Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as  Protein phosphorylation provides one of the primary means of transducing cellular signals, and as such has been utilized by a majority of organisms that span all domains of life Many variables contribute to the likelihood of a kinase targeting a given protein in the cell including i) temporal expression of the kinase and substrate, ii) subcellular localization of the kinase and substrate, iii) physical interactions between the kinase, substrate and often other proteins, and iv) the existence of sequence specificity determinants  on the substrate protein. Given that kinase specificity motifs can vary widely . Importantly, the ProPeL methodology represents the first use of a live bacterium  acting as an in vivo peptide library for thousands of simultaneous phosphorylation reactions carried out by an exogenous kinase.Here we address the aforementioned deficiencies of both the combinatorial and “proteome-based” approaches to kinase specificity motif determination by presenting a novel and simple strategy that we call ProPeL  can phosphorylate the native E. coli proteome in a manner that is consistent with the sequence specificity of the kinase. Typically, such modifications to the host proteome would go unmeasured, however, in our approach they serve as a convenient readout of the kinase motif. In order to detect these phosphorylation sites, the bacteria are lysed, proteins are digested using trypsin, phosphopeptides are enriched using SCX/IMAC E. coli peptides are not natural substrates for the expressed kinase, phosphorylation motifs are statistically identified using the motif-xE. coli selected as a background database to account for the proteomic environment within which the reaction occurs.Briefly, a kinase of interest is cloned and expressed in E. coli, i) lacks any eukaryotic-like serine/threonine kinases, ii) has only two kinases with known serine/threonine activity, and iii) has very low levels of endogenous serine and threonine phosphorylation Importantly, the ProPeL strategy is made possible by the fact that E. coli expressing PKA, and 467 phosphorylation sites in E. coli expressing CK II. By comparison, negative controls (untransformed E. coli and E. coli expressing empty plasmid) led to the identification of only 23 endogenous phosphorylation sites, consistent with the known low background phosphorylation levels in E. coliE. coli phosphorylation As a proof of principle, we applied the ProPeL approach to two human kinases, Protein Kinase A (PKA) and Casein Kinase II (CK II), both of which have well-defined motifs p-value of 10−55, the hydrophobic cluster (I/L/M/V/F) at the +1 position, albeit smaller, still has a highly significant p-value of 10−10. Motif deconvolution using the motif-x algorithm E. coli phosphorylation sites . Similarly, scoring 348 known human CK II substrates with the CK II pLogo, and an equivalent number of random human phosphorylatable residues with the same CK II pLogo, also yielded a highly statistically significant difference in average scan-x score . These results both demonstrate that the pLogos obtained via the ProPeL methodology can be used to accurately discern the difference between a random serine or threonine residue and a true PKA or CK II phosphorylation site, and in turn that the pLogos are a strong representation of known PKA and CK II specificities.To quantitatively assess the accuracy of the motifs obtained through the ProPeL method, we used a previously devised scoring strategy, the scan-x to identify potential PKA and CK II native kinase targets in the human proteome using these same pLogos . Within just the top 20 predictions , and 4 proteins had associations with PKA either directly or through protein family members. In the case of CK II . In addition, the highest scoring candidate CK II substrate in the human proteome  has also been shown to be phosphorylated at our predicted site (Ser 312) by 27 independent tandem mass spectrometry studies p-value of <10−17. Given the limited present knowledge of the phosphorylation state of proteins, it is also striking that 80% (16/20) of the top 20 predicted CK II phosphorylation sites were previously shown to be phosphorylated ; most, in dozens of independent experiments. The remaining 4 of the top 20 predicted CK II phosphorylation sites had no prior experimental evidence of phosphorylation. However, these 4 predictions are all contained within tryptic peptides that are longer than 35 amino acids, and are thus also unlikely to be detected using standard high-throughput tandem mass spectrometry workflows.We then used e pLogos  and 3. Idictions , eight sof CK II , the 3rdscan-x as an efficient tool to predict kinase substrates within their native proteomes.The aforementioned results demonstrate that the motifs obtained via the ProPeL methodology can be used to scan whole proteomes in order to predict new high-confidence phosphorylation sites specific to a given kinase. Therefore, in addition to uncovering the motifs for kinases with unknown sequence specificities, by using a bacterial expression system, the ProPeL methodology can be used in conjunction with scan-x predictions, and to compare these results to those obtained using the combinatorial peptide library based Scansite predictor scan-x and Scansite PKA and CK II specific predictors  or to purify large quantities of intact and active recombinant kinases. Additionally, when compared to the most current combinatorial peptide library approaches Aside from its simplicity, the ProPeL approach has a number of other benefits over the well-established combinatorial peptide library approaches and the recent proteome-derived library approaches . Most siE. coli intracellular environment.In the present study, large portions of the ProPeL methodology were carried out independently by two different laboratories (one lab for each kinase), using slightly varying interpretations of the SCX/IMAC protocol (see Methods section for details). The fact that these independent experiments yielded hundreds of phosphorylation sites, both of which accurately recapitulated the expected motifs, provides evidence for the robustness of the approach. Additionally, the use of acidophilic (Casein Kinase II) and basophilic (Protein Kinase A) kinases from opposite sides of the kinase phylogeny suggests a potential for applicability to intermediate kinases without significant bias from the E. coli, we demonstrate here that the diversity of the E. coli proteome is more than sufficient for the discovery of protein kinase motifs, which typically contain no more than two to three major specificity-determining positions motif-x we were able to detect numerous motifs with 3 fixed positions  as a way to interrogate different sequence repertoires available for motif determination.While the human proteome is much larger than that of ositions , thus inE. coli, including kinases, may result in a range of toxicities to the bacterial host. Because of this, as with other protein expression systems, some kinases will likely require some fine-tuning of expression and culture conditions. Additionally, there exist kinases that require specific ligand binding . In this way, recombinant kinases may be expressed, purified, and activated in any appropriate system, while still realizing the benefits of the low background phosphorylation levels in the E. coli proteome.It is well established that expressing a variety of exogenous proteins in We also wish to note that while compiling this manuscript we became aware of work by the Huber lab at the University of Illinois demonstrating the use of bacterial phosphorylation to determine the specificity of several plant receptor-like kinases (RLKs). Their successful results (simultaneously under review at the time of this manuscript’s submission) further suggest that the methodology is broadly applicable and can be used to query the specificity of kinases from a wide range of species – the overwhelming majority of which remain yet unknown.Even in this post-genomic era, elucidation of cellular interactions remains a significant bottleneck for our understanding of molecular mechanisms in humans and other species. ProPeL can rapidly decode the sequence-based determinants of kinase specificity and provide experimentalists with the first line of hypothesis generation necessary to fully annotate the breadth of kinase-substrate interactions. In turn, we can better understand the role of protein phosphorylation and how it affects cell physiology in health and disease.Escherichia coli OverExpress C41(DE3) cells from Lucigen were transformed and plated on Luria-Bertani (LB) plates supplemented with 50 µg/mL carbenicillin, with an untransformed control. Colonies were inoculated in LB broth supplemented with 50 µg/mL carbenicillin and grown up overnight at 37°C with shaking at 200 rpm. Overnight cultures were diluted 10 fold into fresh media and grown under the same conditions until OD600 reached 1, at which point protein expression was induced with 1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and grown overnight. Cultures were centrifuged at 3000 g for 20 minutes and cell pellets were stored at −80°C until lysis.Human his-tagged full-length PRKACA/pBEV construct was provided by Vertex Pharmaceuticals Inc Cell lysate was prepared according to Villén and Gygi Phosphopeptide enrichment was performed according to Villén and Gygi. Fifteen mg of each protein sample was reduced, alkylated and digested with trypsin. Peptides were desalted with 500 mg 3 cc tC18 SepPak Vac solid-phase extraction cartridges (Waters) and dried in a SpeedVac. Samples were fractionated by HPLC using a Resource S column  with 8 fractions collected according to Macek et al. A plasmid containing the human CSNK2A1 gene in an Invitrogen Gateway donor vector (pDONR223) was kindly provided by The Broad Institute and was transferred using the standard Gateway protocol to the pDEST17 backbone for bacterial expression (Life Technologies). Escherichia coli OverExpress C41(DE3) cells (Lucigen) were transformed and plated on Luria-Bertani (LB) plates supplemented with 100 µg/mL ampicillin, with the empty vector pUC19 (New England Biolabs) serving as a control. Colonies were inoculated in LB broth supplemented with 100 µg/mL ampicillin and grown up overnight at 37°C with shaking at 250 rpm. Overnight cultures were diluted 50 fold into fresh media and grown under the same conditions until OD600 reached 0.6–0.7, at which point protein expression was induced with 1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and grown for a further 3 hours. Cultures were pelleted by centrifugation at 3000 g and 4°C for 10 minutes. Pellets were stored at −70°C until lysis.Cell lysate was prepared according to Villén and Gygi Phosphopeptide enrichment was performed according to Villén and Gygi. Samples were reduced, alkylated and digested with trypsin. Peptides were desalted with 500 mg 3 cc tC18 SepPak Vac solid-phase extraction cartridges (Waters), snap-frozen in liquid nitrogen and lyophilized. Samples were fractionated by HPLC using a polySULFOETHYL A SCX column (PolyLC) with 12 fractions collected, snap frozen in liquid nitrogen, partially lyophilized to remove acetonitrile, and desalted using 100 mg 1 cc SepPaks (Waters). Phosphopeptide enrichment was performed with PhosSelect iron affinity gel (Sigma) and desalted with StageTips made from C18 material (3M), as described in Rappsilber et al. For each of PKA and a negative control (untransformed cells), 8 SCX fractions were collected and enriched with IMAC as described above.For each of CK II and a negative control , 12 SCX fractions were collected and enriched with IMAC as described above.Before analysis with mass spectrometry, each fraction (either from a set of 8 or a set of 12) was dried down and resuspended in 30 uL of loading Buffer A (see below). Fractions 5, 6, 7 and 8 for a given starting sample were combined, dried and then resuspended to 30 uL loading buffer. For the samples with 12 fractions, fractions 9, 10, 11 and 12 were similarly combined before mass spectrometry was performed. For each fraction (or combined fractions), 4 uL was loaded (using buffer A) onto a C18 nanocapillary column with a pulled tip that sprayed directly into the inlet of a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer.Peptides were eluted from the column into the inlet of the mass spectrometer using an Agilent 1200 HPLC binary pump with a gradient that changed solvents from 100% to 65% Buffer A (0% to 35% Buffer B) over a 48 minute time period .During the course of the injection, the mass spectrometer was run using a TOP10 method .E. coli proteome which included decoy database entries Each fraction’s spectra were searched using SEQUEST Following SEQUEST analysis, peptides from spectra containing predicted serine and threonine phosphorylated peptides were summarized for motif analysis. In order to minimize false positives, for each of the two classes of peptide charges z = +2 and z = +3 and greater, minimum XCORR thresholds were chosen to be above the value of the highest XCORR for a decoy hit from the database. The deltaXCORR (the difference between the first and second hits to the databases) was always required to be 0.08. This corresponded to a predicted False Discovery Rate (FDR) of 0%, however it was still subject to statistical variations and may have included some small contamination of false positives.For each input sample, peptides from each fraction (or combined fractions) were identified with a predicted FDR of 0% as described, and then the peptides were combined into a single list of non-redundant peptides for each fraction. Redundant peptides occurring across fractions, but not across samples were highly specific for a particular kinase, and many peptides were identified in more than one independent spectrum. Negative controls occasionally shared peptides with positive samples and with other negative controls.E. coli proteome The final lists of peptides used for each kinases’ motif analysis consisted of phosphopeptides that were not contained in controls nor previously reported to be found in the normal http://plogo.uconn.edu) are currently in preparation.pLogo images were generated using in-house software. Specifically, pLogos depict residues proportional to the log-odds of their binomial probabilities with respect to a given background motif-x analyses for both the PKA and CK II MS/MS peptide identification results were carried out using an internal version of the motif-x web tool scan-x analyses of known and random substrates were carried out using an internal version of the scan-x software (described in detail in reference http://phosphosite.org), while random substrates were obtained by randomly choosing an equivalent number of serine/threonine 15 mers from the human proteome. A whole proteome scan was also carried out using the PKA and CK II pLogos against the entire SwissProt Human proteome containing nearly 1.17 million serine- and threonine-centered 15 mers to generate a ranked list of the highest scoring predicted substrates for those respective kinases.single human kinase were retrieved. Sites shown to be phosphorylated by PKA were called “positive PKA sites” while those known to be phosphorylated by a different kinase were called “negative PKA sites”. Similarly, sites shown to be phosphorylated by CK II were called “positive CK II sites” while all other kinase specific phosphorylation sites were called “negative CK II sites”. scan-x analyses were carried out using an internal version of the scan-x software (described in detail in reference http://scansite3.mit.edu) with either the CK II or PKA matrices selected under minimum stringency setting . To plot the ROC curves, sensitivity and specificity were calculated as previously described To obtain a “gold-standard” data set for ROC curve generation all human phosphorylation sites within the PhosphoSitePlus database which were only known to be phosphorylated by a Table 1Raw mass spectrometry phosphorylated peptide sequence results for the Protein Kinase A (PKA), Casein Kinase II (CK II), and control ProPeL experiments.(XLS)Click here for additional data file.


Treatment of bovine aortic endothelial cells (BAECs) with OA-NO2 induced a significant increase in both AMPK-Thr172 phosphorylation and AMPK activity as well as upregulation of heme oxygenase (HO)-1 and hypoxia-inducible factor (HIF)-1α. Pharmacologic inhibition or genetic ablation of HO-1 or HIF-1α abolished OA-NO2-induced AMPK phosphorylation. OA-NO2 induced a dramatic increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation that was abrogated by the HO-1 inhibitor, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG). Inhibition of ERK1/2 using UO126 or PD98059 reduced but did not abolish OA-NO2-induced HIF-1α upregulation, suggesting that OA-NO2/HO-1-initiated HIF-1α induction is partially dependent on ERK1/2 activity. In addition, OA-NO2 enhanced endothelial intracellular Ca2+, an effect that was inhibited by the HIF-1α inhibitor, YC-1, and by HIF-1α siRNA. These results implicate the involvement of HIF-1α. Experiments using the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609, the selective CaMKII inhibitor KN-93, and an isoform-specific siRNA demonstrated that OA-NO2-induced AMPK phosphorylation was dependent on CaMKKβ. Together, these results demonstrate that OA-NO2 activates AMPK in endothelial cells via an HO-1–dependent mechanism that increases HIF-1α protein expression and Ca2+/CaMKKβ activation.The AMP-activated protein kinase (AMPK) is an important regulator of endothelial metabolic and functional homeostasis. Here, we examined the regulation of AMPK by nitrated oleic acid (OA-NO The combined blood levels of the free and esterified fatty acid derivatives exceeds 1 µM 2-UFA derivatives in hyperlipidemic patients are elevated relative to levels in normolipidemic subjects in vivo promote cardiovascular homeostasis and compensate for impaired vascular endothelial function in the context of dyslipidemia, obesity, and diabetes. The molecular target(s) and mechanisms underlying the vascular-protective and anti-inflammatory effects of NO2-UFAs remain poorly defined.Plasma free and esterified nitrated oleic acid (OA-NO2+) calmodulin-dependent kinase kinase (CaMKK) 2+ concentrations ([Ca2+]i), and this kinase phosphorylates and activates AMPK in an AMP-independent manner 2+]i in response to nutrients, drugs, or physiological stimuli. While the AMPK pathway is traditionally thought to be a regulator of metabolism, recent studies have demonstrated that AMPK may also act to maintain endothelial function The serine/threonine kinase, AMP-activated protein kinase (AMPK), is a member of the Snf1/AMPK protein kinase family that is found in all eukaryotes 2-UFAs are similar to those observed in response to AMPK activation, we hypothesized that AMPK activation is an important mediator of NO2-UFA activity. As such, AMPK activation may explain the pleiotropic beneficial effects of NO2-UFAs on the cardiovascular system in obesity and diabetes. Whether NO2-UFAs activate AMPK and, if so, by what mechanism(s) has yet to be determined. In the present study, we examined the effects of NO2-UFAs on the AMPK upstream kinases, specifically LKB1 and CaMKKβ. We also investigated whether NO2-UFAs modulate the eNOS/NO pathway, which is known to be an indicator of endothelial function and an important property of AMPK in cardiovasculature. We report that treatment with NO2-UFAs induces CaMKKβ-dependent AMPK activation through an HO-1/HIF-1α/Ca2+ pathway in vitro and show that NO2-UFAs promote p-eNOS and NO production via activation of the AMPK pathway in endothelial cells.Since many of the metabolic changes and endothelial-protective effects attributable to NO2 in endothelial cells, we treated confluent BAECs with different concentrations of OA-NO2 for 2–16 h. AMPK activation was indirectly assessed by western blot analysis of AMPK Thr172 phosphorylation, which is essential for AMPK activity 2  resulted in a dose- and time-dependent increase in AMPKα phosphorylation at Thr172. AMPK Thr172 phosphorylation in BAECs gradually increased beginning 6 h after incubation with 2.5 µM OA-NO2 and reached peak levels after 16 h. This increased AMPK phosphorylation was associated with elevated AMPK activity, as measured by the SAMS peptide assay  at similar potencies, we performed the majority of our experiments with BAECs.To investigate activation of AMPK by OA-NOde assay . OA-NO2 2 concentrations resulted in an upregulation of HO-1 that was initially observed at 4 h with peak induction at 16 h. HO-1 induction and AMPK activation were specifically due to the nitroalkene moiety of OA-NO2, because oleic acid (OA) did not induce HO-1 protein expression or AMPK phosphorylation  to suppress AMPK activity. AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and metformin, two well characterized AMPK activators, were used as positive controls for AMPK phosphorylation. OA-NO2 significantly elevated AMPK phosphorylation  induced AMPK phosphorylation on Thr172 in Hela-S3 cells , at Ser79 , providing an additional indication that AMPK was activated in the absence of LKB1. To further explore the role of LKB1, we investigated the effect of siRNA-mediated LKB1 downregulation on AMPK activation induced by OA-NO2. As shown in 2-induced AMPK phosphorylation was not significantly altered in cells pre-treated with LKB1 siRNA. The suppression of LKB1 protein expression by LKB1 siRNA (∼60%) was confirmed by western analysis. Taken together, these results strongly suggest that OA-NO2-induced phosphorylation of AMPK Thr172 is independent of its upstream kinase, LKB1.We next determined whether OA-NO2 induced both HO-1 expression and AMPK activation, we determined whether OA-NO2 also influenced HIF-1 expression. As predicted, OA-NO2 induced an increase in HIF-1α in HUVECs with a time course similar to OA-NO2-induced HO-1 expression and AMPK phosphorylation  and HO-1-specific siRNA. As shown in 2, suggesting that OA-NO2 acts through the induction of HO-1 to increase HIF-1α expression and subsequently activate AMPK. The efficacy of the HO-1 siRNA in the downregulation of HO-1 expression was confirmed by western analysis (Hypoxia-inducible factor (HIF)-1 is a dimeric protein complex that plays an integral role in the body's response to hypoxia rylation . To explanalysis .2 did not activate PI3-K/Akt (data not shown). To determine whether OA-NO2-mediated HIF-1α induction is dependent on the MAPK pathway, we examined p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation following stimulation with OA-NO2. Treatment with OA-NO2 induced a dose-dependent increase in the phosphorylation of ERK1/2 and p38 MAPK , and the MAPK/ERK kinase (MEK)-specific inhibitor PD98059 (50 µM). Pre-treatment of BAECs with UO126 or PD98059 decreased HIF-1α levels by approximately 50% relative to cells treated with OA-NO2 alone  recognition We next determined whether ERK1/2 was a potential upstream mediator of OA-NOO2 alone , suggest2-mediated AMPK activation, we employed the widely used HIF-1α inhibitor, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) and HIF-1α siRNA. Pre-treatment of BAECs with YC-1 or HIF-1α siRNA significantly inhibited the OA-NO2-induced increase in AMPK Thr172 phosphorylation levels relative to OA-NO2 treatment alone  significantly inhibited OA-NO2-stimulated AMPK phosphorylation  2  2 , suggestrylation . Downregtibodies . Moreoveet al. demonstrated that infection of endothelial cells with a recombinant adenovirus expressing constitutively active AMPK resulted in eNOS activation and increased NO production 2 increased eNOS Ser1177 phosphorylation in a dose-dependent manner that was very similar to that for AMPK phosphorylation. The time course of OA-NO2–induced eNOS phosphorylation was also similar to that for AMPK phosphorylation . As expected, phosphorylation of both AMPK and eNOS was increased after treatment of control BAECs (Ad-GFP-infected or non-infected BAECs) with 2.5 µM OA-NO2 for 16 h , suggesting that OA-NO2-stimulated eNOS phosphorylation does not require Akt but depends on activation of AMPK.Both Akt and AMPK are capable of phosphorylating eNOS at Ser1179 2-induced eNOS phosphorylation was associated with increased NO release. Treatment with OA-NO2 significantly increased NO release, an effect that was inhibited by Ad-DN-AMPK transfection  by LNO2, which shows a higher potency in this context than other established stimuli, including oxidized fatty acids and hemin. This induction of HO-1 expression by NO2-FA is not mediated by NO, NF-κB, or PPARγ et al.2-FA requires synergy between the cAMP-dependent response element and the AP-1 sequences in the −4.5 kb HO-1 promoter region HO is a rate-limiting enzyme in heme degradation and functions to convert heme to biliverdin, carbon monoxide (CO) and iron. Human HO occurs in two main isoforms, the inducible HO-1 form and the constitutive HO-2 form. Previous studies demonstrated that HO-1 exerts anti-inflammatory effects 2 induces HIF-1α expression via HO-1 under non-hypoxic conditions. HIF-1, which exists as a heterodimer composed of the HIF-1α and HIF-1β subunits, has been shown to mediate numerous physiological and pathophysiological responses to hypoxia. Under normoxic conditions, however, HIF-1α is ubiquitinated and rapidly degraded et al.The novel finding of this study is that OA-NO2+]i, which is responsible for CaMKKβ-dependent AMPK activation  and elevates NO bioavailability, and these effects may not only protect against early events in atherogenesis, such as white cell adherence in vitro and are widely considered to be a possible endogenous source of NO in vivo may actually play a compensatory role, providing an adjustable supply of NO to compensate for the impaired NO bioactivity and endothelial-dependent vasorelaxation that is characteristic of the early steps of vascular disease. Second, AMPK signaling acts as a novel regulator of angiogenesis and is specifically required for endothelial cell migration and differentiation under conditions of hypoxia de novo synthesis of diacylglycerol Results from previous studies indicated that endothelial AMPK may play an important physiological role in the function of both endothelial cells and the cardiovascular system as a whole; thus, activation of AMPK may provide an explanation for the beneficial effects of OA-NO2-UFAs activate AMPK in endothelial cells by a mechanism that depends on an increase in HO-1 followed by HIF-1α protein expression and Ca2+/CaMKKβ activation. Our results also indicate that AMPK activity is required for eNOS/NO production in endothelial cells , and AICAR was purchased from Toronto Research Chemicals . The MEK inhibitors PD 98059 and UO 126 were obtained from Calbiochem . 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was purchased from AG Scientific Inc. . Antibodies against phospho-ACC (Ser79), phospho-AMPK (Thr172), AMPK, phospho-LKB1 (Ser428), LKB1, and phospho-eNOS (Ser1177) were purchased from Cell Signaling . The antibodies against ACC were obtained from Alpha Diagnostic International . All other chemicals and organic solvents were of the highest grade and were obtained from Sigma.BAECs and cell culture media were obtained from Clonetics Inc. . HUVECs and cell culture media were purchased from Cascade Biologics . FFA-free bovine serum albumin (BSA) and oleic acid were obtained from Sigma . ZnBG was purchased from Porphyrin Products, Inc. . OA-NO2/95% air at 37°C. BAECs were infected with adenovirus encoding a dominant-negative mutant form of AMPKα (Ad-DN-AMPK) or green fluorescence protein (Ad-GFP) as a control. Infections were performed in 80% confluent cultures of BAECs in media containing 0.1% fetal bovine serum and recombinant adenovirus at the indicated multiplicity of infection (MOI). Cultures were incubated with adenoviruses for 48 h before experimentation. Using these conditions, infection efficiency was typically at least 80%, as determined by GFP expression.BAECs were grown in EBM supplemented with 2% fetal bovine serum and growth factors. HUVECs were maintained in Medium 200 supplemented with a low-serum growth supplement before use. All culture media were supplemented with both penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells between passages 5 and 10 were used for all experiments. All cells were incubated in a humidified atmosphere of 5% CO2 or vehicle for 24 h.HUVECs or BAECs were transfected with LKB1 siRNA, HO-1 siRNA, HIF–1α siRNA, CaMKKβ, or the corresponding scrambled siRNA (negative control) for 48 h using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. Infected cells were starved in serum-free medium for 6 h, then exposed to the indicated concentrations of OA-NOFor NO detection, BAECs grown in 24-well plates were incubated for 30 min in the presence of 15 µM 4,5-diaminofluorescein diacetate (DAF-2 DA) in PBS or in PBS alone (control) in the dark at 37°C. Cells were then washed with PBS to remove excess DAF-2 DA, and the change in fluorescence over 15 min was measured with excitation and emission wavelengths of 485 and 530 nM, respectively, at room temperature using a microplate reader . Changes in fluorescence were also visualized with a fluorescence microscope (Olympus IX71), and images were captured for analysis 2+]i was measured using a Fluo-4 NW kit from Invitrogen according to the manufacturer's instructions. In brief, BAECs were treated with OA-NO2, control or HIF-1α siRNA, YC-1, or BAPTA-AM. The culture medium was then aspirated, cells were washed once with Hepes buffer (pH 7.4), and 1 ml of Hepes buffer with fluorescent dye was added to the cells. After the cells were incubated for 30 min, the fluorescence intensity was measured with excitation and emission wavelengths of 485 and 520 nM, respectively.[CaBAECs, HUVECs, or Hela-S3 cells were lysed in cold RIPA buffer. Protein concentrations were determined using a bicinchoninic acid protein assay system . Proteins were analyzed by western blotting with ECL-Plus detection as described previously AMPK activity was assayed in the presence and absence of AMP (200 µM) using the SAMS peptide, as previously described P-values less than 0.05 were considered significant.Statistical comparisons of vasodilation were performed using a two-way analysis of variance (ANOVA), and intergroup differences were analyzed using Bonferroni's post-hoc test. Time-course studies were analyzed using a repeated-measure ANOVA. All other results were analyzed using a one-way ANOVA. Values are expressed as mean ± SD.


Literature about the value of diagnostic tests for hazelnut allergy in children is scarce. For peanut allergy cutoff levels of specific IgE with a 95% positive predictive value (PPV) were published. To evaluate current diagnostics for hazelnut allergy in children, data of 151 children, who underwent a double-blind placebo-controlled food challenge (DBPCFC) for hazelnut were analyzed. The PPV or negative predictive value (NPV) of the level of specific IgE (CAP) for hazelnut and the size of the skin prick test (SPT) for hazelnut was determined. The influence of spiking of the CAP for hazelnut with rCor a 1 was analyzed. The level of specific IgE for hazelnut was a moderate predictor for a positive DBPCFC for hazelnut. No cutoff levels of specific IgE for hazelnut with a 95% PPV could be determined. Before Cor a 1 spiking the maximum reached PPV was 73% for a cutoff level of 26 kUA/L, after spiking the maximum reached PPV was 64% for a cutoff level of 31 kUA/L. The spiking increased the NPV from 91% to 100% for a cutoff level of 0.35 kUA/L. SPT was a better predictor for a positive DBPCFC compared to the level of specific IgE. When the SPT >16 mm, the PPV was 100%. By combining both tests, the PPV reached 100% when the level of specific IgE for hazelnut was >5 kUA/L and the level of SPT was >12mm. However, the PPV of 100% for SPT alone and the combination of CAP and SPT accounted for only 11% respectively 13% of the children undergoing a DBPCFC for hazelnut. So, the level of specific IgE and reactivity of SPT are moderate predictors for the outcome of a DBPCFC for hazelnut in children. New diagnostic tools are needed to replace the DBPCFC which is burdensome, expensive and limited available.


Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles  were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.Apical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity.  Plasmodium falciparumThere is strong need for vaccines against malaria to combat the global burden of disease, particularly in light of increasing drug resistance Plasmodium falciparum laboratory strains and provide limited insight into the number and type of AMA1 alleles required for an effective vaccine.Currently, there is a limited understanding of antigenic diversity of AMA1 and the requirements of AMA1 vaccines to give broad population coverage. Approximately 10% of the amino acid residues in AMA1 are polymorphic Clusters of polymorphisms that might contribute to antibody escape have been identified on all three domains of AMA1 P. falciparum isolates to i) measure the cross-inhibitory capacity of antibodies to different AMA1 alleles generated by immunisation, ii) understand antigenic diversity of inhibitory epitopes, iii) assess relationships between sequence polymorphisms and immune escape, and iv) investigate whether a multi-allele vaccine may be an effective and feasible strategy to overcome antigenic diversity. Our studies focussed on the growth-inhibitory activity of AMA1 antibodies because this is thought to be the major mechanism of action P. falciparum with defined mutations in AMA1 sequences to identify key polymorphisms, or polymorphic regions, responsible for escape from growth-inhibitory antibodies. Using this approach, we quantified the importance of residues in the C1-L for antibody escape.In this study, we aimed to address these gaps in our knowledge and advance the development of a broadly effective AMA1 vaccine. We compiled a diverse array of ma1 alleles were codon optimised for expression in E.coli (Genscript). The DNA sequence encoding amino acids 25 to 546 of the codon optimised ectodomain was PCR amplified using the oligonucleotide primer sets A-D . Anti-3D7, anti-HB3 and anti-FVO AMA1 antibodies were adjusted to 20 mg/ml of total IgG after purification on protein-G sepharose. Anti-W2Mef #1and anti-FVO#2 rabbit antisera were generated as previously described and tested as whole serum in GIAs P. falciparum asexual stage parasites were maintained in culture in human erythrocytes (blood group type O+) at a hematocrit of 4% in RPMI-HEPES supplemented with 0.25% (w/v) Albumax™ (Invitrogen) and 5% (v/v) heat inactivated human serum P. falciparum isolates used in this study are listed in P. falciparum growth inhibition assays (GIA) were performed as described 5 red blood cells counted per well using a BD FACSCantoII flow-cytometer. FACS counts were analysed using FlowJo™(Ver 6.4.7) software (Treestar). Percent growth inhibition = (1−[parasitemia in test well/mean parasitemia in pre-immunization rabbit controls wells])×100. All GIAs were run in a 96-well plate format, with each antibody or peptide tested in triplicate wells. Parasite growth inhibition is represented as the combined mean of two separate triplicate well assays set up on different days. Growth inhibition by rabbit antibodies is expressed relative to non-immunized control rabbits; there was no significant inhibition of growth by IgG or whole serum from non-immunized control rabbits. Purified antibodies were tested at a final concentration of 2 mg/ml total IgG; normal physiologic concentration of total IgG is 10–15 mg/ml. Since it is not known how the levels of inhibition in GIAs in vitro relate to the inhibitory or protective activity of antibodies in vivo, the levels of inhibition by individual antibodies and antibody pools tested in GIAs should be regarded as relative inhibition, rather than absolute levels of inhibition, allowing comparisons of the inhibitory activity between antibodies and isolates. For analysis of the relationship between antibody inhibition and polymorphisms in AMA1, results were standardized such that the level of inhibition of the homologous isolate by anti-allele antibodies was designated as 100% . This was done to facilitate comparisons of the inhibitory effects of different antibodies against the panel of different isolates; the standardisation did not affect the correlation coefficients or p values.ama1 allelic exchange cassette consisting of a W2Mef AMA1 targeting domain, the W2Mef AMA1 promoter and an E.coli codon-optimised form of the new AMA1 allele to be introduced. The W2Mef AMA1 targeting domain was amplified by PCR using the oligonucleotide primer set E (Plasmodium reichenowi C1-L (aa196–206) were constructed by SOE PCR using a codon-optimised W2Mef AMA1 template, flanking oligonucleotide set L and internal oligonucleotide sets M to R respectively  as a loading control. Horseradish peroxidase-coupled (HRP) goat anti-rabbit Ig (1∶2000) or sheep anti-mouse Ig (1∶2000) Millipore) were used as secondary antibodies.SDS-PAGE and Immunoblot analysis of parasite proteins was performed as previously described Amino acid sequence alignments and relative amino acid identity scores were performed using Genious 5.5.6. software (Biomatters). Phylogenetic trees were generated using ClustalW. Statistical analysis was performed using Prism 5 (Graphpad Software). Correlations between growth inhibition and sequence identity scores were calculated by linear regression. Paired two-tailed t-tests were used for comparisons between the level of inhibitory activity between antibodies or between different isolates.P. falciparum isolates that would broadly represent AMA1 global diversity , which represented different levels of polymorphism naturally present in populations , whereas parasites expressing the XIE or Pr C1-L did not escape inhibitory activity, and were inhibited by 10% more than W2Mef (P<0.001, To further assess the importance of C1-L residues, we used a second transgenic model based on W2Mef-AMA1 allele. The significance of C1-L in the W2Mef allele has not been reported, but our preliminary analyses suggested that C1-L polymorphisms may be important. We constructed plasmids containing W2Mef AMA1 with the C1-L from 3D7, HB3, FVO, XIE, or Pf2006 alleles, or from the related species, ulations . Testingulations . W2Mef hP<0.001, . This suP<0.001, , furtherP<0.001, .Our findings provide important new knowledge on AMA1-based vaccines and understanding immune escape. The diversity of AMA1 inhibitory epitopes is lower than expected from sequence analyses, with substantial antigenic overlap between different alleles. Our results support the concept that, when combined, antibodies raised against four different alleles increases median growth inhibition across all isolates. As such, a vaccine containing four AMA1 alleles may cover allelic-diversity, provided the right alleles are selected. Additionally, we developed novel tools and approaches to dissect the importance of specific polymorphic epitopes and quantified the importance of polymorphisms in the C1-L region.P. falciparum isolates. Our findings suggest that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. Correlations between the level of sequence polymorphism and cross-inhibitory activity against different isolates were modest and inconsistent across the four anti-allele antibodies, when the whole ectodomain, separate domains or discrete regions were considered. Not all polymorphisms mediate the same level of immune escape (The premise that immune selection drives the evolution of AMA1 diversity suggests that an AMA1 vaccine must encompass this diversity to be broadly cross-protective. Recent studies have attempted to estimate global AMA1 sequence diversity and group AMA1 haplotypes into clusters of related sequences e escape  and 6 [4By generating parasites with modified AMA1 alleles, we demonstrated that while the polymorphic C1-L epitope is a significant target of some inhibitory antibodies in GIA, these antibodies only constitute a minor proportion of the total inhibitory antibody repertoire induced by immunisation. W2Mef-AMA1 antibodies targeting the C1-L epitope accounted for 38% of total inhibition, and C1-L also appeared important for HB3. In contrast, C1-L polymorphisms did not have a major effect with the 3D7 allele. This conflicts with previous studies using modified recombinant proteins of 3D7-AMA1 Prior studies on the inhibitory activity of antibodies induced by immunization with a mixture of three or four different AMA1 alleles also suggest that broadly inhibitory antibodies are generated, although the number of different isolates tested in these studies has been very limited Our findings support the concept and feasibility of developing a multi-allele AMA1 vaccine for malaria and suggest that the promising strain-specific protective effects shown in the recent phase 2b trial of a mono-allelic vaccine can be broadened using a multi-allele approach. While the four AMA1 alleles used in this study were useful for establishing this concept, further studies are needed to determine the optimum formulation of specific alleles required in a multi-allele vaccine that maximizes coverage in different populations, and to determine concentrations of inhibitory AMA1 antibodies required for protective immunity. The novel approaches developed in this study using transgenic parasites could be valuable to define the importance of specific polymorphic residues and regions for vaccine escape, thereby aiding the selection of specific alleles for vaccine inclusion.


PMP22 gene in chromosome 17p11.2. Clinically, it presents with painless pressure palsies, typically in the 2nd and 3rd decades of life, being a rare entity in childhood. We present the case study of a six-year-old male child who presented with left hand drop that he kept for over four weeks. Electrophysiological studies suggested HNPP and genetic studies confirmed it. With this paper, we pretend to create awareness to this entity as a diagnosis to be considered in a child with painless monoparesis and to emphasize the importance of electrophysiological studies in the diagnosis.Hereditary neuropathy with liability to pressure palsy (HNPP) results from the deletion of the  PMP22  codifying gene. This leads to myelin instability with liability to nerve pressure palsy, being the superficial nerves the most affected ones [Hereditary neuropathy with liability to pressure palsy (HNPP) is an autosomal dominant disease characterized by recurrent mononeuropathies with focal sensory or motor disturbance precipitated by minor trauma or compression, similar to those entrapment or compression neuropathies commonly acquired in adulthood, that is, median nerve at carpal tunnel, ulnar at the elbow, and peroneal at fibular head. There is a deletion in chromosome 17p11.2, corresponding to the ted ones .PMP22 gene was demonstrated in 1993 [The relation between HNPP and the  in 1993 ; howeverThe prevalence of this disease is unknown, mainly due to the inexistence of detailed epidemiological studies, and it has been estimated in 2–5/100000 (European study) and 16/100000 (population study of southwestern Finland) .It usually occurs between the 2nd and 3rd decades of life, being a rare entity in childhood, with about 15 cases published in children less than 10 years old, between 1975 and 2006 [The first description of HNPP was made in 1947 by De Jong  and it wIt is a benign course entity, concomitant with electrophysiological anomalies characterized by mild generalized demyelinating motor and sensory polyneuropathies that may, inclusively, be detected in asymptomatic patients , 7. MostTherefore, it is extremely important not only to perform a complete clinical history, but also electrophysiological and genetic studies .In the majority of cases, there is in each episode a complete resolution of the symptoms. However, situations with a precocious onset seem to be associated with a higher risk of recurrence .We present the case study of a 6-year-old Azorean male child, Caucasian, and previously healthy, adopted, with unknown familiar history.He was admitted in our Emergency Department  complaining of left hand drop, which he developed after sleeping in his grandmother's lap.He denied any past history of trauma, infection, or physical exercise. He had no pain or other symptoms apart from palsy.Physical examination showed left hand drop, with reduced muscle strength of wrist and fingers extensors. Superficial and deep sensitivities were normal, and deep tendon reflexes were brisk and normal, without muscular atrophies. The remaining physical exam was unremarkable.Initial therapeutics included vitamin B12 and physiotherapy.Since there was no improvement after one month, he was sent to a central hospital in Lisbon. At this time, an electrophysiological exam was performed by one of us, confirming not only the radial mononeuropathy but also showing a mild generalized slowing of the sensory conduction velocities with an ulnar conduction block at the elbow, suggesting HNPP.PMP22 gene, thus confirming the diagnosis.Genetic study was performed (polymerase chain reaction amplification and multiplex ligation-dependent probe amplification—MLPA) and a deletion in the chromosome 17p11.2 was detected, corresponding to the The recovery was complete and when seen after 4 months he had no neurological deficits.With this case study, we pretend to create awareness to HNPP and to the fact that its diagnosis can be missed, mostly during the first decade of life. HNPP can present with multiple phenotypes, and it is this clinical heterogeneity that contributes to the difficulty in reaching a diagnosis. The electrophysiological findings observed in HNPP patients (both children and adults) seem to have no relation with clinical severity . On the other hand, many relatives clinically do not have symptoms, and only by electrophysiological and genetic studies they were demonstrated to be HNPP affected .In this concrete child, despite the difficulty in obtaining a detailed family history, the electrophysiological study, together with the clinical history, led us to think about HNPP, being this diagnosis posteriorly confirmed by the genetic study.It has been suggested  that theIn fact, early onset HNPP seems to be eventually associated with relapsing and further to the evolution of chronic polyneuropathies .Therefore, it is extremely important to obtain a timely diagnosis, in order to prevent recurrent palsy episodes in the patient and his descendents, namely, in situations that imply prolonged immobilizations with nervous compression .In spite of being a rare entity in childhood, HNPP hypothesis should be raised upon a child with a focal, acute painless neuropathy. As it was demonstrated by this paper, therapeutic approach should include physiotherapeutic treatment, being the recovery slow, but usually total.


Here, we used proteases to examine the channel’s composition and function. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3×Dd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function. They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake.The plasmodial surface anion channel mediates uptake of nutrients and other solutes into erythrocytes infected with malaria parasites. The  Malaria parasites develop and replicate within erythrocytes, contributing to disease severity and pathogen success P. falciparumP. knowlesi, a phylogenetically distant malaria parasite, induces remarkably similar channels on rhesus monkey red blood cells P. falciparum clones yield channels with modest, but reproducible differences in gating or pharmacology, even when erythrocytes from a single donor are used clag3.1 or clag3.2 in the parasite genome, is trafficked to the host membrane. Consistent with functional studies, this protein is conserved in all malaria parasites and absent from other organisms Patch-clamp studies have implicated the plasmodial surface anion channel (PSAC) in increased uptake by human erythrocytes infected with in trans. As a peripheral membrane protein in host cytosol, it could modulate channel activity via transient interactions with the pore or, more indirectly, through soluble second messengers How CLAG3 functions to alter erythrocyte permeability is unknown. This 160 kDa protein has no homology to known ion channels and lacks conventional transmembrane domains for formation of stable aqueous pores. The protein belongs to a small conserved family restricted to malaria parasites; some members of this family have been implicated in erythrocyte invasion or cytoadherence Here, we have examined these uncertainties with externally applied proteases. We found that multiple proteases cleave the CLAG3 protein within an extracellular variable loop, but there are significant differences in their effects on transport. Our studies establish a causal link between proteolysis within this loop and reduced PSAC-mediated transport. Site-directed mutagenesis of CLAG3 identified a single variant residue as the responsible protease cleavage site. These studies indicate an involvement of integral membrane CLAG3 at the host membrane and contribute to an evolving structural model for parasite-induced nutrient permeation.P. falciparum clones were cultivated in human erythrocytes  and maintained under 5% O2, 5% CO2, 90% N2 at 37°C. Trophozoite-infected cells were enriched by percoll-sorbitol density gradient centrifugation, washed and resuspended at 5% hematocrit and 25 uL volume in 150 mM NaCl, 20 mM Na2HPO4, 0.6 mM CaCl2, 1 mM MgCl2, pH 7.4. Protease treatment was initiated by adding an equal volume of the buffer with freshly dissolved protease at 2 mg/mL (chymotrypsin or trypsin) or 1 mg/mL (pronase E). In each experiment, a no-protease control was identically diluted in protease-free buffer. After a 1 h incubation at 37°C, 2 mM phenylmethylsulfonyl fluoride (PMSF) was added to inhibit serine proteases.p – τo)/τp, where τp and τo are the interpolated time to 50% hemolysis for protease-treated and matched untreated cells, respectively. Estimates of transport inhibition using this approach match those obtained with tracer flux and patch-clamp methods These cells were immediately used to quantify PSAC activity with osmotic lysis as described Protease protection assays examined whether ISPA-28 binding to CLAG3 prevents chymotrypsin-induced inhibition. These experiments were performed as above with the addition of 2.5 μM ISPA-28 during chymotrypsin exposure. Chymotrypsin treatments were increased to 2 h for Dd2-infected cells to yield detectable transport inhibition. After addition of PMSF, infected cells were washed to remove PSAC inhibitor before initiating transport.Immunoblots were performed with a specific anti-CLAG3 antibody raised against a recombinant C-terminal fragment as described previously http://www.rqtl.org/) as described P = 0.05 significance threshold was determined by permutation analysis. A secondary scan to search for additional QTL was carried out by controlling for the primary chromosome 3 locus as described in the R/qtl software package.QTL analysis to identify genomic loci responsible for differences in chymotrypsin sensitivity was performed as described previously TATACAACTGGACAACATT-3′5′-TTAGTTGGACACACGCT and ATAAGCGTGTGTCCAACTAA-3′5′-AATGTTGTCCAGTTGT to introduce the I1105Y mutation; TTTACAGATCCTGAATACG-3′5′-CAACATTTAATTCCCCAA and AAATTGGGGAATTAAATGTTG-3′5′-CGTATTCAGGATCTGT for the L1115F mutation. After DpnI digestion of template plasmid, the PCR product was used to transform chemically competent XL-10 gold E. coli. After DNA sequencing to confirm the desired mutation, digestion with NotI and PstI released the clag3.1 gene fragment. The fragment was transferred to fresh pHD22Y backbone and used for allelic exchange transfection of Dd2 parasites. The transfected culture was cycled on and off 5 nM WR99210 every three to four weeks for 4 months prior to PCR detection of homologous recombination of the L1115F mutant plasmid. All experiments were performed with a limiting dilution clone The pHD22Y-120w transfection plasmid, generated previously hdhfr gene on the plasmid backbone was prepared, hybridized to the blot, and washed as described previously Genomic DNA was digested with BamHI or HindIII, resolved on a 0.7% agarose gel, and transferred to nylon membrane. A digoxigenin-dUTP labeled DNA probe complementary to the clag3 genes in each parasite. RNA was harvested from schizont-stage cultures with TRIzol reagent , treated with DNase to remove residual genomic DNA contaminant, and used for reverse transcription . PCR was then performed with gene-specific primers and identical quantities of cDNA template in all reactions Two-step RT-PCR was used to quantify relative expression of the two Treatment of infected cells with chymotrypsin or pronase E reduces PSAC-mediated solute transport, but trypsin has negligible effect P. falciparum clones Because our antibody was raised against a recombinant C-terminal fragment, the additional band’s size corresponds to the distance of the site of proteolysis from the protein’s C-terminus. Then, the observed ∼ 35 kDa distance puts the cleavage site within or near a 15–30 residue domain that varies amongst sequenced We next examined the effects of protease treatment on channel-mediated transport with kinetic measurements of osmotic lysis in sorbitol, a sugar alcohol with high PSAC permeability Trypsin’s inability to reduce transport is in sharp contrast to its proteolytic action against CLAG3. This, along with the large number of host and parasite proteins on the erythrocyte surface, suggests that CLAG3 hydrolysis might not be directly responsible for reduced transport after treatment with any of these proteases. One possibility is that other proteins define the PSAC pore; selective cleavage of these other unknown channel components by chymotrypsin or pronase E, but not by trypsin may then account for the lack of trypsin effect on transport. Another possibility is that proteolysis of CLAG3 is fully responsible for transport inhibition, but that the extent of inhibition depends on the specific cleavage site(s) on the polypeptide.P<10−9, Student’s t test, determined from 10 trials each). While this difference appears to parallel a greater efficacy for CLAG3 proteolysis in HB3 , it was necessary to identify conditions for proteolytic PSAC inhibition on this parasite. We found that increasing the duration of chymotrypsin treatments to 2 h yielded reproducible inhibition of Dd2 channels . Parallel experiments using HB3 parasites revealed no protection by ISPA-28 , consistent with this inhibitor’s inability to bind and block HB3 channels , suggesting that the polymorphic 1115 residue is a primary determinant of chymotrypsin-induced transport inhibition. We also found that channels produced by Dd2L1115F were less well inhibited by ISPA-28 than those of the parental Dd2 line .In a third model, there may be additional proteolytic sites required for loss of PSAC function; these additional sites may be either on CLAG3 or on other proteins that are subunits of the channel; these other proteins are unknown but may be encoded by either the parasite or the host. In this scenario, the L1115F residue implicated by our studies would need to play a permissive role in light of the quantitatively complete change in chymotrypsin susceptibility observed in the Dd2clag genes in malaria parasites and the greater accessibility of bloodstream antimalarials to this target on the host cell surface should stimulate further interest in drug discovery or vaccine development Precise determination of which, if any, of the above models is correct remains critical for understanding permeation through this unusual channel and uncovering how the parasite remodels its host cell. In addition to the Dd2-specific ISPA-28, inhibitors that interact with conserved sites on the channel have been found through high-throughput screens. Because these compounds kill malaria parasites in culture and have supported an essential role of PSAC in parasite nutrient acquisition


These effects were not due to changes in regulators of general translation initiation machinery nor mediated via the 5′ or 3′ UTR sequences of Nanog alone. The data we present provide both new conceptual insight into the mechanisms regulated by Gsk-3 that may contribute to ESC self-renewal and, importantly, establish control of protein translation as an additional mechanism involved in modulation of ESC pluripotency.Maintenance of embryonic stem cell (ESC) self-renewal and pluripotency are controlled by extrinsic factors, molecular signaling pathways and transcriptional regulators. While many of the key players have been studied in depth, how the molecular signals interact with transcription factors of the pluripotency network to regulate their action remains less well understood. Inhibition of glycogen synthase kinase 3 (Gsk-3) has been implicated in the maintenance of mouse ESC pluripotency, although there is contradictory data on its role, with enhancement of cell survival and metabolism, stabilisation of c-Myc and activation of Wnt signalling proposed as potential mechanisms. We have discovered that suppression of Gsk-3 activity leads to enhanced protein levels of key transcriptional regulators of the pluripotency network, notably Nanog, Tbx3 and c-Myc. Protein stability was unchanged following Gsk-3 inhibition, although interestingly, Nanog and Tbx3 proteins were found to have half-lives of 1–3 h, while that of Oct4 protein was longer, at 6 h. We demonstrate that the effects on protein levels seen following inhibition of Gsk-3 are due to both enhanced  ESC pluripotency is regulated by the coordinated action of extrinsic factors, signaling pathways and an intrinsic network of transcription factors The transcription factors Oct4, Sox2 and Nanog have been termed ‘master regulators’ owing to the important role they play in specifying and maintaining ESC pluripotency The dynamic transcriptional control of Nanog and other ESC-expressed factors such as Rex1 and Esrrb has been reported The mESC lines, E14tg2a 5 cells/5 cm dish in GMEM supplemented with FBS and LIF, N2B27 supplemented with LIF and BMP4 or N2B27 with DMSO (Control), 3 µM Gsk-3 inhibitor (CHIR) or 1 µM MEK inhibitor (PD0325901) for 3 days. Dishes were counted in triplicate using Trypan Blue dye.Cells were plated at 2×104 cells/cm2 onto gelatin-coated dishes in the presence of serum and LIF, and incubated in the presence of 3 µM CHIR99201 or DMSO (as a control). Cells were followed for 24 h, during which cell morphology and the percentage of Nanog:VNP positive cells were analysed . E14tg2a cells were used as a negative control, to obtain the positive gate region. Nanog:VNP analyses were performed on a FACS Calibur flow cytometer (Becton Dickinson).For time-lapse confocal microscopy, 24 h after plating in serum plus LIF Nanog-TNG mESCs To assess protein stability, ESCs were cultured in GMEM supplemented with LIF and serum or N2B27 supplemented with BMP4 and LIF overnight before adding 10 µg/ml of cycloheximide. Protein extracts were obtained after 0, 1, 3 and 6 hours of cycloheximide addition using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) following the Manufacturer’s instructions. Protein concentrations were measured using the Bio-Rad protein assay reagent following the Manufacturer’s instructions. To investigate protein re-synthesis, cells were cultured as above, treated with 10 µg/ml cycloheximide for 4 hours after which cells were washed extensively before addition of fresh medium free of cycloheximide. Protein extracts were obtained using the NE-PER nuclear and cytoplasmic extraction reagent (Pierce).Cell lysates were separated by SDS-PAGE using 10 and 12% polyacrylamide gels and transferred to nitrocellulose membranes by immunoblotting in semi-dry transfer buffer as described previously 2 and 10% (w/v), 15% (w/v), 20% (w/v), 35% (w/v), 40% (w/v) and 50% (w/v) sucrose, 1 mM DTT and 100 µM cycloheximide. Sedimentation was performed by centrifugation at 130,000×g for 1.5 hr at 4°C in an SW40-Ti swinging bucket rotor (Beckman) ESCs grown in GMEM supplemented with LIF and serum were treated as required and polysomal fractionation carried out by differential sucrose density centrifugation as described in 4, 10 mM CaCl2) (Promega) at 37°C for 30 minutes. DNase was heat inactivated at 65°C for 10 minutes with 1 µl DNase stop solution (20 mM EDTA pH8.0). Reverse transcription was carried out as previously described Total RNA was extracted using Trizol reagent (Invitrogen) following the manufactureŕs instructions. To remove contaminating genomic DNA, RNA samples were treated with RQ1 DNase. 1 µg of RNA was incubated with 1 U DNase (Promega) in DNase buffer  as previously described http://rna.tbi.univie.ac.at/cgi-bin/AREsite.cgi].Potential IRES sequences were identified by searching for polypyrimidine tracts (≥10) and then analysing for sequence motifs e.g. CCTCC, often found within such tracts in functional IRES. Potential upstream open reading frames (uORFs) were identified by looking for a start codon  in the 5′ UTR, an in-frame stop codon before the end of the coding sequence and a length of at least 9 nucleotides. All analyses were performed using in-house Perl scripts. AU-rich elements (ARE sites), that bind proteins such as TTP, HuR, and Auf1 to stabilise/destabilise mRNA, were searched for using AREsite [GCTAGCTCTATCGCCTTGAGCCGTTGGAATA 3′ and Nanog 5′UTR reverse primer 2 sequence with Xhol1 restriction site underlined - 5′ CTCGAGGTCAGTGTGATGGCGAGGGAAATAAA 3′) and cloned into NheI/XhoI restricted pGL3’ ACTAGTGACTTACGCAACATCTGGGCTATAATT 3′ and Nanog 5′ UTR reverse primer 1 5′ GAATTCCGACTGCTCTTCCGAAGGTCTTATA 3′ to generate 222 bp segment (R1) or reverse primer 2, to generate 1048 bp segment (R2), with EcoR1 site underlined) and cloned into SpeI/EcoRI restricted pGL3-MCS The 215bp 5′ UTR region of mouse Nanog gene (RefSeq NM_028016), was amplified with the following primers  had been inserted into the endogenous Nanog locus Nd-ESCs; . In thisde novo protein synthesis.Our data demonstrate that the increase in Nanog protein observed between 1 and 3 h in Gsk-3 DKO ESCs following release of CHX block does not directly correlate with increases in Nanog RNA. However, at later time points, for example 24 h following addition of 1 m, we have observed modest increases in Nanog RNA  and we aNext, we were interested to investigate the mechanism mediating the effects of Gsk-3 inhibition on Nanog translation. A previous report has suggested that general translation rates are increased as mouse ESCs differentiate Another pathway that regulates protein synthesis is the mTor pathway. Gsk-3 can inhibit the mTor pathway by phosphorylating TSC2 at Ser1337 and Ser1341 leading to TSC2 activation and the subsequent inhibition of Rheb and mTor activity Given that Gsk-3 inhibition does not appear to be acting via regulation of the general translation initiation machinery, we were interested to examine whether the 5′ or 3′ untranslated regions of Nanog are responsible for mediating the effects of Gsk-3 inhibition. Our bioinformatic analyses demonstrate that the 215 base pair 5′UTR region of the mouse Nanog gene contains three polypyrimidine tracts, which represent potential internal ribosomal entry sites, and one potential upstream open reading frame (uORF) with an ATG start codon, shown in de novo synthesis of Nanog protein is increased following Gsk-3 inhibition. We also report corresponding increases in polyribosome association of Nanog and Tbx3 RNAs following Gsk-3 inhibition, consistent with enhanced translation. Based on our data, we propose that Gsk-3 is involved in the regulation of dynamically expressed pluripotency-associated factors in mouse ESCs, including Nanog and Tbx3, by altering their translation. This represents a novel additional mechanism by which Gsk-3 may contribute to modulation of ESC self-renewal and highlights the role that control of translation can play in fine-tuning protein levels to reinforce the network of pluripotency factors. Furthermore, our study provides additional evidence regarding the coupling of molecular signals to transcriptional control in ESCs.The data we present here provide new insight into the mechanisms regulated by Gsk-3 in ESCs. We show that inhibition or ablation of Gsk-3 activity enhances the protein levels of the pluripotency-associated transcription factors Nanog, Tbx3 and c-Myc. The stability of these proteins were not altered, but rather we demonstrate, using two approaches, that the A number of possible mechanisms have been proposed to account for the ability of Gsk-3 inhibition to enhance self-renewal of mouse ESCs, including promotion of ESC survival and metabolism Previous reports have suggested that Nanog binds to its own promoter, enhancing its transcription In our study we were interested to determine the mechanism underlying the ability of Gsk-3 to modulate protein translation. Interestingly, when we investigated whether inhibition of Gsk-3 in ESCs led to a change in regulators controlling general (CAP-dependent) initiation of translation, no alteration in phosphorylation of either Ser51 on eIF2α or Ser 539 on eIF2Bε phosphorylation were observed. These results suggest that general translation initiation is not perturbed following Gsk-3 inhibition in ESCs, consistent with the fact that only a subset of pluripotency factors are targeted by Gsk-3, e.g., Nanog and Tbx3. These results are interesting in view of the fact that general translation has been reported to be lower in undifferentiated ESCs, increasing following differentiation into embryoid bodies The fact that translation of other genes, including Oct4 and Cyclin D1, are not increased following Gsk-3 inhibition and the lack of effect on regulators of CAP-dependent initiation of translation suggests that the increase in translation observed with Nanog and Tbx3 are, at least partially, selective. Translation of specific RNA transcripts, without an increase in general translation or in conditions where the CAP-dependent translation is compromised, can occur via a number of different mechanisms including IRES-based translation, control by miRNAs or via RNA binding protein-mediated mechanisms. Our bioinformatic studies predicted that the 5′UTR of Nanog contains three possible IRES and a potential in-frame upstream open reading frame, so we were interested to investigate whether it mediated the effects of Gsk-3 inhibition observed. Our functional analyses show that while the Nanog 5′UTR enhances reporter gene expression, by itself it does not confer responsive to Gsk-3 inhibition. Our further analyses demonstrate a lack of conservation in the Nanog 5′UTR sequences across a range of species. We also discovered that Nanog 3′UTR sequences do not appear to independently mediate the effects of Gsk-3 inhibition, thus, it may be that a combination of 5′ and 3′ UTR sequences are required. Another alternate possibility is that ß-catenin mediates the effects of Gsk-3 inhibition, given its reported role in maintaining ESC self-renewal In this study we present evidence supporting a role for Gsk-3-dependent regulation of translation of selected pluripotency-associated transcription factors that show dynamic protein turnover. We propose that this represents a novel additional mechanism that may contribute to modulation of ESC pluripotency by enabling rapid responses to changes in signals, leading to alteration of protein levels that act to reinforce the network of pluripotency-associated transcription factors. This study highlights the importance of interrogating the control of protein dynamics and translational regulation to gain further insight into the mechanisms contributing to the control of ESC fate.Figure S1Colony morphology of E14 mouse ESCs cultured in N2B27 medium in the different conditions indicated.(TIF)Click here for additional data file.Figure S2Gsk-3 inhibition does not alter Nanog, Tbx3, Oct4 or c-Myc protein stability in the presence of serum and LIF. ESCs were grown in GMEM plus serum plus LIF. (A) E14tg2a WT ESCs were pre-incubated for 24 h without (CTL) or with 2 µM 1 m. (B) WT and Gsk-3 DKO ESCs were cultured for 24 h before addition of cycloheximide (CHX) to halt protein synthesis. (i) Protein samples were extracted after 0, 1, 3 and 6 hours of CHX treatment and immunoblotting performed with the indicated antibodies. (ii) Antibody signals were quantified and protein levels normalised to Gapdh. In each case a value of 100% was given to the untreated (t = 0) samples to enable direct comparisons to be made. The data are the average and S.E.M of triplicate experiments.(EPS)Click here for additional data file.Figure S3Gsk-3 inhibition increases Nanog protein synthesis. E14tg2a mouse ESCs were incubated with cycloheximide (CHX) for 4 h to halt protein synthesis or left untreated (UT) as a control. Cells were washed thoroughly to remove CHX and fresh media supplemented with serum and LIF and vehicle (DMSO/D) or 2 µM 1 m (1 m) added back. Cells were harvested and protein extracts prepared at the times indicated after CHX wash-out. (ii) Immunoblotting was performed with the antibodies indicated. (iii) Nanog and ß-catenin expression levels normailsed to Gapdh and expressed relative to the UT control are shown following the 16 hr treatment. The values are the average and S.E.M. from 3 independent experiments.(TIF)Click here for additional data file.Figure S4Gsk-3 inhibition increases Nanog protein synthesis in Nd ESCs. Nd ESCs were sorted and the VNP-low sub-population collected and incubated in GMEM supplemented with LIF and serum in the presence or absence of DMSO (controls) or in the presence of 3 µM CHIR99201. (A) Representative bright field images of Nd ESCs at 4 h and 24 h after plating. Scale-bar = 100 µm. (B) Representative dot blots for Nanog:VNP expression at 4 h and 24 h after plating. VNP-low FACS sorted populations of Nd ESCs were plated in the conditions indicated and reporter expression measured after 4 and 24 h. At 4 h no differences were observed, while at 24 h a statistically significant increase in the number of Nanog:VNP positive cells was observed. (C) Relative Nanog:VNP expression after plating of low-Nanog sorted population. In the presence of CHIR99201, Nanog:VNP expression increases approximately 30-fold relative to time 0 h, while in DMSO only a 10-fold increase is observed. No differences were observed between GMEM supplemented with LIF and serum in the presence or absence of DMSO. The data are the average relative Nanog:VNP expression levels (relative to time 0 h) and Standard Deviations of at least 3 biological replicates. E14tg2a cells were used as a negative control, to obtain the positive gate region. All p-values were calculated using a two-tailed distribution, two-sample equal variance t-test. For 8, 12 and 24 h time points p<0.05.(TIFF)Click here for additional data file.Figure S5Gsk-3 inhibitors do not alter T389 phosphorylation of S6K1 in Gsk-3 DKO ESCs. Gsk-3 DKO ESCs were cultured in serum plus LIF for 24 h before LIF was removed from the cells for 4 h. Cells were then pre-treated for 30 mins with 5 µM LY294002 (PI3k inhibitor), 10 nM Rapamycin (mTor inhibitor) or 2 µM 1 m (GSk-3 inhibitor). Cells were then treated for 10 mins with 1000 U/ml LIF prior to generation of cell extracts. Immunoblotting was performed with the antibodies indicated.(EPS)Click here for additional data file.Table S1Sequences of primers used in this study for quantitative PCR.(DOC)Click here for additional data file.Video S1Nanog-TNG (GFP-reporter) ESCs were plated in GMEM supplemented with Serum and LIF overnight and then equilibrated for 1 h on the confocal microscope at 37°C, prior to addtion of DMSO. Time-lapse was conducted for a period of 24 h, with images taken every 10 mins. The video shows 10 frames per second.(AVI)Click here for additional data file.Video S2Nanog-TNG (GFP-reporter) ESCs were plated in GMEM supplemented with Serum and LIF overnight and then equilibrated for 1 h on the confocal microscope at 37°C, prior to addtion of 3 µM CHIR99201 (corresponding to CHIR-2 in (AVI)Click here for additional data file.


A) receptors are tonically activated by low and consistent levels of ambient GABA, mediating chronic inhibition against neuronal excitability (tonic inhibition) and the modulation of neural development. Synaptic (phasic) inhibition is spatially and temporally precise compared with tonic inhibition, which provides blunt yet strong integral inhibitory force by shunting electrical signaling. Although effects of acute modification of tonic inhibition are known, its pathophysiological significance remains unclear because homeostatic regulation of neuronal excitability can compensate for long-term deficit of extrasynaptic GABAA receptor activation. Nevertheless, tonic inhibition is of great interest for its pathophysiological involvement in central nervous system (CNS) diseases and thus as a therapeutic target. Together with the development of experimental models for various pathological states, recent evidence demonstrates such pathological involvements of tonic inhibition in neuronal dysfunction. This review focuses on the recent progress of tonic activation of GABAA conductance on the development and pathology of the CNS. Findings indicate that neuronal function in various brain regions are exacerbated with a gain or loss of function of tonic inhibition by GABA spillover. Disturbance of tonic GABAA conductance mediated by non-synaptic ambient GABA may result in brain mal-development. Therefore, various pathological states  may be partly attributable to abnormal tonic GABAA conductances. Thus, the tone of tonic conductance and level of ambient GABA may be precisely tuned to maintain the regular function and development of the CNS. Therefore, receptor expression and factors for regulating the ambient GABA concentration are highlighted to gain a deeper understanding of pathology and therapeutic strategy for CNS diseases.High-affinity extrasynaptic gamma-aminobutyric acid A (GABA A)-mediated synaptic transmission is particularly important because its fast, ligand-gated inhibitory conductance allows fine homeostatic tuning of excitation-inhibition balance  are vital in pursuing specific therapeutic strategies. In inhibitory neurotransmitter systems, gamma-aminobutyric acid (GABA) A receptor  existing in the extrasynaptic space  concentration is high in immature neurons due to the balance of Cl- transporters , and its involvement in their therapeutic strategies. Furthermore, we review the latest findings of tonic depolarization underlying brain mal-development .A receptor subtypes are formed from two copies of a single α, two copies of a single β, and one copy of another subunit . Each subunit combination has a distinct distribution pattern in terms of subcellular domains. The δ subunit, generally partnered with the α4 (forebrain predominant) and α6 (cerebellum predominant) subunits, are shown to be exclusively localized in extrasynaptic membranes in various neuronal cells, including cerebellar granule cells (CGCs), dentate gyrus granule cells, neocortical layer 2/3 pyramidal cells (A (δ-GABAA) receptor shows high affinity and slow desensitization to GABA  and form mostly hetero-oligomeric pentamers , which mainly mediate synaptic inhibition, also exist in the extrasynaptic membrane  and can  neurons . Neverthg humans .A receptor activation can be masked by compensatory mechanisms pyridin-3-ol  was used as an hypnotic drug, reaching a phase 3 trial before its cessation because of side-effects, such as hallucination and disorientation . However, THIP has been recently shown to be effective against behavioral and motor dysfunction in mouse models of autism spectrum disorders  and astrocytic GAT3 (corresponding to GAT4 in mice) synergistically modulate the ambient concentration of GABA via its uptake from the extrasynaptic space . In vivorization . In generization , and thurization  and prevrization . Insuffirization  or excesrization  amounts Figure 1.A Schematic drawing of the regulation of tonic inhibition with some implications for CNS disorders possibly associated with dysfunction of tonic inhibition (details will be shown below) is shown as Tonic GABA conductances exist within ascending sensory pathways on each level, including spiny neurons of the dorsal horn in the spinal cord , thalamiA receptor null mutant mice pyridin-3-ol (THIP) acts as an anti-nociceptive in rodent models of acute pain . The antant mice , indicatant mice . Tonic Gant mice . Studiesant mice . This re KO mice . TherefoA receptors and down-regulation of glutamic acid decarboxylase (GAD) 67 have been found to be possible mechanisms underlying this difference  reduces learning and memory. Mice deficient in the α5 subunit show better performance of cognitive functions compared with wild-type  neurons, biphasically modulating their excitability depending on the presence or absence of stress , in CRH neurons, switching tonic GABA conductance from inhibitory to excitatory and resulting in increased activity of the HPA axis and elevated levels of corticosterone  axis plays an important role for mediating physiological responses by promoting stress-induced steroid synthesis  recognizably distinct syndromes caused by mutations in specific genes or chromosomal loci, and (2) more common, genetically heterogeneous subjects, referred to as idiopathic autism . The numA receptor subunits, particularly the δ subunit, are targeted substrates of FMRP , indicating that tonic inhibition is a potent therapeutic target for FXS. In contrast, increased GAD65 and GAD67 expression was also observed in the cortex and hippocampus of FMR1 KO mice (Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by malfunction of the fragile X mental retardation 1 (FMR1) gene on the X chromosome, which encodes the FMR protein FMRP; . Common  of FMRP . Dendrit of FMRP , suggest of FMRP . Decreas of FMRP . Olmos-S KO mice , thus inSimilar presynaptic dysfunction in GABAergic neurotransmission has been proposed as a mechanism underlying the autism spectrum-related disorder, Rett syndrome. Mice lacking the causal gene, methyl CpG binding protein 2, from GABAergic neurons recapitulate phenotypes as those seen in Rett syndrome, with decreased GABA levels, reduced expression of GAD65/67, and miniature inhibitory postsynaptic current amplitudes in cortical pyramidal neurons . TherefoUBE3A), encoding the Ube3a protein , the reduction of tonic inhibition could thus underlie cerebellar ataxia in Angelman syndrome. Expression of GAT1 and Ube3a is increased during development  in idiopathic autism spectrum disorders , and thus the developmental switch of GABA-mediated depolarization to hyperpolarization is induced by changes in the transmembranous Cl- gradient, from its efflux to influx, respectively. This change is regulated by cation-Cl- cotransporters . Furthermore, in immature neurons the release mechanism of GABA is non-vesicular and non-synaptic, and therefore GABA-mediated activity is generally tonic. These tonic depolarizing (and excitatory on occasion) GABA actions are necessary for neurogenesis, differentiation, migration, and synaptogenesis .Gamma-aminobutyric acid induces depolarization and hyperpolarization in the immature and adult brain, respectively. The GABAA receptor subunits exhibit a completely different expression pattern  tangentially migrate into the cerebral wall , resulting in the modulation of intracellular Ca2+ oscillations. These oscillations could differentially affect the migratory status of GABAergic cells (“go”) and cortical plate cells (“stop”). Therefore, immature cortical plate neurons and GABAergic neurons migrate in distinct patterns to form the microgyrus and thus, abnormally induced tonic functions of GABA could be involved in migration disorders that ultimately result in cortical malformations.Neocortical malformations, such as polymicrogyria show the presence of cortical cells in heterotopic positions; often caused by a disruption in neuronal migration . TherefoFigure 1). In addition to pharmacological progress, animal model studies are presently opening the door for practical medicine. However, an overview of these studies also presents problems for clinical application. For example, the same phenotype may result from a decrease or increase of tonic inhibition. Therefore, the net shift from excitation to inhibition may not always occur with a modulation of tonic inhibition. This paradox is possibly due to the complexity of network regulation by tonic conductance. For example, simultaneous receptor activation in either inhibitory or excitatory neurons, or multiple mechanisms can be responsible for changing membrane properties, such as shunting inhibition and a shift in intracellular Cl- concentration. Although temporal characteristics of tonic inhibition are weak, maintaining network functions can possibly regulate its strength, thus suggesting a narrow therapeutic range for such modulators. A detailed assessment of these mechanisms would therefore be needed to develop a therapeutic strategy.This review has focused on the recent progress in understanding the impact of dysregulated tonic inhibition in CNS diseases. Accumulating evidence highlights the pathophysiological significance of dysregulated tonic inhibition in a number of neurodevelopmental disorders, psychiatric disorders, and cognitive dysfunctions . Thus far, very few studies have addressed this series of events as a potential risk factor for CNS diseases. Therefore, research focused on the pathological effects of multimodal functions of GABA during neurodevelopment may shed light on understanding the pathology of neurodevelopmental disorders in which GABA is known to play a role, for example in schizophrenia and autism spectrum disorders.This review also discussed tonic depolarization induced by ambient GABA during early development, where the release of GABA is possibly non-vesicular and occurring before synaptic organization (see The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The development of Kaposi’s sarcoma (KS), the most common malignancy in AIDS patients, results from Kaposi’s sarcoma-associated herpesvirus (KSHV) infection of endothelial cells and subsequent induction of proliferation, angiogenesis, and inflammation. Previously, we demonstrated that KSHV infection of human umbilical vein endothelial cells (HUVEC) induced a transcriptional induction of Angiopoietin-2 (Ang-2), a pro-angiogenic and pro-inflammatory cytokine that is highly present in KS tumors. This transcriptional up-regulation of Ang-2 started at 12 h and peaked at 54 h post-infection. In difference to our previous data, here we demonstrate that KSHV infection of HUVEC induces rapid release of Ang-2 within minutes of viral contact. Pre-made Ang-2 is stored in the Weibel-Palade body in endothelial cells and is released through regulated exocytosis. KSHV binding to HUVEC is responsible for rapid Ang-2 release because blockade of viral binding inhibits this cytokine exocytosis. We find that KSHV binding to its integrin receptors on endothelial cells activates the integrin tyrosine kinase receptors signaling pathways, including tyrosine phosphorylation of the kinases FAK and Src, and triggers rapid calcium mobilization. This mobilization likely plays a key role in mediating Ang-2 release, as its inhibition by various calcium chelators and calcium channel blockers substantially reduces Ang-2 release. We also demonstrate a direct interaction and association of the kinase Src with the alpha1C subunit of L-type calcium channel. Indeed, specific inhibitors of protein tyrosine phosphorylation not only disrupt this interaction but also abolish Ang-2 release. Finally, preliminary data from in vitro cell adhesion assays suggest that this rapidly released Ang-2 enhances migration and adhesion of monocytes to the infected endothelial cells. To our knowledge, this is the first demonstration of interaction between KSHV and its integrins receptors in regulating rapid cytokine release. This study also uncovers a novel mechanism of KSHV induction of angiogenesis and inflammation, which is much faster, and could likely play important roles in the early event of KS tumor development.