Mn-dependent activation of ERK1/2 and JNK1/2/3 in striatal and hippocampal slices with no significant changes in cell viability. This finding indicates that MAPKs activation may represent an important primary event that precedes the deleterious effects of Mn on the cellular viability. Extrapolating our data to


Plasmodium falciparum Merozoite Surface Protein 4 (MSP4) is a vaccine candidate that has been selected for development for inclusion in an asexual stage subunit vaccine against malaria.Malaria remains a global public health challenge. It is widely believed that an effective vaccine against malaria will need to incorporate multiple antigens from the various stages of the parasite's complex life cycle. Escherichia coli-expressed recombinant MSP4 protein and characterized. These Mabs were used to develop an MSP4-specific competition ELISA to test the binding specificity of antibodies present in sera from naturally P. falciparum-infected individuals from a malaria endemic region of Vietnam. The Mabs were also tested for their capacity to induce P. falciparum growth inhibition in vitro and compared against polyclonal rabbit serum raised against recombinant MSP4Nine monoclonal antibodies (Mabs) were produced against in vitro in a manner proportionate to the antibody titre. By contrast, polyclonal antisera raised to individual recombinant fragments rMSP4A, rMSP4B, rMSP4C and rMSP4D gave negligible inhibition. Similarly, murine Mabs alone or in combination did not inhibit parasite growth.All Mabs reacted with native parasite protein and collectively recognized at least six epitopes. Four of these Mabs recognize reduction-sensitive epitopes within the epidermal growth factor-like domain found near the C-terminus of MSP4. These sera were shown to contain antibodies capable of inhibiting the binding of the six Mabs indicating infection-acquired responses to the six different epitopes of MSP4. All of the six epitopes were readily recognized by human immune sera. Competition ELISA titres varied from 20 to 640, reflecting heterogeneity in the intensity of the humoral response against the protein among different individuals. The IgG responses during acute and convalescent phases of infection were higher to epitopes in the central region than to other parts of MSP4. Immunization with full length MSP4 in Freund's adjuvant induced rabbit polyclonal antisera able to inhibit parasite growth P. falciparum growth inhibition in vitro.The panel of MSP4-specific Mabs produced were found to recognize six distinct epitopes that are also targeted by human antibodies during natural malaria infection. Antibodies directed to more than three epitope regions spread across MSP4 are likely to be required for  Plasmodium falciparum continues to be a major cause of morbidity and mortality in tropical countries. There is an urgent need for the development of efficacious control measures, one component of which could be a safe, effective and affordable malaria vaccine against P. falciparum. It is widely believed that any such vaccine will need to incorporate multiple antigens from the various stages of the parasite's complex life cycle  × 100, where I = inhibition (%); ODL = OD405 of the labelled antibody alone and ODU+L = OD405 of the labelled antibody where unlabelled antibody had been added.Microtiter plates were coated with rMSP4D and other steps performed as described previously . Briefly405 values as the mean plus the standard deviations (2SD) of the OD405 values of the 30 negative control sera. Sera with a titre ≥ 20 were considered positive. The percentage inhibition of each serum at 1/10 dilution was calculated using the following equation: I (%) = [(ODIFS - ODTS)/ ODIFS] × 100, where I = inhibition (%); ODIFS = OD405 of the inhibitor-free sample and ODTS = OD405 of the test sample. The antibody levels between sera at different time points were compared using the two-tailed Wilcoxon matched-pairs ranked sign test. To compare the prevalence of antibody inhibition between different groups of subjects, the two-tailed Mann-Whitney test was used.Microtiter plates were coated with rMSP4B, rMSP4C or rMSP4D, blocked as above and antigen reacted with serial two-fold dilutions of human sera as described previously . After wA relative affinity assay (potassium thiocyanate inhibition) was performed according to a technique described previously . Brieflyin vitro growth inhibition of P. falciparum was assayed as previously described  × 100, where GI = growth inhibition (%); PC = parasitaemia in control and PT = parasitaemia in presence of test antibody . Differences between the test and control cultures were assessed using the two-tailed student t test.The escribed  with modP. falciparum using indirect IFA further supported the specificity of the Mabs. In addition, all nine Mabs recognized a protein resolving at approximately 40 kDa when immunoblotted against blood stage P. falciparum lysates consistent with that previously observed for MSP4. The sequence of MSP4 suggests that the theoretical molecular weight of this protein is 27 kDa, but has previously been reported to show aberrantly slow electrophoretic migration, resolving at 40-45 kDa by SDS-PAGE [1 as determined by an isotype specific ELISA.A panel of nine MSP4-specific Mabs was generated and characterized Table . The speInitial epitope recognition was carried out by immunoblotting each Mab against the recombinant MSP4 fragment proteins rMSP4A, rMSP4B, rMSP4C and rMSP4D Table . Each frThe reduction sensitivity of epitopes was examined in more detail by reacting each of the Mabs against rMSP4 that was either non-reduced or reduced and alkylated prior to SDS-PAGE. This treatment destroys conformational epitopes that require disulfide bonds for their integrity. The MSP4D region contains three such bonds Figure . The binThe affinity of the Mabs was determined by the ability of thiocyanate to interfere with antigen-antibody binding in a concentration dependent manner, and are shown in Table To further define the epitopes of the C-terminal region of MSP4, the ability of the six anti-MSP4D Mabs to block each other's binding to rMSP4D was assessed using competition ELISA Table . BindingPlasmodium merozoite surface proteins, such as that of Plasmodium vivax Merozoite Surface Protein 1 (PvMSP1) have been resolved [Overlapping synthetic peptides spanning the entire length of the MSP4 molecule were used in an ELISA to further map Mab recognition sites to 20-amino acid linear epitopes Figure . Mab H12P. falciparum parasitaemia. Sera were collected at three different time points: T0 at the beginning of the survey and prior to drug treatment, T1 at the time of re-infection and drug treatment and T28 collected from the same individuals 28 days later. The four regions of MSP4 represented by rMSP4A, rMSP4B, rMSP4C and rMSP4D were recognized by the sera from these individuals suggesting that the human immune response to MSP4 is directed to at least four distinct epitopes [It was of interest to determine whether antibodies induced during natural infection of humans recognized similar epitopes to those seen by mice immunized with recombinant MSP4. Human sera were sampled from individuals who had demonstrable epitopes ,7.0). A pool of human sera were able to block the binding of all four Mabs in a dose-dependent manner, indicating that C-terminal conformational and linear MSP4 epitopes are recognized by both the Mabs and the human immune sera , during infection (T1) and convalescent phase (T28) of infection were examined in competition with Mabs recognizing epitopes within the central (MSP4C/D) and C-terminal (MSP4D) regions of MSP4  and linear (1H2 and 6G11) epitopes within rMSP4D were tested in the presence of human serum collected at the beginning of the survey  showed a titre of over 1 × 106. The other two antisera raised against full length rMSP4 (S193 and S1620) had an antibody titre of 3 × 104 and showed no detectable growth inhibition. These results suggest a positive correlation between the in vitro growth inhibitory capacity of antisera and antibody titre. The growth inhibitory capacity of antisera raised against fragments of the MSP4 molecule was also examined. In contrast to full-length protein, antisera raised against fragments rMSP4A, rMSP4B, rMSP4C and rMSP4D gave negligible parasite growth inhibition in vitro. This suggests that multiple MSP4 epitopes spanning the entire protein need to be targeted in order to significantly inhibit parasite growth in vitro. These results were consistent with those obtained using individual anti-MSP4 Mabs to inhibit parasite growth. The extent of growth inhibition obtained with the purified Mabs was not significant. To determine if three antibody specificities in three different fragments would be able to demonstrate inhibition, Mabs 6G10 (anti-MSP4C), 1B7 (anti-MSP4B) and 4C10 (anti-MSP4D) were tested in combination. Significant growth inhibition was not obtained by this combination of antibodies. This suggests that antibodies directed to more than such three epitopes spread across the molecule are necessary to result in parasite growth inhibition.Rabbit polyclonal antisera raised against full-length rMSP4 inhibit parasite growth el Table . Of the P. falciparum infection [19 antibodies is further dependent on their fine specificity, rather than mere prevalence or titre [The natural immune response to malaria is highly complex involving both antibodies and cell mediated immunity . The clinfection . Howevernfection . The antnfection . For exanfection ,24. The or titre . Similaror titre ,27. In cor titre , as wellEscherichia coli were produced and characterized. These Mabs recognize six distinct epitopes of MSP4, including two conformational epitopes in the C-terminal region containing the EGF-like domain. The remaining Mabs recognize epitopes that are not affected by reduction and alkylation and are presumably linear, with two in the C-terminal region of MSP4 and two in the central region of the protein. The reactivity of Mab H12 was interesting in that it reacted only with the reduced and alkylated form of full-length MSP4 but mapped to a region of the molecule, MSP4A, that has no disulphide bonds. This suggests that there are long range conformational effects which alter the shape of the N-terminus of MSP4 when disulphide bonds in the C-terminus are interrupted. This is in accord with previous observations in which the redox state of the EGF-domain was crucial for the antigenicity of the entire protein, including regions that are not immediately adjacent in the primary structure and did not contain disulphide domains such as the N-terminus [In this study, nine monoclonal antibodies specific for recombinant MSP4 protein expressed in terminus .This panel of antibodies was used in competition ELISA to analyse the binding specificities of naturally acquired antibodies to MSP4 in individuals from a malaria-endemic region of Vietnam. The three tested regions of MSP4 were readily recognized by the sera examined. The existence of persistent heterogeneity was observed among individuals in the level of antibody reactivity and the spread of epitope recognition. The reactivity of anti-MSP4 antibodies in the sera tested was considerably higher for epitopes in the central region of MSP4, in particular the MSP4B region. This result is also in agreement with our previous findings  that theMSP4 is a surface exposed protein that is highly conserved. The finding that antibodies target multiple epitopes spread across the entire length of the molecule may provide some insight into the mechanisms for co-evolution of parasite with its human host. It is possible to speculate that the sequence conservation of MSP4 maybe partly caused by the fact that immune selective pressure needs to target multiple epitopes simultaneously rather than individual epitopes. In addition to helping in understanding the nature of the antibody response to MSP4 during infection, the panel of monoclonal antibodies will also serve as valuable reagents for testing product integrity, purity, antigenicity and immunological activity during the highly complex and stringently regulated process of MSP4 vaccine development and manufacture.in vitro. In contrast, rabbit antisera specific for MSP4 fragments corresponding to three of the regions of MSP4 were unable to inhibit parasite growth. Similarly, the individual Mabs had no inhibitory effect on the growth of the parasite. These results are consistent with those previously obtained using an in vivo P. yoelii murine malaria challenge model in our laboratory. Immunizations carried out with recombinant PyMSP4/5 were able to induce immune responses that protect mice against challenge with a lethal dose of P. yoelii parasites [It is shown that polyclonal rabbit antisera raised against the full length MSP4 protein are able to inhibit parasite growth arasites . In contFollowing schizont rupture and merozoite release, re-invasion occurs within seconds. Therefore, antibodies will only be useful if they are available at very high concentrations and/or are of high affinity. Consistent with this picture, the observations in this study showed that growth inhibitory capacity is directly proportional to antibody titre. Similar observations have been made in field studies in malaria endemic areas; under natural exposure, immunity to malaria results from high titre antibodies to multiple antigenic targets . These oin vitro is currently being used as a criterion for selection of blood stage antigens that are to be incorporated into a subunit malaria vaccine. Growth inhibition in vitro is considered a functional assay because it measures the capacity of antibodies to bind and inhibit parasite invasion and/or growth. AMA-1 [in vitro [19 antibodies show more moderate inhibitory activity [The capacity to induce experimental antibodies with growth inhibitory activity (GIA) h. AMA-1  and MSP1h. AMA-1  are the in vitro ,31 and mactivity ,32,33. IPlasmodium infections is still unclear. The validity of this in vitro assay may be confirmed once vaccine trials are carried out in humans and in vitro inhibition is shown to correlate with in vivo protection. A recent Phase IIa trial of AMA-1 has shown disappointing results in this regard [Despite extensive work the value of using GIA as the sole correlate of immune protection from s regard . Antibods regard .in vitro assay that can be used to determine antibody efficacy in co-operation with monocytes to limit P. falciparum growth [in vitro growth inhibition by antibodies alone. In view of this finding, our antisera raised against MSP4 are currently being tested for ADCI activity.The Antibody Dependent Cellular Inhibition or Cytotoxicity assay (ADCI or ADCC) is another m growth ,36. Intem growth  and GLURm growth , despiteApart from antibody-mediated immunity, cellular mechanisms such as the production of IFNγ-producing T cells are also important in mediating protection against blood stage malaria ,40. T ceP. falciparum MSP4 were generated, characterized and used to develop an MSP4-specific competition ELISA to test antibody specificity in sera from naturally infected individuals. All epitopes recognized by the Mabs were readily recognized by human immune sera, with heterogeneity in the intensity of the humoral response among different individuals. Growth inhibition assays were carried out comparing growth inhibitory activity of the Mabs and polyclonal rabbit sera. The results demonstrate that multiple MSP4 epitopes spanning the entire protein need to be targeted to significantly inhibit P. falciparum growth. These findings provide insight into the MSP4 specific antibody response and have important implications for the design of blood stage vaccines.A panel of Mabs specific for The authors declare that they have no competing interests.RLC conceived the study. HdeS and SS designed and performed the experiments and analysed the data. HdeS wrote the manuscript. MP and SK helped with revision of manuscript. LW, CB and MP contributed information and reagents. All authors read and approved the final manuscript.


Although the inhibitory effect of cAMP on mTOR could be mediated by impinging on signalling cascades  that inhibit TSC1/2, an upstream negative regulator of mTORC1, we show that cAMP inhibits mTORC1 in TSC2 knockout (TSC2−/−) MEFs. We also show that cAMP inhibits insulin and amino acid-stimulated mTORC1 activation independently of Rheb, Rag GTPases, TSC2, PKB, MAPK and AMPK, indicating that cAMP may act independently of known regulatory inputs into mTOR. Moreover, we show that the prolonged elevation in [cAMP]i can also inhibit mTORC2. We provide evidence that this cAMP-dependent inhibition of mTORC1/2 is caused by the dissociation of mTORC1 and 2 and a reduction in mTOR catalytic activity, as determined by its auto-phosphorylation on Ser2481. Taken together, these results provide an important insight into how cAMP signals to mTOR and down-regulates its activity, which may lead to the identification of novel drug targets to inhibit mTOR that could be used for the treatment and prevention of human diseases such as cancer.cAMP and mTOR signalling pathways control a number of critical cellular processes including metabolism, protein synthesis, proliferation and cell survival and therefore understanding the signalling events which integrate these two signalling pathways is of particular interest. In this study, we show that the pharmacological elevation of [cAMP] ► cAMP inhibits mTORC1 and mTORC2. ► cAMP causes the dissociation of mTORC1 and 2. ► cAMP inhibits mTORC1 independently of TSC2, Rheb, Rag GTPases, PKB, MAPK and AMPK. ► cAMP inhibits TORC1 via a PKA-dependent mechanism. We provide evidence that this is mediated by a novel PKA-dependent mechanism which is independent of TSC2, Rheb and Rag GTPases. This report provides new insights into how cAMP talks to mTOR and could lead to the discovery of new anti-cancer drug targets.22.1All chemicals were purchased from Sigma-Aldrich , unless specified.2.2+/+/p53−/− MEFs (mouse embryonic fibroblasts), TSC2−/−/p53−/− MEFs ) 2 and 95% air.TSC23, 2.5 mM MgCl2, 2.5 mM CaCl2, 20 mM HEPES, pH 7.4), supplemented with 0.5× MEM amino acids solution (50× stock), 0.5× MEM non-essential amino acids solution (100× stock) and 0.5×l-glutamine (100× stock).Prior to treatments (unless specified in the figure legends) the media were removed and the cells were washed twice and incubated with modified Krebs–Ringer bicarbonate buffer (KRB) . Normalized lysates were stored at − 20 °C until further analysis.After treatment (unless specified in the figure legends), cells were lysed by the addition of ice cold lysis buffer (1% (v/v) Triton, 10 mM β-glycerophosphate, 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM benzamidine–HCl, 0.2 mM phenylmethylsulfonyl fluoride, 1 μg/ml each of leupeptin and pepstatin, 0.1% (v/v) β-mercaptoethanol and 50 mM NaF). Lysates were then centrifuged for 10 min at 16,0002.3Recombinant adenovirus expressing dominant negative AMPK was kindly provided by Dr. Ian Salt  2.4GTPQ99L and RagCGDPS75L were described previously 2 method as previously described HA tagged S6K1 (HA-S6K1) pRK7 vector was kindly provided by Prof. John Blenis  2.5g. The supernatants were kept, and total protein concentrations were determined by the Bradford assay. Lysates containing 1.2 mg of protein were incubated with anti-myc antibody (Sigma) for 16 h at 4 °C with rotation, followed by the incubation with protein-G sepharose beads for a further period of 2 h at 4 °C with rotation. Beads were washed three times with 0.3% (w/v) CHAPS buffer, and then resuspended in 2× Laemmli sample buffer.The immunoprecipitation of mTORC1 and 2 complexes was essentially performed as previously described 2.6SDS-PAGE and Western blotting were performed as described previously 2.7i was measured by interpolation of a standard curve and related to cellular protein, which was assessed by Bradford assay.Cells were grown in 24-well tissue culture plates. After treatment, buffer was removed and ice cold 0.5 M trichloroacetic acid was added. cAMP was extracted and determined using a radioreceptor assay with binding protein purified from calf adrenal glands as previously described 33.1i, . In these cells, insulin also stimulated mTORC1 as determined by the phosphorylation of its downstream targets S6K1 on Thr389 and 4EBP1 on Ser65 and the phosphorylation of rpS6 on Ser240/Ser244 and a decrease in the electrophoretic mobility of S6K1  MEFs were pretreated with increasing concentrations of forskolin plus IBMX in the absence or presence of insulin  and 4EBP1 (Ser65) and a decrease in the electrophoretic mobility of S6K1 and 4EBP1  were incubated with forskolin and IBMX prior to stimulation with serum, myc-mTOR and associated proteins were then immunoprecipitated from these cells. In serum repleted cells mTORC1 was active as determined by the phosphorylation status of S6K1. In addition, mLST8 and the mTORC1 specific components RAPTOR and PRAS40, and the mTORC2 specific component RICTOR were co-immunoprecipitated with myc-mTOR, indicating the integrity of mTORC1 and mTORC2 under these conditions. As expected, the preincubation of cells with forskolin/IBMX inhibited serum-stimulated mTORC1 activity, as determined by the phosphorylation status of S6K1. Moreover, preincubation of cells with forskolin/IBMX caused the dissociation of the mTORC1 specific component RAPTOR and PRAS40. In addition, forskolin/IBMX caused the dissociation of the mTORC2 specific component RICTOR from myc-mTOR. The association of mLST8 to myc-mTOR was unaffected   During cancer development, the mTOR pathway is often abnormally up-regulated, which favours cancer cell survival, growth, replication, angiogenesis and metastasis i suppresses mTORC1/2 by promoting mTOR complex disassembly and inhibiting mTOR's intrinsic catalytic activity. These observations provide new insights into the crosstalk between cAMP and mTOR, which may also contribute to the design of novel mTOR inhibitors for future strategies in the fight against cancer.In conclusion, we show that elevation of [cAMP]


Xenopus tadpole spinal cord and hindbrain, which generate swimming rhythms. Silencing activity on one side led to rapid cessation of activity on the other side. Analyses reveal that this resulted from the depression of reciprocal inhibition connecting the two sides. Although critical neurons in intact tadpoles are capable of pacemaker firing individually, an effect that could support motor rhythms without inhibition, the swimming network itself requires ∼23 min to regain rhythmic activity after blocking inhibition pharmacologically, implying some homeostatic changes. We conclude therefore that reciprocal inhibition is critical for the generation of normal locomotor rhythm.Alternating contractions of antagonistic muscle groups during locomotion are generated by spinal “half-center” networks coupled in antiphase by reciprocal inhibition. It is widely thought that reciprocal inhibition only coordinates the activity of these muscles. We have devised two methods to rapidly and selectively silence neurons on just one side of  ► Silencing activity on one side stops fictive swimming ► Silencing activity on one side depresses reciprocal inhibition ► Motor rhythms resume ∼23 min after pharmacological block of inhibition ► Reciprocal inhibition is essential for maintaining swimming It is widely thought that reciprocal inhibition merely coordinates the activity of antagonistic muscles. Using new methods which can silence nerve cells in milliseconds, Moult et al. show that reciprocal inhibition is also essential for generating locomotor rhythms. Reciprocal inhibition is present in various neural circuits  and has Xenopus tadpole swimming is controlled by neural circuits in the spinal cord and caudal hindbrain, which are symmetrical on the left and right sides connected by reciprocal inhibitory commissural interneurons (cINs) (s (cINs) . We haveHalorubrum sodomense) complementary RNA (cRNA) into one blastomere in the two- to eight-cell stage embryos. Blastomere lineage fate is uniquely predetermined in Xenopus laevis from the one-cell stage -tagged Archaerhodopsin-3   . dINs poThe one-sided silencing experiments (light illumination or −DC injection) therefore show that swimming rhythms on one side are critically dependent on the activity in the other. We next investigated mechanisms that could enable one-sided silencing to stop swimming. We asked whether the activity stopped on a particular side first. The neuronal activity stopped first on the suppressed side in most cases . We defined “cycle 0” as the period (∼100 ms) immediately after the last m.n. burst. Cycles −1 and −2 were the last and second last cycles, respectively. One-sided silencing reduced firing probability in neurons recorded from the suppressed side in all three cycles. In cycle 0, it was 6.9% for light illumination  and 0% for −DC injections . In cycles −1 and −2, they were 56.6% ± 11.2% and 68.3% ± 11.3% for light illumination and 70% ± 8.5% and 85.5% ± 5.4% for −DC injections, respectively. In the opposite side, firing probability only dropped in cycle 0. It was 0% for light silencing  and 0% for −DC injections  and tonic inward currents . We clamn-methyl-d-aspartate (NMDA) receptor-dependent pacemaker firing, if inhibition is pharmacologically blocked -resistant 10 Hz oscillations, which underlie repetitive dIN pacemaker firing, could be recorded in intact tadpoles as soon as 100 μM NMDA was microperfused  via reciprocal inhibition. Light activation of ArCh or −DC injections into single dINs: (1) stops neuronal firing on the suppressed side, (2) weakens cIN inhibition from the suppressed side, (3) results in dIN rebound failures and (4) leads to the cessation of swimming on the opposite side A and 8B.Redundant mechanisms or homeostatic plasticity have been found in many systems , and theHomeostatic changes can complicate the interpretation of experimental results, especially under different experimental conditions. For example, strychnine application was initially shown to cause tonic irregular m.n. activity . This waIn the tadpole swimming circuit, rebound mechanisms were proposed for the maintenance of swimming based on analyses of synaptic events during swimming and the firing property of dINs at rest . When heThe discrepancy between the one-sided fast silencing experiments and previous hemicord studies may be explained by the occurrence of homeostatic changes after hemisectioning, which facilitate pacemaker mechanisms to mediate motor rhythms. Previous hemicord studies normally leave at least a 20 min gap between the surgery and recording. We show here that, when the inhibition was blocked by strychnine and SR95531, the usual single-skin stimulation for evoking swimming failed to initiate any motor rhythm. Double-skin stimulation, which also evokes swimming in control conditions, led to seizure-like depolarization in most cases. The occasional rhythms, observed during the recovery period, suggest that the swimming circuit is still capable of generating rhythms, most likely via pacemaker firing in dINs. The delay in recordings after hemicord sections in previous studies is comparable to the recovery time revealed here (∼23 min), though the rhythms in the former were initiated by stimulating the hindbrain directly. It is not known whether the changes following hemisectioning and disinhibition by strychnine are similar or not. Whatever occurs during the recovery period to allow the resumption of rhythms, based on dIN pacemaker properties, is also unknown. As we show, individual dINs are capable of pacemaker firing throughout A and 6B.The two methods used in this study, optogenetics and −DC injections, enabled us to depress neuronal activity, including cIN inhibition, on a millisecond time scale, potentially leaving little time for reliable dIN pacemaker firing to become established in a substantial number of dINs to support the normal swimming rhythm. It is hard to exclude the presence of pacemaker firing during normal swimming, because dINs can, very occasionally, fire extra action potentials after the activity on the targeted side has stopped due to one-sided silencing. But such firing is very rare C and 3D.In another, more established swimming vertebrate model, zebrafish larvae, reciprocal inhibitory interneurons have been recently categorized . A subtyRapid progress has been made recently in unravelling the mammalian locomotor circuits . AlthougOur study provides strong evidence that reciprocal inhibition is not only important in coordinating activity between the left and right sides of the spinal cord, but is also essential in the maintenance of normal swimming rhythm. This view is based on silencing neuronal activity very rapidly, at a speed that cannot be achieved with other approaches, such as lesioning, pharmacological blockade, and genetic ablations.Xenopus embryos were collected and raised after regular human chorionic gonadotropin injections to pairs of adult Xenopus. All experimental procedures were approved by a local Animal Welfare Ethics committee and comply with UK Home Office regulations. Tadpoles at 2 days old (stage 37/38) were immobilized using α-bungarotoxin . Recording saline contained 115 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2.4 mM NaHCO3, 1 mM MgCl2, 10 mM HEPES, with pH adjusted to 7.4 with NaOH. Dissections were made to expose neuronal cell bodies in the tadpole spinal cord and hindbrain for whole-cell recordings. Tadpole hindbrain was cross-sectioned at the fifth and sixth rhombomere levels to remove all higher brain inputs to the swimming circuit in most experiments. Electrode solution  contained 0.1% neurobiotin (Vector Labs) for final identification of neurons after recordings. Whole-cell recordings were made in either current-clamp or voltage-clamp mode with an Axon Multiclamp 700B, digitized with a Power 1401 mkII, and sampled with Signal . We applied electrical stimulus (0.2 ms) to the tail skin of immobilized tadpoles to start fictive swimming (defined as swimming throughout the text). m.n. recordings were made with glass suction electrodes from the middle trunk region on the left. A swimming cycle is the period from one m.n. burst to the next one. For injecting −DC into single dINs via the recording electrode, the −DC (rectangular pulses) level was progressively increased without membrane destabilization. In the dINs in which −DC could stop swimming, the absolute amplitude of DC was about three to six times the threshold current for evoking dIN firing at rest. The dIN membrane potential was hyperpolarized by a maximum of 150 mV.To evoke rebound firing in dINs at rest, we placed a suction electrode on one side of the spinal cord surface to stimulate cINs directly and dINs were recorded on the opposite side. Saline containing a combination of 6 μM NBQX , 60 μM D-AP5 (D-(-)-2-amino-5-phosphonopentanoic acid, NMDAR, Tocris), and 2 μM DHβe  was microperfused close to the recorded dIN to block excitatory synaptic transmission from sensory pathway neurons. To test dIN pacemaker firing in intact tadpoles, we applied NMDA  within 50 μm upstream to the recorded neuron using microelectrode iontophoresis . TTX 0.. StrychnXenopus expression vector, incorporating a T7 promoter, a restriction site for linearization, and both 3′ and 5′ Xenopus globin-flanking sequences that aid translation and stabilize the message. cRNA was transcribed in vitro using Ambion mMessage mMachine. cRNA concentration was measured using a NanoDrop ND-1000 spectrophotometer. ArCh cRNA (0.5–1 ng) was injected into one blastomere of two- to eight-cell stage embryos showing regular cleavage patterns, using a TooheySpritzer (Toohey Company). The embryos then were raised to stage 37/38 and their GFP expression examined. Tadpoles with good GFP expression on the right side were chosen for light-silencing tests and electrophysiological recordings. Yellow light for ArCh activation and blue light for GFP observation from LED arrays  were controlled by Power 1401 mkII using Signal software. A Nikon E600 FN or an Olympus BX51WI microscope was used for visually guided patch-clamp recordings. Yellow light  was applied through a 40× water-immersion objective with a maximum intensity of 10 mW/mm2 onto the caudal hindbrain area of tadpoles. The intensity of light was gradually increased until we found a level that could reliably stop swimming. Fluorescent images were captured using a Veho VMS-004 USB microscope or a Scientifica BFWCAMXM camera mounted on the recording microscope.ArCh complementary DNA  was obtaData processing and analyses were carried out using Dataview  and Excel. Statistical analyses were carried out using IBM PASW statistics 18 (SPSS). For normally distributed data, mean was given with standard error and statistical differences between groups were examined using Student’s t test. For nonnormally distributed data, median and range were given and measurements were compared using the Wilcoxon signed-rank test. The effectiveness of one-sided silencing (either yellow light illumination or −DC injections into dINs) was assessed in trials alternated with control episodes at the beginning of each recording. A side was judged active if regular m.n. bursts were recorded or neurons received rhythmic excitatory synaptic potentials/currents.


Acarus siro, a medically important storage mite.Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from A. siro produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found.We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity. Acarus siro, one of the most frequent and abundant species in central Europe.Storage mites are global pests of stored food products of increasing medical and economical impact. In agricultural environments, they cause occupational allergy in farmers and grain handlers. Storage mites are also found in house dust from rural and urban dwellings and are important contributors to the allergen content, which expands their clinical significance. The storage mites belong to the Acaridae and Glycyphagidae families; our work focuses on http://www.allergen.org. Allergens from house dust mites of Dermatophagoides spp. have been extensively studied; however, much less is known about allergens from storage mites  . Theriew, see -9. A detDermatophagoides pteronyssinus, Euroglyphus maynei, and Blomia tropicalis, and their sequences have been determined .,11.10,11A. siro demonstrated that Aca s 4 of ~56 kDa is a significant IgE-reactive component although it is a protein of low abundance in the A. siro extract. Aca s 4 is likely recognized by serum IgE due to (i) a cross-reactivity with homologous α-amylase allergens from house dust mites . To gain insight into the structural basis of a possible cross-reactivity, we compared Aca s 4 with Der p 4, an α-amylase allergen from D. pteronyssinus with known sequence . Figure AD38942; ). Both sA. siro. The results give new insights into the biochemistry of the group 4 allergens of mites and suggest that the interaction of Aca s 4 with patients' IgE may be relevant to allergic hypersensitivity to mites.Our work provides the first comprehensive protein-level analysis of the α-amylase allergen Aca s 4 from A. siro and D. farinae originated from laboratory cultures that were maintained and mass-reared as previously described -agmatine.Aca s 4: a group 4 allergen from JP carried out the biochemical and immunological studies. ML carried out the molecular modeling. MS performed the proteomic analysis. PT participated in the immunological analysis. LM participated in the enzyme inhibition analysis. MM designed the study and wrote the manuscript. All authors read and approved the final manuscript.


The ghrelin-induced up-regulation in mucin synthesis, like that of cPLA2 activity, was subject to suppression by Src inhibitor, PP2, ERK inhibitor, PD98059, as well as Akt inhibitor, SH-5 and ascorbate. Moreover, the loss in countering effect of ghrelin on the ethanol cytotoxicity and mucin synthesis was attained with cNOS inhibitor, L-NAME as well as COX-1 inhibitor, SC-560. Furthermore, while the effect of L-NAME was also reflected in the inhibition of the acinar cell capacity for NO and PGE2 generation, and cPLA2 S-nitrosylation, the COX-1 inhibitor caused the inhibition in PGE2 only. Our findings demonstrate that ghrelin protection of the acinar cells against ethanol cytotoxicity and the impairment in salivary mucin synthesis involves Src kinase activation of the Akt/cNOS pathway that leads to up-regulation in cNOS activity. We also show that cNOS-derived NO induction of the cPLA2 activation through S-nitrosylation, for the increase in PGE2 generation, is an essential element of the protective mechanism of ghrelin action.Recent advances in identifying the salivary constituents of significance to the maintenance of soft oral tissue integrity have brought to focus the importance of a 28-amino acid peptide hormone, ghrelin. Here, we report on the role of ghrelin in countering the disturbances in salivary mucin synthesis caused by ethanol cytotoxicity in rat sublingual gland acinar cells. We show that the countering effect of ghrelin on mucin synthesis was associated with the increase in NO and PGE2 production, and the enhancement in cytosolic phospholipase A Among the recognized consequences of alcohol abused on the health of oral cavity are the diminished secretion of saliva, high incidence of microbial and fungal infections, mucosal inflammatory changes, and the increased risk of cancer of the oral cavity –3. The sRecent advances in identifying the salivary constituents of significance to the maintenance of soft oral tissue integrity have brought to focus the importance of a 28-amino acid peptide hormone, ghrelin . This en2 (cPLA2) enzyme  enzyme , 21. Thebstrates , 20, 22.sylation .In this study, using rat sublingual salivary gland mucous acinar cells, we investigated the influence of ghrelin on the disturbances in salivary mucin synthesis caused by ethanol.7 cell/ml, transferred in 1 ml aliquots to DMEM in culture dishes containing [3H] glucosamine (100 µCi), used as a marker of mucin synthesis, and incubated under 95% O2 – 5% CO2 atmosphere at 37ºC for 16h  Eagle’s minimal essential medium (DMEM), supplemented with fungizone (50 µg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% fetal calf serum, and gently dispersed by trituration with a syringe, and settled by centrifugation . Followi for 16h . After w3H-labeled mucin were treated at 4ºC with 10 volumes of 2% phosphotungstic acid in 20% trichloroacetic acid for 4h and the formed precipitates were collected by centrifugation. The crude glycoprotein precipitates were dissolved in 6 M urea and chromatographed on a Bio-Gel A-1.5 column, and the mucin fractions eluted in the excluded volume were subjected to analysis for incorporation of radiolabel and protein content  and 100 µl aliquots of the spent medium supernatant, according to the manufacturer’s instruction. To assess NO production in the acinar cells, we measured the stable NO metabolite, nitrite, accumulation in the culture medium using Griess reaction .3H]arginine to L-[3H]citrulline. The cells following various experimental conditions were washed with phosphate-buffered saline and incubated at 37°C in HEPES buffer, pH 7.4, containing 124 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.2 mM Cl2, 20 mM HEPES, 10 mM glucose, 10mM cold L-arginine, and 3.5 µCi/ml L-[3H]arginine. Each incubation was performed without and in the presence of 400 µM cNOS inhibitor, L-NAME (+), and the formed l-[3H]citrulline contained in the flow through was quantified by liquid scintillation counting. Values obtained in the presence of L-NAME were subtracted from each sample.The assay of cNOS activity in the intact salivary gland acinar cells was carried out by measurement of the conversion of L-[, L-NAME . After 12 activity in the acinar cells following various experimental conditions was carried out using cPLA2 assay kit (Cayman). Following experimental treatments, the cells were homogenized in 1 ml of 50 mM HEPES buffer, pH 7.4, containing 1 mM EDTA, centrifuged at 10,000× g for 15 min at 4°C and the supernatants filtered through an Amicon YM30 filter concentrators, followed by 15 min incubation with 5 µM of calcium-independent PLA2 inhibitor, bromoenol lactone, and the aliquots (10 µl) of such prepared cell lysates were subjected to cPLA2 assay  .The acinar cells, collected by centrifugation, were resuspended for 30 min in ice-cold lysis buffer , and folAll experiments were carried out using duplicate sampling and the results are expressed as means ± SD. Analysis of variance (ANOVA) was used to determine significance and the significance level was set at P<0.05.To examine the role of ghrelin in countering the detrimental effect of alcohol abuse on salivary mucin production, we employed rat sublingual salivary gland mucous cells exposed to incubation with ethanol at the dose range 3%) that impairs the acinar cell capacity for mucin synthesis and prostaglandin generation –6. We de% that imFurther, our results revealed that significant loss in the countering effect of ghrelin on the ethanol-induced toxicity and the acinar cell capacity for mucin synthesis occurred with cNOS inhibitor, L-NAME as well as COX-1 inhibitor, SC-560, while selective iNOS inhibitor, 1400W and a specific COX-2 inhibitor, NS-398 had no effect (Fig. To gain an additional insight into the involvement of cNOS in ghrelin-induced signaling leading to up-regulation in NO production in response to ethanol, we examined the effect of ghrelin on the acinar cell cNOS activity. We observed that ghrelin countering effect on the ethanol-induced toxicity and the capacity for mucin synthesis was manifested by a 2.4-fold increase in the acinar cell cNOS activity Fig. , the aci2 (2 enzymatic activity. We found that ghrelin countering effect on the ethanol-induced toxicity and the acinar cell capacity for mucin synthesis was reflected in a 68.3% increase in cPLA2 activity (Fig. 2 activity, like that of mucin synthesis, was inhibited by Src kinase inhibitor, PP2 and MAPK/ERK1/2 inhibitor, PD98059, as well as the inhibitor of cNOS, L-NAME, and Akt inhibitor, SH-5. However, COX-1 inhibitor, SC-560, while not causing discernible alteration in the cPLA2 activity, exerted the inhibitory effect on mucin synthesis (Fig. We next sought additional leads into the role of ghrelin in regulation of the acinar cell PGE2 generation. As the initial and rate limiting step in prostaglandin production is the liberation of arachidonic acid from membrane phospholipids by highly selective cPLA2 , 21, we 2 activity and the capacity for mucin synthesis. The results revealed that ghrelin-induced up-regulation in cPLA2 activity, PGE2 generation as well as that of mucin synthesis showed susceptibility to ascorbic acid (Fig.2, revealed that ghrelin prevention of the ethanol-induced cytotoxicity was reflected in the increase in cPLA2 protein S-nitrosylation. Preincubation with L-NAME, on the other hand, caused the blockage in the ghrelin-induced cPLA2 S-nitrosylation (Fig. Finally, we analyzed the influence of S-nitrosylation on the acinar cell cPLAacid Fig., which iacid Fig., 31. Morion Fig. .Diminished secretion of saliva, high incidence of microbial and fungal infections, oral mucosal inflammatory changes, and the increased risk of cancer of the oral cavity are well-recognized consequences of alcohol abuse on the health of oral cavity –3. The dEmploying rat sublingual salivary gland mucous acinar cells exposed to ethanol–6, we de1177 with the involvement of Src/Akt pathway (Further, we found that the countering effect of ghrelin on the ethanol-induced impairment in mucin synthesis, and NO and PGE2 production, were subject to suppression by Src kinase inhibitor, PP2, whereas the inhibitor of ERK1/2, PD98059, elicited suppression in mucin synthesis and PGE2 generation, but had no effect on NO production. Hence, the activation of tyrosine kinase Src appears to be triggering event whereby ghrelin is capable of affecting the acinar cell capacity for mucin synthesis as well as NO and PGE2 generation. The results also point to the role of MAPK/ERK in the processes of PGE2 generation and mucin synthesis. Moreover, the inhibition of ghrelin-induced acinar cell capacity for mucin synthesis, and NO and PGE2 generation by Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME, and only that of PGE2 and mucin synthesis by COX-1 inhibitor, SC-560, suggest that up-regulation in NO generation through Src kinase activation of the Akt/cNOS pathway plays an essential role in the regulation of PGE2 production and mucin synthesis in response to ghrelin. This interpretation of our results is in concordance with the reports showing that post-translational regulation of cNOS activity occurs through a rapid enzyme protein phosphorylation at the critical Ser pathway , 26, 32.Indeed, in our studies, we observed that ghrelin countering effect on the ethanol-induced toxicity and the acinar cell capacity for mucin synthesis was reflected in the increase in cNOS phosphorylation, while the suppression of ghrelin effect of NO production by Akt inhibition was manifested in a drop in cNOS phosphorylation. Moreover, we found that the stimulatory effect of ghrelin on cNOS activity in the intact acinar cells was sensitive to Src kinase inhibitor, PP2 as well as Akt inhibitor, SH-5, but not to ERK1/2 inhibitor, PD98059. Thus, the activation of Src kinase by ghrelin plays an essential role in Akt mediated rapid up-regulation in cNOS activity through phosphorylation.2 (2 activation. We found that ghrelin countering effect on the ethanol-induced toxicity was reflected in up-regulation in the acinar cell cPLA2 activation, which like that of mucin synthesis and PGE2 generation was subject to suppression by Src kinase inhibitor, PP2, and MAPK/ERK inhibitor, PD98059, as well as the inhibitor of cNOS, L-NAME. However, COX-1 inhibitor, SC-560, while not causing discernible alteration in the cPLA2 activity, exerted the inhibitory effect on mucin synthesis. Hence, the activation of the acinar cell cPLA2 by ghrelin for the increase in mucin synthesis to counter ethanol cytotoxicity occurs with the involvement of cNOS and requires Src kinase-dependent MAPK/ERK participation. This interpretation of our results is supported by the literature data indicating that post-translational MAPK/ERK-dependent cPLA2 phosphorylation on the critical Ser505 residue facilitates the enzyme translocation from cytosol to membrane to gain access to phospholipid substrates (We next sought additional leads into the role of ghrelin in regulation of the cross-talk between cNOS and COX-1 enzyme systems leading to up-regulation in the acinar cell PGE2 generation. As the initial and rate limiting event in prostaglandin production is the release of arachidonic acid from membrane phospholipids by highly selective cPLA2 , 20, 21,bstrates , 20, 22.526 residue has been linked to the NO-induced enhancement in COX-2 activity (2 activation through S-nitrosylation at cysteine152 was reported in human epithelial cells (2 activation in the acinar cells, we analyzed the influence of S-nitrosylation on the cPLA2 activity and the capacity of mucin synthesis. We found that, In keeping with well known susceptibility of S-nitrosylated proteins to reduction by ascorbic acid (2 activity and PGE2 generation, as well as the acinar cell capacity for mucin synthesis showed susceptibility to suppression by ascorbate. Furthermore, Western blot analysis of the acinar cell lysates subjected to biotin switch procedure revealed that ghrelin countering effect of the ethanol-induced cytotoxicity was reflected in the increased acinar cell cPLA2 protein S-nitrosylation. The suppression of ghrelin effect on NO production with cNOS inhibitor, L-NAME, led to the blockage in the cPLA2 protein S-nitrosylation, thus supporting the role of S-nitrosylation in the enzyme activation by ghrelin.While, the post-translational activation through phosphorylation of the key enzymes for prostaglandin and NO generation is well recognized, recent evidence indicates that the increase in prostaglandin formation may also result from NO-induced enzyme protein S-nitrosylation , 23, 31.bic acid , 29, 31,2 through S-nitrosylation plays an essential role in the mechanism of ghrelin protection against the ethanol-induced disturbances in salivary mucin synthesis.Taken together, our findings demonstrate that the activation of the acinar cell cPLA