Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends. Saccharomyces cerevisiae, we explored the functional role of polo kinase Cdc5 in regulating the DSB–induced checkpoint. Polo kinases have been previously involved in checkpoint inactivation in all the eukaryotes, and they are frequently overexpressed in cancer cells. We found that elevated levels of Cdc5 affect the cellular response to a DSB at different steps, altering DNA processing and overriding the signal triggered by checkpoint kinases. Our findings suggest that Cdc5 likely regulates multiple factors in response to a DSB and provide a rationale for a proteome-wide screening to identify targets of polo kinases in yeast and human cells. Such information may have a practical application to design specific molecular tools for cancer therapy. Two related papers published in PLoS Biology—by Vidanes et al., doi:10.1371/journal.pbio.1000286, and van Vugt et al., doi:10.1371/journal.pbio.1000287—similarly investigate the phenomenon of checkpoint adaptation/overriding.Double strand DNA breaks (DSBs) are dangerous chromosomal lesions that can lead to genome rearrangements, genetic instability, and cancer if not accurately repaired. Eukaryotes activate a surveillance mechanism, called DNA damage checkpoint, to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to DSBs, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Budding yeast is a good model system to study checkpoint, and most of the factors involved in the DSBs response were originally characterized in this organism. Using the yeast  Saccharomyces cerevisiae cells suffering a double stranded DNA break (DSB) activate a robust Mec1-dependent checkpoint response when DSB ends are processed to expose single-stranded DNA (ssDNA), and progression through the cell cycle is arrested prior to anaphase. Several well conserved factors are recruited at the DSB lesion, and contribute to the activation of a signaling pathway based on sequential phosphorylation events driven by the upstream kinases Tel1/ATM and Mec1/ATR which, in turn, activate the transducer kinases Rad53/Chk2 and Chk1 SAE2, KU70/80, RAD51, RDH54, SRS2, affect the inactivation of the DSB-induced checkpoint response Drosophila Polo kinase CDC5 is an essential gene and the point mutation cdc5-ad  causes the inability to adapt to one irreparable DSB lesion and to turn off Rad53 kinase cdc5-ad cells can recover from checkpoint when the DSB is repaired, suggesting that adaptation and recovery are two genetically separate processes cdc5-ad mutation in Plks has not yet been isolated in mammals; however, it was found that Plk1 depletion severely blocks checkpoint recovery and adaptation Therefore, the activity of Plks must be finely regulated during the DNA damage checkpoint response, and it is worth mentioning that the expression of a constitutively active Plk1 protein variant overrides the G2/M arrest induced by DNA damage To further characterize the functional link between Plks and the DNA damage checkpoint and, possibly, to understand why Plks are frequently overexpressed in cancer cells, we used budding yeast as a model system to study DNA damage related events in the presence of high levels of Cdc5.Here, we show that overproduction of Cdc5 impairs the Mec1-signaling pathway in response to an inducible DSB lesion, altering phosphorylation of Ddc2, Rad9, Rad53 and other Mec1 targets. We also found that elevated levels of Cdc5 slow down DSB ends processing, although it does not prevent the formation of ssDNA, which triggers the recruitment of checkpoint factors. Consistently, we observed that overexpression of Cdc5 does not alter the loading of the apical Mec1 kinase checkpoint complex and recruitment of the checkpoint mediator Rad9, but surprisingly it physically interact with the checkpoint inhibitor Sae2, inducing its hyper-phosphorylation and an increased and persistent binding onto a DSB lesion.We propose that high levels of polo kinase Cdc5 override Mec1-induced checkpoint response to DSB lesions, likely by regulating multiple factors, previously phosphorylated by CDK1, involved in both DSB processing and checkpoint signaling. Our work may represent a simple model to further understand why polo kinases are frequently overexpressed in cancer cells.DNA damage checkpoints represent a barrier to oncogenesis; in fact, loss of these surveillance mechanism is a characteristic of early tumor development cdc13-1 mutant cells with uncapped telomeres has been reported to override the checkpoint-dependent cell cycle block in the G2 phase of the cell cycle In budding yeast, overproduction of the polo kinase Cdc5 in CDC5 is placed under the control of the GAL1 promoter, the DNA damage-induced inhibition on overproduced Cdc5 is not complete. This is likely due to the elevated Cdc5 levels, which are higher than the endogenous amount  in which one irreparable DSB can be induced at the MAT locus by expressing the site-specific HO nuclease CDC5 and the two cdc5-ad and cdc5-kd mutant alleles  carrying the MYC or HA epitope tags at their carboxyl-terminal end. Quantitative multiplex PCR was then used to monitor co-immunoprecipitation of DNA fragments located either 66 kb centromere-proximal to the MAT locus (CON) or 1 kb away from the HO-cut site (DSB) , whether overexpression of te (DSB) .CDC5 overexpressing cells blocked by nocodazole treatment  it binds Sae2 protein, causing its hyper-phosphorylation and leading to its increased and persistent binding onto DSB.In conclusion, in the present study we further explored the role of the polo kinase Cdc5 in attenuating the DNA damage checkpoint in budding yeast. We found that overproduction of Cdc5 affects different parameters of the cellular response to an inducible DSB: i) it overrides Mec1 signaling and prevents the phosphorylation of various Mec1 targets ; ii) it causes a slower resection of DSB ends in a The emerging scenario suggests that Cdc5 may target multiple factors involved in various aspects of the cellular response to DSB lesions and DNA damage checkpoint signaling. Indeed, Cdc5 is a fundamental regulator of cell cycle progression and targets many proteins throughout a normal cell cycle Recently, a proteome-wide screening led to the identification of novel Cdc5 targets in a normal cell cycle MATα, hmldelta::ADE1, hmrdelta::ADE1 ade1-100, trp1delta::hisG, leu2-3, leu2-112, lys5, ura3-52, ade3::GAL::HO), with the exception of strain Y38, which was generated from strain Y5 . To construct strains, standard genetic procedures for transformation and tetrad analysis were followed. Y38 and Y210 were obtained by integration of ApaI-digested plasmid pJC57 (pGAL1::CDC5-3HA) at the URA3 locus. Y215 was derived by integration of ApaI-digested pJC59 (pGAL1::CDC5-3myc) at URA3 locus. Y220 was obtained by integration of ApaI-digested plasmid pJC62 (pGAL1::cdc5-K110A-3HA) at URA3 locus. Y222 was obtained by integration of ApaI-digested plasmid pJC69 (pGAL1::cdc5-L251W-3HA) at URA3 locus. Deletions and tag fusions were generated by the one-step PCR system Mata his3 ura3 trp1 6lexAOP-LEU2; lexAOP-lacZ reporter on plasmid pSH18-34) as the host strain 340–705 for the two-hybrid assay, expressing lexA fusion with polo box domain of Cdc5, was obtained by amplifying the corresponding coding sequence of CDC5 gene (aa 340 to 705) from genomic DNA and ligating the resulting fragment into pEG202 (kind gift from R. Brent). Prey plasmids pJG4-5-Swe1173–400 and pJG4-5-SAE2, expressing B42 activating domain fusions, were obtained by amplifying the corresponding coding sequence of SWE1 (aa 173 to 400) and SAE2 (full length) from genomic DNA and ligating the resulting fragments into pJG4-5.Strains are listed in The TCA protein extraction and the western blot procedures have been previously described It was performed as previously described 3, cocktail proteases inhibitors (Roche)). HA-tagged proteins were immunoprecipitated using anti HA monoclonal antibody (12CA5) conjugated to protein G Agarose.Yeast whole cell extracts were prepared by FastPrep  in NP-40 lysis buffer , 1 mM dithiothreitol (DTT), 60 mM β-glycerophosphate, 1 mM NaVOE. coli cells as previously described GST and GST-PBD were induced in BL21 Crude extracts were prepared as described 6 cells/ml were arrested with nocodazole (20µg/ml). A DSB was produced by adding 2% galactose and inducing the production of the HO endonuclease. The maintenance of the arrest was confirmed by FACS analysis and monitoring of nuclear division. Genomic DNA was isolated at intervals, and the loss of the 5′ ends of the HO-cleaved MAT locus was determined by Southern blotting SspI sites, we represented the length of the minimal resection for each time point in the graph  were grown in YEP+3%raffinose. The cell cultures were treated with nocodazole to block and maintained the cells in G2/M. Galactose was then added to induce the overproduction of Cdc5 and sample have been taken at the indicated times. (A) The cell cycle block in G2/M was analyzed by FACS. (B) Cdc5 protein was analysed by western blotting with polyclonal antibody, which recognized both the endogenous Cdc5 and the overproduced Cdc5-myc tagged protein.Cellular levels of endogenous and overproduced Cdc5 protein. (A) Exponentially (L) growing culture of the strain Y79 (wild type) and Y114 ((0.96 MB TIF)Click here for additional data file.Figure S2GAL1::CDC5-MYC), exponentially (L) growing in YEP+3%raffinose were blocked in G2/M by nocodazole treatment (N) and zeocin (50 µg/ml) was then added to cause DSBs formation. After 30 min. of treatment with zeocin, 2% galactose was added and samples were taken after 1 hour. Overproduced Cdc5 protein has been immunoprecipitated with anti MYC antibody. Cdc5-MYC and Rad53 proteins were analysed by western blotting with monoclonal antibodies 9E10 (αMYC) and Ma.EL7 (αRad53).Overproduced Cdc5 co-immunoprecipitates with Rad53. (A) Cultures of the strains Y79 (wild type), Y114 ((0.32 MB TIF)Click here for additional data file.Figure S3KpnI sites. After the HO cleavage, DNA is resected. When the left and right leu2 sequences have been converted to ssDNA, repair by SSA can take place and can be monitored by the appearance of a SSA product in a Southern blot.  YEP+raffinose nocodazole-arrested cell cultures of wild type YMV80 and isogenic GAL1::CDC5 strain were transferred to nocodazole-containing YEP+raffinose+galactose (time zero). (B) KpnI-digested genomic DNA, prepared from cells collected at the indicated times, was analysed by Southern blotting with a LEU2 probe. Two fragments, 8 and 6 kb long  are evident in the absence of HO cut, whereas the HO-induced DSB causes the disappearance of the 6-kb species and the formation of a 2.5-kb fragment (HO-cut fragment). Repair by SSA converts such fragment to a repair product of 3.5-kb (SSA-product). (C) Western blot analysis of protein extracts with anti-Rad53 antibodies (Mab.EL7).Overproduction of Cdc5 does not prevent DSB repair by Single Strand Annealing (SSA). (A) Schematic representation of the YMV80 system used to detect DSB repair by SSA. Vertical bars show the relevant (0.81 MB TIF)Click here for additional data file.Table S1Yeast strains used in this study.(0.05 MB DOC)Click here for additional data file.


This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca2+ levels. In this study, we aimed at characterizing 1. expression and functions of KCa channels (including BKCa beta-subunits), and 2. biophysical properties of BKCa channels.Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a CaGCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique.Ca types in human GCs, the intermediate- (IK) and the small-conductance KCa (SK). Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by KCa blockers . Functional IK channels were also demonstrated by electrophysiological recording of single KCa channels with distinctive features. Both, IK and BKCa channels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BKCa channel revealed the presence of mRNAs encoding several BKCa beta-subunits  in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca2+ dependence of individual BKCa channels which we observed in electrophysiological recordings.We identified two KCa, they express all three KCa types known. We suggest that the plurality and co-expression of different KCa channels and BKCa beta-subunits might allow differentiated responses to Ca2+ signals over a wide range caused by various intraovarian signalling molecules . The knowledge of ovarian KCa channel properties and functions should help to understand the link between endocrine and paracrine/autocrine control in the human ovary.Functional and molecular studies indicate the presence of active IK and SK channels in human GCs. Considering the already described BK Ca channel of large (big) conductance (BKCa) in human GCs has a part in endocrine-regulated progesterone production -1H-pyrazole; Sigma-Aldrich) was dissolved in DMSO (10 mM) and final DMSO concentration in the cell culture medium did not exceed 0.01% (v/v).Progesterone concentrations were measured in supernatants of human GCs cultured in 24-well plates. On day three of culture, cells were treated in triplicates with the respective compounds. After 24 h the supernatants were collected and the progesterone concentrations were measured using an ELISA . Experiments were repeated with cells from independent cell preparations (each pooled from up to 3 patients) to account for interpatient variability. After normalization to the respective control (untreated) values, data were statistically analyzed by a repeated measures ANOVA followed by Newman-Keuls multiple comparison test.2, 1 MgCl2, 10 Hepes, and 10 glucose (pH 7.4). The intracellular solution (IC) contained (in mM) 130 K-gluconate, 5 NaCl, 2 EGTA, 1 MgCl2, and 10 Hepes (pH 7.4). Single channel currents were recorded in the inside-out configuration. To assess the Ca2+-sensitivity of the BKCa channel the free Ca2+ concentration [Ca2+]i of the IC solutions was adjusted by using varying concentrations of CaCl2 according to calculations performed by means of the software "Calcium" [2 concentrations were used: 1.000 mM ([Ca2+]i = 100 nM), 1.870 mM ([Ca2+]i = 1 μM), 1.977 mM ([Ca2+]i = 5 μM), 1.996 mM ([Ca2+]i = 10 μM), 2.013 mM ([Ca2+]i = 20 μM), 2.100 mM ([Ca2+]i = 100 μM), and 3.000 mM ([Ca2+]i = 1 mM), respectively. To obtain channel current-voltage relationships a voltage protocol ranging from -80 mV to +90 mV in 10 mV steps was used with each potential applied for 200 ms. For evaluation of channel open probability Po, single channel currents were recorded at +60 mV, +70 mV, +80 mV, and +90 mV for 1600 ms in each case. For analysis, frequency histograms of current traces were calculated and the two amplitude peaks corresponding to open and closed channel state were fitted using Gaussian distributions. The single channel current amplitude was measured as differences between the two peaks. Integration of the area under the curves and division of the area under the open channel peak by the area under the entire curve yielded Po. In the case of two or more active individual channels in an inside-out recording, the open probability per one channel was calculated by converting the values according to the number of simultaneously active channels at each time point. [Ca2+]i values for half-maximal activation (EC50) were determined by fitting Po as a function of [Ca2+]i with a sigmoidal dose-response-curve with variable Hill slope and a fixed bottom value of zero. The voltage-dependence of Po was fitted using a Boltzmann sigmoidal function with the bottom value fixed to be 0 and potentials for half-maximal activation (V50) as well as slope values were obtained. The values are given with the respective 95% confidence interval (C.I.).Human GCs were grown on glass cover slips for 2–12 days and currents were recorded at room temperature by means of an EPC-9 amplifier  . PositivCalcium" . The folTotal RNA was isolated from several human GC preparations, and reverse transcribed using Superscript-RT II  in combination with either a 18-mer polydeoxythymidine primer or random hexamers of polydeoxynucleotide primers. PCR amplification was carried out with oligodeoxynucleotide primer pairs, which spanned at least one intron of the genomic sequence . The cDNA array was analyzed by means of a chemiluminescent detection method  .Ca α subunit proteins in human ovarian sections (5 μm) was examined according to standard procedures i > 1 μM , and because K+ was the only permeant ion present in sufficiently high concentrations on both sides of the membrane. The maxiCl can be excluded due to the low symmetrical Cl--concentrations of 9 mM. In almost all recordings exhibiting the IK channel, at least one BKCa channel was simultaneously active  = 20 μM , Hill slope = 1.0 μM (0.5 to 1.5 μM), and EC50 (70 mV) = 12 μM , slope = 0.8 μM (0.3 to 1.4 μM). Comparing individual cells, heterogeneity of EC50 values – even in the same cell preparation – was observed. The values covered a range of 2 to 118 μM  and of 1 to 91 μM , respectively. At high [Ca2+]i (1 mM) and high positive potentials (+70 mV) inactivation of BKCa channels was observed for time periods ranging from several hundred ms to several s  and a slope factor of +12 mV (+9 to +15 mV) were observed at [Ca2+]i = 1 mM  as well as all three known types of small-conductance KCa  points at a complex KCa repertoire. The finding of mRNA alone can be misleading as was shown in a study on glioma cells in which mRNAs for all KCa channels were detected, although only BKCa channels were functional i i at +60 mV. An increase of [Ca2+]i by one order of magnitude is known to shift the potential V50 necessary for half-maximal activation in several other cell types by about 50 mV (30 to 94 mV) to more negative values [2+]i = 1 μM that can be reached by stimulation with ACh, oxytocin, or relaxin i in close proximity to the channel might be much higher than the monitored overall cytoplasmic elevation of [Ca2+]i, and thereby BKCa channels can be opened at more physiological voltages. Pronounced differences in alteration of global [Ca2+]i monitored by standard techniques and of local [Ca2+]i were described in other cell types i -12. There values -45. At a relaxin -10, the oduction . This apll types ,47.50 values for Ca2+-sensitivity of the BKCa channel over two orders of magnitude could be due to different β subunits present since they are known to be important modulators of Ca2+- and voltage-sensitivity of the channel i was reported to occur in the presence of β2 and/or β3 subunits i than the BKCa i range. The question of existence of GCs subpopulations regarding KCa channels and BKCa β subunits has to be studied in the future to understand cellular processes on the level of individual GCs. The rich instrumentation of Ca2+-dependent channels might be seen in relation to the abundance of intraovarian signaling molecules  acting via raised Ca2+ levels. Therefore, we suggest that this channel group has a part in mediating the conjunction between endocrine  and local ovarian signaling systems.In summary, we found expression  of several classes of KCa: big conductance KCa; [Ca2+]i: intracellular Ca2+ concentration; C.I.: confidence interval; CL: Corpus luteum; EC50: [Ca2+]i for half-maximal activation; GC: granulosa cell; gsc: single-channel conductance; hCG: human chorionic gonadotropin; IbTx: iberiotoxin; IK: intermediate conductance KCa; IVF: in vitro-fertilization; KCa: Ca2+-activated K+ channel; Po: channel open probability; SK: small conductance KCa; TRAM-34: 1- [(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole; V50: potential for half-maximal activation.ACh: acetylcholine; BKThe authors declare that they have no competing interests.MHT performed most of the experiments, was involved in conception of the study, contributed to analysis and interpretation of the data and to writing of the manuscript. DB and UB provided human GCs and were involved in study design. AM conceived of the study and contributed to interpretation of the data and to writing of the manuscript. LK conceived of the study, coordinated the experiments, contributed to analysis and interpretation of the data and to writing of the manuscript. All authors read and approved the final manuscript.


Leukemia associated antigens (LAAs) are being increasingly identified by methods such as cytotoxic T-lymphocyte (CTL) cloning, serological analysis of recombinant cDNA expression libraries (SEREX) and mass spectrometry (MS). In additional, large scale screening techniques such as microarray, single nucleotide polymorphisms (SNPs), serial analysis of gene expression (SAGE) and 2-dimensional gel electrophoresis (2-DE) have expanded our understanding of the role that tumor antigens play in the biological processes which are perturbed in acute myeloid leukemia (AML). It has become increasingly apparent that these antigens play a dual role, not only as targets for immunotherapy, but also as biomarkers of disease state, stage, response to treatment and survival. We need biomarkers to enable the identification of the patients who are most likely to benefit from specific treatments  and to help clinicians and scientists improve clinical end points and treatment design. Here we describe the LAAs identified in AML, to date, which have already been shown to play a dual role as biomarkers of AML disease. Acute myeloid leukemia (AML) describes a heterogeneous group of diseases which are typified by the outgrowth of immature hematopoietic cells, known as blasts, in the bone-marrow and peripheral blood. Most patients with AML, who are younger than 60 years of age, achieve complete remission after polychemotherapy and/or stem cell transplantation (SCT) . However+ and CD8+ T cells as well as B-cells. More recently microarray, single nucleotide polymorphisms (SNPs) and 2-dimensional gel electrophoresis (2-DE) have allowed the large scale analysis of gene, RNA and protein differences between patient and normal donor samples.Immunotherapy offers an opportunity to remove residual diseased cells in remission and can potentially reduce or eliminate the risk of relapse. During the last 20 years significant improvements have occurred in our ability to identify tumor antigens. Originally leukemia associated antigens (LAAs) were identified by virtue of their role in leukemogenesis and were subsequently shown to be immunogenic. The serological analysis of recombinant cDNA expression libraries (SEREX) technique allowed the identification of LAAs due to their recognition by humoral responses but also led to the demonstration that these antigens were recognized by CD4We have had a particular interest in identifying LAAs which may act as targets for the immunotherapy of AML. Tumor antigens such as Wilm’s tumor (WT1) , prefereApart from acute promyelocytic leukemia (APL), none of the remaining subgroups of AML have a single cytogenetic aberration associated with all samples from that subtype . The PMLHowever, some genes are overexpressed in AML and are associated with specific subgroups of AML, such as PRAME. PRAME was first identified as an antigen in human melanoma by virtue of patient CTLs . PRAME iHeat shock proteins (HSPs) may be referred to as molecular chaperones and are found in both normal and cancerous cells. These proteins are immunogenic, with the specificity of the immune response being dictated by the peptides they chaperone  is involved in telomere elongation and is frequently found to be expressed in human cancers and rarely in normal tissues. It has been the target of a number of immunotherapy clinical trials  of childhood AML patients and that the rates of overall and disease-free survival were higher in patients with elevated levels of expression. They also found that PRAME expression was significantly higher in patients with t. Conversely, in adult AML Paydas et al.  could add important prognostic information in distinguishing patients who have a poor prognosis from those who would respond well to chemotherapy and achieve long term remission . In addition these studies identified known leukemia antigens such as RHAMM, MAGE-A1 as well as novel targets for leukemia immunotherapy such as PASD1, SSX2IP and BAGE.We and others have used the SEREX technique  to ident+ cells, could be distinguished based on protein expression and mass spectrometry (MS) peak patterns corresponding to post-translational modification differences.Microarray analysis of gene expression has provided a powerful tool to characterize the molecular mechanisms underlying many cancers. With regards to AML, cDNA microarray analysis has allowed the identification of genes that are differentially expressed in leukemic blasts as compared to normal hematopoietic lineages, indicate prognosis, show the gene expression effects of varying treatment regimes and identify the genes involved in the processes underlying disease , heat shock proteins and redox regulators , multidimensional protein-identification technology (MudPIT) and field effect transistor (FET)-based protein detection are being applied to the identification of cellular and serum biomarkers (reviewed in + cells (Down regulation of eukaryotic initiation factor 5A has been recently described as a novel target for hypusination inhibitors (HI) and, furthermore, the combination of Imatinib and HI produced a synergistic effect that was restricted to Imatinib resistant BCR-ABL+ cells . The ide+ cells . In factSNPs analysis provides a rapid way in which regions of DNA within the genome with an abnormal gain or loss can be identified, enabling the detection of oncogenes and/or tumor suppressor genes, respectively, which are involved in the pathogenesis of a particular disease. Fitzgibbon et al.  used SNPWe have described the expanding field of AML antigen identification and our increasing understanding of the role these gene products play in leukemogenesis. We detail the dual role that some of these LAAs play as biomarkers of prognosis, MRD and survival while a number of preceding reviews have described in detail the role of these antigens as potential targets for the immunotherapy of AML. It remains to be determined whether these antigens will be predictive of disease progression, response to treatment and whether they can play a similar role in predicting outcome to treatment in the same way that cytogenetic findings do. Immunotherapies continue to be developed to target these LAAs as treatments for AML and our growing understanding of the role LAAs play in the pathogenesis of leukemia can only aid in the development of effective therapies.


It has also been suggested that increased intracellular free Ca2+ levels as well as MAO-A may be contributing to the oxidative stress associated with Alzheimer disease (AD).Calcium , a Ca2+-binding protein in brain that is greatly reduced in AD, decreases MAO-A activity. The effects of A23187 and CB-28K are both independent of any change in MAO-A protein or gene expression. The toxicity  associated with either A23187 or the AD-related β-amyloid peptide, which also increases free intracellular Ca2+, is attenuated by MAO-A inhibition in HT-22 cells as well as in primary hippocampal cultures.Incubation with Ca2+ availability could contribute to a MAO-A-mediated mechanism with a role in AD-related oxidative stress.These data suggest that increases in intracellular Ca If it is not detoxified by antioxidant systems such as glutathione peroxidase – one of the most abundant such systems in brain -5-HT (NEC-225) and [14C]-PEA (NEC-502) were purchased from PerkinElmer Life Sciences. The MAO-A and CB-28K antibodies were purchased from Santa Cruz Biotechnology. IgG-HRP conjugates were obtained from Cedarlane Laboratories. All other reagents were purchased commercially.5-Hydroxytryptamine (5-HT), β-phenylethylamine (PEA), the β-actin antibody and protease inhibitor cocktail were bought from Sigma-Aldrich Co. -5-HT (for MAO-A activity) or 50 μM [14C]-β-PEA (for MAO-B activity). The incubation was terminated by acidification and the labeled metabolites were extracted into ethyl acetate/toluene , an aliquot of which was subjected to scintillation spectrometry to determine radioactive content. MAO-A substrate kinetics was determined using protein (100 μg per reaction) from HT-22 cells pre-incubated in the absence or presence of Ca2+  and subsequently incubated with increasing concentrations of [14C]-5-HT [15 μM-4 mM]. Incubation in oxygenated potassium phosphate buffer  and extraction of labeled metabolites proceeded as described above. Data were analyzed for estimates of Km and Vmax using the Prism v3.01 software.MAOA and -B activities (nmol/hr/mg protein) in HT-22 cell cultures were estimated radiochemically . Briefly5 cells/well) using ExGen™500 (Fermentas) according to the manufacturer's directions. Expression of eGFP fluorescent protein revealed a transfection efficiency of approximately 50% using this technique. Cells were routinely harvested 24 h post-transfection.The pREP-CB-28K  plasmid expression vector was kindly  provided by Dr. A. Pollock . HT-22 cells were seeded in log phase and transfected with plasmid DNA  was visualized by enhanced chemiluminescence. Depicted immunoblots are representative of two-three independent experiments.Treated HT-22 cells were washed twice with ice-cold PBS and proteins were extracted in ice-cold lysis buffer , containing protease inhibitor cocktail and 1 mM orthovanadate. Standard denaturing (SDS-PAGE) conditions were used to resolve proteins, which were then transferred to nitrocellulose. Protein expression in total cell lysates . The resultant semi-quantitative PCR products were electrophoresed on agarose gel and visualized using ethidium bromide staining.Total RNA from treated HT-22 cells was prepared with TRIZOL reagent according to the manufacturer's protocol and digested with RNase-free DNase to clear residual genomic DNA. First strand cDNA was reverse-transcribed from 2 μg of total RNA using oligo-(dT) . The cDNA was amplified using HT-22 cells were loaded with the fluorescent dye Fluo3-acetoxymethyl ester  at 4 μM for 30 min at 37°C . Fluores2-DA  or DMSO, vehicle). The cells were washed in prewarmed HEPES-buffered (20 mM) HBSS (pH 7.0) containing 5 mM glucose, and prepared for DCF fluorescence .2',7'-Dihydrodichlorofluorescein diacetate (DCFH2-DA)  permeate2+- and Mg2+-free Hank's balanced salt solution (HBSS) supplemented with 15 mM HEPES and penicillin (100 U/ml)-streptomycin (100 μg/ml) (Gibco). Collected tissues were digested at 37°C with 0.25% trypsin-EDTA 15 min. The reaction was quenched with fetal bovine serum  and tissues were rinsed 3–4 times with HBSS to remove FBS. Following centrifugation at 800 × g for 10 min, the medium was removed and cells were resuspended in a chemically defined serum-free NeuroBasal medium supplemented with 1% N2, 2% B27, 50 μM L-glutamine, 15 mM HEPES, 10 U/ml penicillin and 10 μg/ml streptomycin. Neurons were then plated on coverslips (coated with 25 μg/ml poly-D-lysine), and grown at 37°C with 5% CO2-humidified atmosphere. The medium was replaced 24 h later with fresh NeuroBasal medium lacking L-glutamine and antibiotics. Medium was replaced after 4–5 days in vitro (DIV). Neurons were treated on DIV 7, following which they were fixed with 4% paraformaldehyde in 0.01 M PBS for 20 min at room temperature, washed several times with PBS, and stained with Hoechst 33258  for microscopic visualization of chromatin condensation (a characteristic of apoptotic cell death).Animal care followed protocols and guidelines approved by University of Saskatchewan Animal Care Committee and the Canadian Council on Animal Care. Rat hippocampal cultures were prepared from E18 fetuses  as described before with some modifications . In brieP < 0.05) was assessed by unpaired t-tests or by one-way ANOVA with post hoc analyses relying on Bonferonni's Multiple Comparison Test (GraphPad Prism v3.01). Data are represented as mean ± standard deviation (SD).Significance (set at 2+ (range: 0.1–10 mM) was increased, with a peak (~20% above baseline) around 0.5–1 mM Ca2+  , indicating that Ca2+ facilitates the enzymatic reaction. Vmax (nmol/h/mg protein) remained unchanged , suggesting that Ca2+ was acting via an allosteric mechanism  was changed [0.0441 ± 0.0009 (without Ca2+: r2 = 0.9973) vs. 0.0328 ± 0.0007 (with Ca2+: r2 = 0.9967)], whereas the y-intercept (e.g. 1/Vmax) was not [3.13 ± 0.21e-4 (without Ca2+) vs. 3.20 ± 0.18e-4 (with Ca2+)] ] Fig. .2+ ionophore A23187  so as to examine the effect of increasing Ca2+ availability on MAO-A activity in living cells. Fluorescence microscopy   was also not affected by A23187 treatment   was observed, which was partly blocked by pretreatment with the MAO-A inhibitor clorgyline   Fig. . MAO-A p 4) Fig. . The exp 4] Fig. . Neitherur) Fig. .2+ and decreased constitutive ROS levels   or protein  or the pREP-CB-28K expression plasmid Fig. . Overexpels Fig. . Overexp 6] Fig. . CB-28K  6] Fig.  and it dein Fig. . Neither2+  = 69.16, P < 0.0001]. Post-hoc analysis revealed that the effect of Aβ(1–42) [P < 0.001] was greatly attenuated by pre-treatment with the MAO-A inhibitor clorgyline  [P < 0.01]  peptide  had significantly more free Ca2+ Fig.  [F01] Fig. .2O2 is a natural by-product of MAO-A-mediated degradation of amines [2O2.Hf amines . The norf amines  and Alzhf amines ,41 could2+  occurs tagonist , mouse [tagonist , rat [31er et al  have rec2+ are necessary to reveal this effect, the fact that MAO-B activity remains unaffected clearly supports differences in MAO-A and MAO-B regulation. The need for millimolar concentrations also suggests pathological relevance. Interestingly, the mitochondria can modulate cytoplasmic Ca2+ homeostasis by accumulating Ca2+ into the very high micromolar range [2+ concentrations localized to microdomains may also represent unique means of modulating localized mitochondrial membrane and/or matrix function [2+ during cell stimulation to help clear large Ca2+ loads [2+ may be involved in several pathological conditions, including ischemia-reperfusion lesions, neurotoxicity and neurodegenerative diseases, where ATP depletion, overproduction of ROS and release of apoptotic factors lead to cell damage [While low millimolar levels of Caar range . Furtherfunction , includifunction . In neur2+ loads -48. The l damage .2+ ionophore A23187, we now confirm that increasing free intracellular Ca2+ well above normal levels selectively increases MAO-A activity in living hippocampal HT-22 cells. The associated increase in peroxyradicals is moderately decreased by MAO-A inhibition, supporting a contribution by the enzyme to A23187-associated toxicity. Intracellular Ca2+ levels can also be quenched by specific proteins including calbindin-28K (CB-28K), the predominant Ca2+-binding protein in brain [2+ levels and significantly decreased MAO-A activity. Again MAO-B activity was spared. The effects of both A23187 and CB-28K occurred independent of any change in the expression level of MAO-A protein (or mao-A gene), which, in combination with our demonstration of affinity changes of the MAO-A enzyme in the presence of Ca2+, suggests a functional modification of the MAO-A protein itself. These combined data implicate a CB-28K-dependent, Ca2+-sensitive component to MAO-A function, which could be exacerbated when CB-28K expression levels are compromised, such as in normal aging or, more importantly, during neurodegenerative processes [2+ being considered as a core factor in the pathogenesis of AD [2+ signalling can occur early in AD, well before any obvious extracellular Aβ deposition [2+ channel blockers, which are known to block the selective increase in MAO-A in the senescence-accelerated mouse model [2O2 production in an increasingly toxic Aβ environment, leading to localized cell death. Treatment with Aβ(1–40) increases intracellular levels of Ca2+ [in vivo assessment of the effect of Ca2+ on MAO-A as well as a closer examination of the relation between Ca2+/CB-28K and MAO-A in AD tissues is warranted.Using the Cain brain . Overexprocesses ,34,51. Iis of AD . This isis of AD -58 and bposition . Furtherse model , may impse model  and may se model ,62. As Cse model ,63, it i of Ca2+  [present of Ca2+ . A patho of Ca2+  as well  of Ca2+  and Aβ-ie.g. H2O2. Given the neuroprotective role of CB-28K in human pathologies such as AD (and in models of AD), in addition to our demonstration that the toxicity of the AD-related peptide, Aβ, is sensitive to MAO-A inhibition, we suggest that part of the oxidative stress associated with AD may rely on a Ca2+/CB-28K-sensitive, MAO-A-mediated mechanism.MAO-A is a risk factor in AD and changes in MAO-A activity parallel changes in the production of ROS, 2+ is positively correlated with the activity of MAO-A, a mitochondrial H2O2-generating enzyme. The influence of Ca2+ on MAO-A function could potentially impact AD-related pathology, which is often associated with altered Ca2+ homeostasis, mitochondrial dysfunction and oxidative stress.The availability of free intracellular CaThe author(s) declares that there are no competing interests.XC carried out the majority of the work. ZW purified and cultured the primary hippocampal cells. GGG participated in the FLUO-3AM microscopy studies. XC, XL and DDM drafted the manuscript. DDM conceived and designed the study, and coordinated data collection and analysis. All authors read, edited and approved the final manuscript.


Stress Granules (SG) are sites of accumulation of stalled initiation complexes that are induced following a variety of cellular insults. In a genetic screen for factors involved in protecting human myoblasts from acute oxidative stress, we identified a gene encoding a protein we designate SGNP (Stress Granule and Nucleolar Protein).A gene-trap insertional mutagenesis screen produced one insertion that conferred resistance to sodium arsenite. RT-PCR/3′ RACE was used to identify the endogenous gene expressed as a GFP-fusion transcript. SGNP is localized in both the cytoplasm and nucleolus and defines a non-nucleolar compartment containing 5.8S rRNA, a component of the 60S ribosomal subunit. Under oxidative stress, SGNP nucleolar localization decreases and it rapidly co-localizes with stress granules. The decrease in nucleolar SGNP following oxidative stress was accompanied by a large increase in nucleolar 5.8S rRNA. Knockdown of SGNP with shRNA increased global mRNA translation but induced growth arrest and cell death.These results suggest that SGNP is an essential gene that may be involved in ribosomal biogenesis and translational control in response to oxidative stress. Translation of “housekeeping” transcripts can be globally repressed following oxidative stress by the formation of cytoplasmic stress granules (SGs) Myoblast transfer has been proposed as a potential therapy for muscular dystrophy In an effort to identify genes that could contribute to the increased survival of cells following transplantation, we used a retroviral gene trap approach to screen immortalized human skeletal myoblasts (LHCNM2) for insertions that lead to resistance to the oxidizing agent sodium arsenite. About 40% of the parental cells failed to exclude propidium iodide, a membrane impermeant dye The subcellular distribution of the GFP-82 fusion protein was examined with anti-GFP antibodies and compared to cells infected with the unfused eGFP cDNA (pBabepuro-eGFP). Free GFP was detected ubiquitously while the GFP-82 fusion protein was present in the cytoplasm but also colocalized with the nucleolar marker nucleolin . After tst exon of Iso1 but initiated from a different upstream exon. Based on the subcellular distribution of the GFP–82 fusion protein under normal and stress condition, we designated this protein Stress Granule and Nucleolar Protein (SGNP).RT-PCR/3′ RACE was performed to amplify the fusion transcripts using primers from the 5′ unique GFP sequences. Sequence analysis indicated that GFP was inserted into the hypothetical LOC26010 locus. Four predicted alternatively spliced isoforms were confirmed by RT-PCR (iso1–4) or 5′RACE . The vector pTrap-GFP had been inserted between the 1st and 2nd exons and in frame with the downstream exons to produce a GFP-82 fusion protein . RT-PCR A rabbit polyclonal antibody against a SGNP peptide showed that the endogenous SGNP exhibited a distribution similar to the GFP82 fusion protein. Nuclear SGNP colocalized with B23, a nucleolar protein . After sWe stably expressed retroviral SGNP isoforms 2, 3 or 4 fused with either an HA peptide tag or a green fluorescent protein (eGFP) to assess potential differences. The N-terminal tagged SGNP iso3  and a DUF1387 domain deletion mutant (GFP-SGNP Iso3 ΔDUF1387) were both primarily localized to the nucleus and nucleolus in unstressed cells . After sSGNP occasionally defined a previously undescribed nucleoplasmic compartment . This SGPyronin Y staining indicated that the paranucleolus contained a much higher concentration of RNA than the nucleolus , arrow. Little is known about whether nuclear export of 5.8S rRNA is affected after oxidative stress. In unstressed cells, the 5.8S rRNA antibody Y10B produced intense labeling of the cytoplasm and faint signals in the nucleolus (stained with fibrillarin) due to rapid transfer of the 5.8S rRNA to the cytoplasm . In contLentiviral shRNAs vectors producing >90% reduction in SGNP mRNA  reduced 35S]methionine for 30 minutes three days after transduction with shGFP or shSGNP. Total protein synthesis was increased two-fold following shSGNP knockdown . One step Reverse-transcriptase PCR (SuperScript™ III One-Step RT-PCR System from Invitrogen) was performed using a 3′RACE Primer Appoly(T)17 according to To make a C-terminal HA tagged SGNPiso2 expression construct, SGNPiso2-HA cDNA was obtained by RT-PCR using primers SGNP hmm22084 and hmm22048-HA-Re. PCR products separated on 1% agarose gel showed two bands, representing iso1 and 2 respectively. The iso2 was subcloned into the PCR2.1TA vector and sequenced. The SGNP iso2-HA fragment was then removed with EcoRI and subcloned into the retroviral vector pWZLblast at the EcoRI site.SGNP iso3 or iso4 cDNA was obtained by RT-PCR using primers SGNPhmm22984 and hmm22984-Re. The DUF1387 fragment was obtained by PCR from SGNP iso3 cDNA with primers DUF1387 Fw and DUF1387 Re. To make an N-terminal GFP tagged SGNP iso3, iso4 or DUF1387, all cDNA fragments were ligated into the PCR2.1TA vector and removed with BamHI/EcoRI, then subcloned into the bglII/EcoRI site of pEGFPC1. The eGFP tagged fragments were then cut out by SnaBI/EcoRI digestion and subcloned into the retroviral vector pBabepuro at the SnaBI/EcoRI site. To the N-terminal GFP tagged SGNP iso3 deletion (GFP-SGNP iso3 ΔDUF), the SGNP iso3 ΔDUF was obtained by ligating two PCR fragments from SGNP iso3 cDNA using the following two pairs of primers: fragment 1 ; fragment 2 (fragment 2 Fw: hmm22984-Re) and subcloning the ligated fragments into the BglII/EcoRI sites of the vector pEGFPC1. The eGFP tagged fragment was then removed with SnaBI/EcoRI and subcloned into the retroviral vector pBabepuro at the SnaBI/EcoRI sites.Hmm22084 and hmm22984 are splicing isoforms predicted in Genbank.The shRNA expression vectors were engineered by PCR using U6sh FW: shSGNP1 Re or U6sh FW: shSGNP2 Re or U6shF:shGFP Re. The PCR products  were digested with SpeI and PstI and cloned into thepHRCMVSin18 vector, generously provided by E.H.Blackburn  Lentivirus was generated as described previously All RT-PCR was performed using SuperScript™ III One-Step RT-PCR System using 1 ug of RNA. To monitor the quantity and quality of RNA samples, RT-PCR of the house keeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was performed in parallel according to Cells grown on coverslips were fixed with 4% paraformaldehyde for 10 min., permeabilized with 0.1% Triton X-100 in PBS, and incubated for 2 h at room temperature with primary antibodies. After three PBS washes, cells were incubated with either FITC, or Texas red, Alexa Fluor 488 [green], or Alexa fluor 594 [red])-conjugated secondary antibodies for 2h, then the coverslips were mounted in Vectorshield mounting media with DAPI.An affinity-purified rabbit polyclonal antibody against SGNP was generated using two peptides (the sequences CGKKKNNKRKRSKSKQHQGNKDAKDKVER and CNGSSNQRRRFNPQYHNNRLNGPAKSQGS). Other antibodies were to nucleolin , TIAR and B23 (Santa Cruz Biotechnology), GFP (Invitrogen), fibrillarin and 5.8S rRNA(Y10B)(Abcam) and Hsc70 and HSP70 (Stressgen). All images were taken by Leica confocal microscope or Zeiss fluorescent microscope.35S]methionine (Amersham Corp). 5×105 cells from each plate were pelleted and washed with ice-cold PBS, then solubilized in 0.5 ml of lysis buffer (RIPA buffer with proteinase inhibitor tablet from Roche). Equal volumes of cell lysate (20 ul) were loaded onto 10% SDS-PAGE gels and analyzed by autoradiography. The amount of radioactivity of newly synthesized proteins was quantified by Phosphorimager analysis.shGFP and shSGNP cells, ∼80% confluent on 60-mm culture dishes, were washed twice with methinone-free DMEM and incubated in methinone-free myoblast culture media (methinone-free DMEM plus 20% FBS with supplements as described) for 1 hour. Cells were then incubated for an additional 30 min after adding 50 uCi/ml [


Yet studies on the identity of the IP3-dependent intracellular Ca2+ stores remain virtually unexplored.The gliotransmitters released from astrocytes are deemed to play key roles in the glial cell-neuron communication for normal function of the brain. The gliotransmitters, such as glutamate, ATP, D-serine, neuropeptide Y, are stored in vesicles of astrocytes and secreted following the inositol 1,4,5-trisphosphate  IP3R isoforms.We have therefore studied the potential existence of the IP2+ storage proteins and chromogranins interact with the IP3Rs to activate the IP3R/Ca2+ channels, i.e., increase both the mean open time and the open probability of the channels, these results imply that secretory granules of astrocytes function as the IP3-sensitive intracellular Ca2+ store.Given that the secretory granule marker proteins chromogranins and secretogranin II are high-capacity, low-affinity Ca However, in recent studies it has been demonstrated that the ER plays only a minor role in the IP3-dependent Ca2+ mobilization system in the cytoplasm of neuroendocrine cells 3-induced Ca2+ release in the cytoplasm of the cells in which they exist 2+ of all subcellular organelles 3R/Ca2+ channels in neuroendocrine cells 3R/Ca2+ channels of secretory granules are ∼7-fold more sensitive to IP3 than those of the ER 2+ in response even to one-seventh the IP3 concentration that is required to induce Ca2+ release from the ER.Similar to other secretory cells, the regulatory secretory pathway in astrocytes is shown to depend on inositol 1,4,5-trisphosphate  and secretogranin II (SgII), in astrocytes. Moreover, in view of the highly concentrated localization of the IP3R/Ca2+ channels in secretory granules of neuroendocrine cells 2+ release through the IP3R/Ca2+ channels in proliferation, migration, and survival of glioblastoma 3Rs in the large dense core vesicles of astrocytes.Taken together, these results clearly indicate that in secretory cells where secretory granules are intrinsically present secretory granules function as the major IP3R isoforms in secretory granules of astrocytes. Hence, in view of the roles of secretory granules in secretory cells as the major IP3-sensitive intracellular Ca2+ store, the existence of secretory granules in glial astrocytes appears to point out the presence and operation of an IP3-sensitive intracellular Ca2+ store role of secretory granules in astrocytes.In the present study, we found the localization of two typical secretory granule marker proteins chromogranin B and secretogranin II 3R peptides specific to terminal 10–13 amino acids of type 1 (HPPHMNVNPQQPA), type 2 (SNTPHENHHMPPA) and type 3 (FVDVQNCMSR) were synthesized with a carboxy-terminal cysteine and anti-rabbit polyclonal antibodies were raised. The polyclonal anti-rabbit antibodies were affinity purified on each immobilized peptide following the procedure described The polyclonal anti-rabbit chromogranin A (CGA), chromogranin B (CGB), secretogranin II (SgII) antibodies were raised against purified intact bovine CGA, CGB and SgII The human brain tissue  samples examined in this study were obtained from patients undergoing surgical treatments following written consent in accordance with appropriate clinical protocols in the Department of Neurosurgery of Seoul National University Hospital. The use of samples for the present study was approved by the Institutional Review Board of Seoul National University Hospital .3) and fixed for 2 h at 4°C in PBS containing 0.1% glutaraldehyde, 4% paraformaldehyde immediately after surgical removal. After three washes in PBS, the tissues were postfixed with 1% osmium tetroxide on ice for 2 h, and washed three times in PBS. The tissues were then embedded in Epon 812 after dehydration in an ethanol series. After collection of the ultrathin (70 nm) sections on Formvar/carbon-coated nickel grids, the grids were stained with 2.5% uranyl acetate (7 min) and lead citrate (2 min). For immunogold labeling experiments, the ultrathin sections that had been collected on Formvar/carbon-coated nickel grids were floated on drops of freshly prepared 3% sodium metaperiodate for 40 min. After etching and washing, the grids were placed on 50 µl droplets of buffer A  for 1 h. The grids were then incubated for 3 h at room temperature in a humidified chamber on 50 µl droplets of polyclonal anti-rabbit CGB or SgII antibody appropriately diluted in solution B , followed by rinses in solution B. The grids were reacted with the 15-nm gold-conjugated goat anti-rabbit IgG, diluted in solution A. Controls for the specificity of CGB- or SgII-specific immunogold labeling included 1) omitting the primary antibody, 2) replacing the primary antibody with the preimmune serum, and 3) adding the primary antibody in the excess presence of purified CGB or SgII.For the electron microscopic study of human brain tissues, the tissue samples were minced into small pieces  antibody appropriately diluted in solution B , followed by rinses in solution B. The grids were then reacted with the 10-nm gold-conjugated goat anti-mouse IgG, diluted in solution A. After extensive washes in PBS, the grids were then incubated with polyclonal anti-rabbit either CGB or SgII antibody as described above, followed by rinses in solution B. The grids were reacted with the 15-nm gold-conjugated goat anti-rabbit IgG, diluted in solution A. After washes in PBS and deionized water, the grids were stained with uranyl acetate (7 min) and lead citrate (2 min). Following washing in deionized water and drying the samples were examined with a JEOL JEM-1011 electron microscope.2 area of secretory granule and mitochondria -labeling gold particles (3R (IP3R3)-labeling gold particles , and a sudden increase of cytoplasmic Ca2+ concentration is the trigger signal for exocytotic processes.Although astrocytes are not traditionally regarded as secretory cells, it is nonetheless evident that they store a variety of molecules that are secreted in a regulated manner and participate in the signaling pathways in the brain. Hence, in spite of the dearth of information regarding exocytosis in glial cells compared to neurons, exocytotic activity in astrocytes is deemed essential in the astrocyte-to-neuron communication that is increasingly considered important for normal function of the brain. Astrocytes are known to contain many gliotransmitters such as glutamate, ATP, D-serine, and regulatory peptides neuropeptide Y (NPY) and atrial natriuretic peptide (ANP) 2+- and SNARE protein-dependent manner 2+ dependent manner 2+]c Large molecules such as regulatory peptides NPY and ANP are primarily stored in the LDCVs 2+ storage proteins, binding 30–93 mol of Ca2+/mol of protein with dissociation constants (Kd) of 1.5–4.0 mM 2+2+ in any subcellular organelles. These proteins are also released, along with other secretory granule contents, in response to stimuli that elevate [Ca2+]c.Chromogranins and secretogranins are marker proteins of secretory granules 2+ concentrations in astrocytes is thought to depend on Ca2+ release from intracellular Ca2+ stores 2+ from intracellular stores 2+ from internal Ca2+ stores of astrocytes 3-dependent intracellular Ca2+ stores in the Ca2+-dependent secretory pathway of these cells. Moreover, in light of the fact that the IP3-senstive intracellular Ca2+ stores provide sufficient amounts of Ca2+ to initiate the secretory processes even in the absence of external calcium 2+ stores to understand not only the intracellular Ca2+ control mechanisms but also the regulated secretory pathway of astrocytes.The elevation in the cytoplasmic Ca3R3, containing 58–69% of total cellular IP3Rs 3Rs at the intragranular pH 5.5 3R/Ca2+ channels, i.e., increase both the mean open time and the open probability of the channels upon IP3 binding, 9–42-fold and 8–16-fold, respectively 3Rs, and that the coupling between the chromogranins and the IP3Rs changes the structure of the IP3R/Ca2+ channels to a more ordered and open-ready state 3-sensitive intracellular Ca2+ store of the cells in which they are localized.Secretory granules of bovine chromaffin cells contain the highest concentrations of all three isoforms of IP3Rs directly and only chromogranin B remains bound to the IP3Rs 3Rs at a near physiological pH 7.5 is significantly weaker than that at the intragranular pH 5.5 3R/Ca2+ channel-activating effect of CGB at this pH is markedly weaker than that shown at pH 5.5 3R/Ca2+ channels are shown to be at least 6–7-fold more sensitive to IP3 than those of the ER 2+ in response to an IP3 concentration that is lower than one-seventh that is required to induce IP3-dependent Ca2+ release from the ER. In other words, the markedly higher IP3 sensitivity of the secretory granule IP3R/Ca2+ channels indicates that secretory granules will be able to sense the arrival of IP3 long before the ER can and respond by releasing the granular Ca2+ ahead of the ER , whereby strongly suggesting the IP3-dependent Ca2+ release from secretory granules of astrocytes. Further, in view of the observation that the cell processes of astrocytes appear to contain more secretory granules than the cell bodies and that the presence of secretory granules in the cell processes appears to be fairly common, the fine control of IP3-dependent Ca2+ signaling mechanism in the cell processes will be all the more important in controlling the exocytotic activity of astrocytes through the cell processes, which is vital in cell-to-cell communication in the brain. Yet the situation will be different in cultured astrocytes. That secretory granules of cultured astrocytes appeared to distribute evenly in the cytoplasm Considering that secretory granules are present in all types of secretory cells , the presence of secretory granules in astrocytes is in line with the already established secretory activity of these cells in the brain. In particular, the rich presence of chromogranin B and secretogranin II and the three IP3-sensitive intracellular Ca2+ store role of secretory granules has been demonstrated with many other types of secretory cells, such as chromaffin cells, pancreatic β- and acinar cells, mast cells and airway goblet cells 3-induced intracellular Ca2+ release from secretory granules in the absence of external Ca2+ has been proven to be sufficient to initiate the exocytotic processes 2+ release through the IP3R/Ca2+ channels of secretory granules in pancreatic acinar cells is primarily responsible for the initiation of alcohol-related acute pancreatitis 3-dependent cytoplasmic Ca2+ release but also the IP3 sensitivity of the cytoplasmic IP3R/Ca2+ channels of the cell 2+ released in the cytoplasm and the high IP3 sensitivity of the cytoplasmic IP3R/Ca2+ channels of astrocytes. In this regard, the recent studies that showed the requirement of IP3-mediated intracellular Ca2+ releases for the increased expression and secretion of fibroblast growth factor-2, which has a size of ∼24 kDa, by astrocytes 2+ release through the IP3R/Ca2+ channels for the proliferation, motility, and invasion of human astrocyte cancer cells 3-dependent Ca2+ signaling in the physiology of astrocytes.The major IP


Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition results in inefficient disassembly of LFA-1 adhesions causing T lymphocyte elongation and shedding of LFA-1 clusters behind the migrating T lymphocytes. We show that calpain 2 is distributed throughout the T lymphocyte, but is most active at the trailing edge as detected by expression of its fluorescent substrate CMAC,t-BOC-Leu-Met. Extracellular Ca2+ entry is essential for the activity of calpain 2 that is constantly maintained as the T lymphocytes migrate. Use of T cells from a patient with mutation in ORAI1 revealed that the major calcium-release-activated-calcium channel is not the ion channel delivering the Ca2+. We propose a model whereby Ca2+ influx, potentially through stretch activated channels, is sufficient to activate calpain 2 at the trailing edge of a migrating T cell and this activity is essential for the turnover of LFA-1 adhesions.The immune cells named T lymphocytes circulate around the body fulfilling their role in immunosurveillance by monitoring the tissues for injury or infection. To migrate from the blood into the tissues, they make use of the integrin LFA-1 which is exclusively expressed by immune cells. These highly motile cells attach and migrate on substrates expressing the LFA-1 ligand ICAM-1. The molecular events signaling LFA-1 activation and adhesion are now reasonably well identified, but the process of detaching LFA-1 adhesions is less understood. The cysteine protease calpain is involved in turnover of integrin-mediated adhesions in less motile cell types. In this study we have explored the involvement of calpain in turnover of LFA-1-mediated adhesions of T lymphocytes. Using live cell imaging and immunohistochemistry, we demonstrate that turnover of adhesions depends on the Ca T lymphocytes circulate continuously in the blood with the purpose of monitoring the tissues for injury or infection. They use the integrin lymphocyte function–associated antigen-1  to migrate from the circulation across vessel walls into lymph nodes and other tissues In migrating fibroblasts and endothelial cells, the cytosolic cysteine protease calpain is responsible for the turnover of integrin-mediated adhesions, promoting adhesion and spreading at the leading edge and deadhesion at the rear of the cell 2+2+ requiring enzymes and, in cell-free assays, calpain activation is Ca2+-dependent, requiring 2-75 µM for calpain 1 and 200–1000 µM Ca2+ for calpain 2 2+ range between 50–300 nM requires further resolution Calpain activity may be modulated, for example, by phosphoinositide/lipid binding 2+ influx following engagement of immunoreceptors such as the T cell or chemokine receptors 2+ stores and subsequent Ca2+ release. The decrease of Ca2+ in the stores is sensed by stromal interaction molecule-1 (STIM1) which then activates the main calcium-release-activated-calcium (CRAC) channel composed of ORAI1 subunits resulting in Ca2+ influx from the extracellular space. The transient receptor potential (TRP) channel superfamily of >20 members comprises a second type of ion channel. The TRP channels have enormous diversity in terms of function and mechanisms of action, but their activity differs from that of the CRAC channel, for instance, in lacking Ca2+ selectivity; whether some TRP channels are activated by depletion of Ca2+ stores is still a matter of controversy 2+ is modulated by potassium efflux through the outwardly rectifying Kv1.3 and KCa channels that generates negative membrane potential and aids Ca2+ influx through the CRAC channel.For lymphocytes, the process of store operated calcium entry (SOCE) brings about Ca2+ signalling.Previously we demonstrated a role for calpain in the adhesion of T lymphocytes to LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) To address whether calpain is involved in the migration of T lymphocytes, the cell permeable calpain inhibitor, calpeptin, that targets the active site of calpain 2+ binding site Calpeptin was titrated to test whether blocking calpain activity affected T cell migration. At a concentration of 50 µg/ml, calpeptin reduced T cell speed by 76.6±5.2%  Fig. 1B.Calpeptin-treated T cells that were fixed after 45 min of migration and stained with high affinity LFA-1 mAb 24 displayed trails of the integrin, highlighting the individual cell trajectories Fig. 1D.We next investigated how calpain inhibition altered the morphology of migrating T cells. Control T cells displayed the classic “hand-mirror” shape with a spreading lamella at the leading edge and a detached, upwardly projecting uropod at the trailing edge Fig. 2A.Movie S1). In contrast, as calpeptin-treated cells migrated on ICAM-1, thin LFA-1-expressing extensions developed from the back of the cell. Eventually fresh adhesions and forward movement caused sufficient membrane tension to rupture the link with LFA-1 and release the trailing edge of the cell .To study the dynamics of this calpain-inhibited effect in more detail, we performed live cell imaging with T cells that were labelled with Alexa488-conjugated Fab' fragments of a non-function blocking LFA-1 mAb. The distribution of LFA-1 on the migrating T cell was again skewed towards the rear in both calpeptin-treated and untreated cells. Control T cells migrated efficiently, detaching their trailing edges and maintaining uropods  we could directly visualize calpain activity in migrating T cells. The cleaved fluorescent product was highly concentrated at the rear of the migrating T cell and at the interface with ICAM-1Fig. 2C.Together these findings support the idea that calpain is involved in the turnover of LFA-1 adhesions at the rear of the cell. When calpain activity is blocked, the cell has difficulty in detaching so that, as the front of the cell moves forward, there is trailing edge elongation and loss of the uropod structure.2+-dependent, but a role for Ca2+, or indeed another divalent cation, in stimulating calpain in intact immune cells such as T cells has not been examined. Stimulating levels of cation could originate from several sources: extracellular influx via cell membrane ion channels, release from the intracellular Ca2+ stores located in the endoplasmic reticulum (ER) or from both sources In cell-free systems activation of calpain is Ca2+ entry 3, a cation channel inhibitor used to block CRAC and TRP channels 2+ signaling 3, 0.9±0.13 µm/min (93.5±0.4% decrease); 2-APB, 3.2±0.55 µm/min (76.5±2.5±2% decrease)  Fig. 3C.When the effect of the inhibitors on the migratory phenotype was viewed microscopically, they all caused cell lengthening Fig. 3D.in vitro has been demonstrated to be Ca2+-dependent, it was important to test whether a relevant ion channel was delivering Ca2+ into the cell. To locate cellular Ca2+, T cells migrating on ICAM-1 were loaded with the Ca2+ binding Fluo-4 dye and examined by fluorescent time-lapse confocal microscopy. The cells displayed elevated Ca2+ that was confined predominantly to the rear of the cell and at the interface with ICAM-1  Fig. 5B.In summary, although calpeptin is considered a specific inhibitor of the calpains, siRNA knockdown of calpain is more selective and allowed discrimination between the two calpains isoforms. We have therefore been able to show that, in spite of the general distribution of both calpain 1 and calpain 2 within the T cell, it is calpain 2 that is active at the rear of the T migrating cell.2+ ion channel activity. Inhibition of calpain 2 by calpain inhibitors and by siRNA silencing, causes reduced speed of migration by interfering with LFA-1 detachment at the trailing edge of the migrating T cell. The T cell can however shed integrin in the absence of efficient adhesion turnover, potentially through tension generated by trailing edge contraction. Over time, this results in trails of released LFA-1 adhesions that highlight the cell's trajectory.In this study we show that calpain 2 has an essential role in releasing the LFA-1 adhesions of migrating T lymphocytes and that this calpain is maintained in an active state by CaThere are conflicting findings over the role of calpains in integrin-mediated adhesion events in T cells. Our previous study and others indicate that calpain has a positive role in terms of promoting LFA-1- and β1- mediated integrin adhesion We show here that the clusters of T cell adhesions containing high affinity LFA-1 require a calpain-dependent step to deadhere. Consistent with these results are single particle tracking studies showing that calpain is responsible for releasing the immobile active form of LFA-1 on the membrane Another issue is that T cells express both calpain 1 and calpain 2 and immunostaining indicates that both isoforms are distributed throughout the T cell. A question is why it is calpain 2, rather than calpain 1, that mediates the release of the LFA-1 adhesions. A major controlling factor may be the exact location of the calpains within the cell. Although the calpains have traditionally been considered to be cytosolic enzymes, there is evidence that calpain 2 is associated with cellular membranes through phosphoinositide binding, and this could give greater access to membrane bound ion channels 2+ required to activate calpain 2. Steady state intracellular Ca2+ levels range between 50–300 nM, whereas in cell free assays calpain 2 is activated by 200-1000 µM levels of Ca2+2+ is highest at the T cell membrane interface with ICAM-1. In terms of activating intracellular calpain-2, Ca2+ could be delivered locally at the membrane in a sufficiently high concentration. Ca2+ levels up to 100 µM have been recorded within 20 nm of Ca2+ channels and this may be sufficient to maintain calpain 2 activity 2+ channel to ensure the high, localized Ca2+ concentrations that are required for its activity.Of relevance is the level of Ca2+-conducting ion channel in regulating both calpain 2 activity and LFA-1-mediated migration. The channel appears to be providing a constitutive or “tonic” signal as the T cells require no added stimulus in order to migrate. A key question is how this activity is initiated and maintained. Cell migration is known to be affected by stretch-activated calcium channels 2+ levels and calpain 2 activity.Our findings demonstrate an essential requirement for a Ca2+ into the cell is the ORAI1-CRAC channel that is activated by depletion of Ca2+ from the ER stores. It is of interest that it is not required for calpain activity or migration in T cells. Another candidate Ca2+ channel in T cells is TRPM7, a non-selective cation channel that is permeable to Ca2+ and also to Mg2+ and is an atypical TRP channel in expressing a C terminal α-kinase 2+ influx and migration in other cell types The major ion channel that delivers Ca2+ influx. Our evidence suggests that calpain 2 is activated close to the T cell membrane and this is consistent with the finding that LFA-1-dependent Ca2+ entry can occur without the release of intracellular Ca2+2+ store depletion giving rise to a more global elevation of Ca2+ within the cell and this distribution may have other consequences. For example Ca2+ influx through the CRAC channel plays a major role in controlling transcription of immune mediators such as cytokines in response to immunoreceptor engagement and resulting in activation of the calcineurin/NFAT pathway The distinction in terms of function between the ORAI1-CRAC channel and the calpain 2-activating ion channel described in this study may have to do with compartmentalisation of Ca2+. The lack of involvement of the major ORAI1-expressing CRAC channel has been unexpected and points to a clear distinction in the roles of Ca2+ delivering ion channels in T lymphocyte function.In summary, we show in this study that calpain-2 regulates the release of LFA-1 adhesions in migrating T lymphocytes, and is maintained in a constitutively active state dependent on influx of Ca3), Sigma; SKF-96365, Sigma; pre-designed siRNAs for calpain 1 (ID 146578), calpain 2 (ID 112796 and 145947), and siRNA control #2 were all from Ambion; CAPN1 13 and 10, CAPN2 1 and 6 and negative control were from Qiagen.LFA-1 mAbs 38 and YTH 81.5 were produced at Cancer Research UK and Fab' fragments of mAb YTH81.5 directly conjugated to Alexa-488 fluorochrome as previously described - leukemic T cell line, HSB2 was maintained in RPMI 1640/10% FCS Peripheral blood mononuclear cells were prepared from single donor leukocyte buffy coats  and T cells expanded as previously described and used between days 10 and 14 7 cells with 200–400 nM siRNAs using a Gene Pulser with Capacitance Extender (Bio-Rad) set at 960 µFD and 260 mV as previously described For siRNA knockdown experiments, HSB2 T cells line were washed twice in OptiMEM+GlutaMAX (Invitrogen) and electroporation was performed on 2×105 cells added to dishes containing HBSS/20 mM HEPES. Cells migrated for 15 min at 37°C prior to initiation of the experiment. Images were captured with an Olympus MTV3 Inverted microscope and a 20× lens or a Zeiss Axiovert 135TV Inverted microscope and a 63× lens plus AQM2001 Kinetic Acquisition Manager software . Cells were tracked using Motion Analysis software (Kinetic Imaging Ltd.) and data analysed using a Mathematica notebook  developed by Daniel Zicha .35 mm glass bottomed petri dishes  or or µ-slides VI  were coated overnight at 4°C with 3 µg/ml ICAM-Fc then blocked for non-specific binding with 2.5% BSA/PBS for 1 h at RT as previously 5/ml) were labelled with Alexa488-conjugated YTH81.5 Fab' fragments (10 µg/ml) for 15 min at 37°C, washed and added to ICAM-1Fc-coated MatTek dishes as described above. T cells were allowed to migrate for 20 min ±50 µg/ml calpeptin, then images were taken at 10 second intervals using a Zeiss Axiovert microscope and a LSM-510 laser scanning system.T cells  for 10 min at 37°C T cells or HSB2 T cells at 5×10T lymphoblasts ± calpeptin, exposed to ICAM-1Fc coated coverslips for 40 min at 37°C, were fixed with 3% formaldehyde in HBSS for 20 min at RT. Coverslips were incubated with 10 µg/ml of LFA-1 mAb in HBSS containing 0.25% BSA for 1 h at RT. Cells were permeabilised with 0.1% Triton-X-100, for 4 min at RT then incubated with 2.5 units/ml Alexa 546-phalloidin and Alexa 488-goat anti-mouse IgG  for 1 h at RT. Finally, coverslips were mounted in Mowiol antifadent (Merck Biosciences) and images collected on a Zeiss Axioplan microscope with a LSM510 laser scanning system.2+ flux by excitation at 480 nm and emission at 516 nm. Images were taken at 30 sec intervals using a Zeiss Axioplan microscope with a LSM510 laser scanning system.T cells were labelled with Fluo-4  plus 10% w/v Pluronic 127 (Invitrogen) for 60 min at 37°C, washed and added to ICAM-1Fc-coated dishes (MatTek) as described above. Cells were visualized by phase contrast and the Ca7/ml for 20 min on ice in 50 mM Tris pH7.4 containing 150 mM NaCl, 1% Triton X-100, 20 µg/ml PMSF and a complete protease inhibitor cocktail tablet (Roche). The lysate was microfuged for 15 min to remove insoluble material. Proteins were separated under reducing conditions by SDS-PAGE. After transfer to nitrocellulose membrane and incubation with antibodies, bound antibody was detected with HRP-conjugated sheep anti-mouse Ig  and ECL Western blotting detection reagents .HSB2 T cells were lysed at 5×10P<0.05; **, P<0.01; ***, P<0.001.The significance of the migration assays was determined using 2 Way Anova (Mathematica notebook (Wolfram Research) further developed by Daniel Zicha (Cancer Research UK)). The significance of other analyses , 3D was Movie S1The distribution of LFA-1 on human T lymphoblasts migrating on ICAM-1. LFA-1 is labelled with Alexa488-conjugated Fab' of non-function-blocking mAb YTH81.5.(AVI)Click here for additional data file.Movie S2The distribution of LFA-1 on human T lymphoblasts migrating on ICAM-1 following treatment with calpeptin at 50 µg/ml. LFA-1 is labelled with Alexa 488-conjugated Fab' of non-function–blocking mAb YTH81.5.(AVI)Click here for additional data file.


Mapping recombination hot and cold spots in yeast has previously relied on mutants, which themselves distort the map. Now a new method promises to overcome that problem. Hic sunt dracones–“Here be dragons.” Text popularly thought to appear on ancient maps to indicate unexplored or dangerous lands, although the only known such instance of the phrase appears on the Lenox Globe, ∼1500–1510 (collection of the New York Public Library) [.Meiosis is the specialized cell division that gives rise to gametes . To complete meiosis, a cell duplicates its chromosomes once but then goes through two rounds of chromosome segregation, reducing the genome by half. The original chromosome number is restored by the fusion of gametes during fertilization to form a zygote. Meiosis is a fundamental bottleneck passed by each generation in a sexually reproducing organism .In the first meiotic division, homologous maternal and paternal chromosomes are separated, whereas sister chromatids—the pair resulting from chromosome replication prior to meiosis—are separate in the second division  Figure . During In most organisms where sufficient information is available, each chromosome can be divided into large regions of relatively high or low recombination rates  , 6 Figu. Within Deriving accurate maps of the peaks and valleys that make up the recombination landscape is of interest for several reasons. First, understanding where recombination happens sheds light on the evolution of genome structure. Second, such maps help to reveal mechanisms behind this uneven distribution. Finally, knowledge of human recombination distribution facilitates association studies to identify disease-related genes.Saccharomyces cerevisiae. Yeast is an outstanding organism that allows researchers to work out molecular details of recombination, because DNA intermediates in the pathway can be analyzed directly, it lends itself to easy genetic manipulation, and most of the proteins involved are evolutionarily conserved.Here we focus on efforts to understand recombination distributions in the budding yeast S. cerevisiae [Meiotic recombination occurs through the repair of programmed DNA double-strand breaks (DSBs), a process best understood in revisiae , 9 , in which Spo11 remains covalently attached to DSB ends [rad50S-like mutants persist, greatly facilitating their detection and quantification.When large-scale sequence information first became available for ot spots . A critiDSB ends ,15  and hybridizing the protein-associated DNA to microarrays containing all 6,200 budding yeast open reading frames. DNA sequences that were highly enriched in the purified fraction relative to total genomic DNA were defined as hot spots. The results for chromosome III agreed well with the manually derived map. Moreover, the general pattern for this chromosome held true across the genome: each chromosome could be divided into hot and cold domains, with regions near centromeres and telomeres almost always low for DSB formation. The same pattern was further confirmed by numerous subsequent microarray studies, all based on enrichment of Spo11-bound DNA in rad50S-like mutants [Schizosaccharomyces pombe [Gerton and colleagues were the first to use microarrays to map global DSB distributions in yeast . They al mutants –20. Simies pombe . Prefererad50S-like mutations made it easier to measure DSBs, but at the cost of altering DSB distributions relative to wild type. One of the first hints came from the observation that rad50S and dmc1 mutants gave different DSB patterns when analyzed by pulsed-field gel electrophoresis [dmc1 mutants, such that Spo11 has been removed from DSB ends and long single-stranded tails have formed .These studies markedly revise the global DSB map for yeast. One major change is the documentation of substantial DSB formation in many regions where DSBs were absent or greatly reduced in elomeres ,26. DSB dmc1 mutants than by DSB maps in rad50S-like mutants. Notably, the dmc1-based maps match better with meiotic recombination maps from wild-type cells, and there is close concordance between distributions of single-stranded DNA purified from dmc1 and from wild-type strains. Moreover, for several locations where a DSB hot spot appeared in dmc1 maps but not in rad50S, Southern blot analysis demonstrated that DSBs do indeed occur in wild-type cells. Overall, more DSBs are observed in dmc1 than in rad50S. Finally, whereas DSBs are under-represented in rad50S-like mutants in regions where DNA replication is delayed by deletion of replication origins, no such DSB suppression is seen in dmc1 mutants [It is likely that the DSB distribution in normal cells is better represented by single-stranded DNA distributions in  mutants . Other i mutants  . Development of novel approaches to DSB mapping will be necessary to solve this issue.Mapping of global DSB patterns in budding yeast has come a long way and has revealed many interesting patterns, but challenges still remain. One challenge revolves around the ability to identify specific hot spots using current microarray methods. As with any methodology, a key issue is the signal-to-noise ratio; in this case, it is the signal from hot spots above background hybridization noise. The problem is easily illustrated by noting that overall DSB distributions were similar in different microarray studies of  studies –20. This studies . By usinot spots . Even wiAn even bigger challenge is to understand the molecular mechanisms that shape the recombination landscape, both the larger hot and cold domains and the more highly localized hot spots within these domains. Continuing to refine the maps of this landscape helps to frame hypothesis-driven functional experiments to test these mechanisms. The latest advances in molecular mapmaking are an important step in this direction.


Aberrant spontaneous, diastolic Ca2+ leak from the SR due to dysfunctional RyR2 contributes to the formation of delayed after-depolarisations, which are thought to underlie the fatal arrhythmia that occurs in both heart failure (HF) and in catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is an inherited disorder associated with mutations in either the RyR2 or a SR luminal protein, calsequestrin. RyR2 shows normal function at rest in CPVT but the RyR2 dysfunction is unmasked by physical exercise or emotional stress, suggesting abnormal RyR2 activation as an underlying mechanism. Several potential mechanisms have been advanced to explain the dysfunctional RyR2 observed in HF and CPVT, including enhanced RyR2 phosphorylation status, altered RyR2 regulation at luminal/cytoplasmic sites and perturbed RyR2 intra/inter-molecular interactions. This review considers RyR2 dysfunction in the context of the structural and functional modulation of the channel, and potential therapeutic strategies to stabilise RyR2 function in cardiac pathology.The cardiac ryanodine receptor-Ca The RyRnstrated . The comnstrated , RyR2 (Nnstrated  and RyR3nstrated . Throughnstrated . Within nstrated .2+ ions from SR Ca2+ stores which then diffuse and bind to the myofilaments to initiate contraction (systole). The reuptake of Ca2+ into the SR Ca2+ store causes relaxation (diastole), to complete the cycle comprising one heart beat. The heart beat therefore is critically dependent on the fidelity of ECC, which in turn is exquisitely regulated by the signal transmission occurring through the microdomain architecture at the regions where the sarcolemma (SL) and its T-tubule invaginations directly abut the junctional SR domains  at the surface of a cardiomyocyte regulates the release of Ca domains . These mrylation .2+ channels preside at the heart of ECC acting as sentinels to the large SR Ca2+ store. This review centres upon the role of the cardiac RyR2 isoform in arrhythmia and SCD. RyR2 dysfunction manifested as tachyarrhythmias is triggered by physical or emotional stress in CPVT patients and similar symptoms causes SCD in ∼50% of heart failure (HF) patients. These arrhythmias are believed to arise as a consequence of delayed after-depolarisations (DADs) that are initiated by diastolic leak of Ca2+ from dysfunctional RyR2. RyR2 function in the context of its normal structural and functional regulation and its role in ECC will be discussed in this review. The mechanism(s) by which RyR2 becomes dysfunctional, including abnormal phosphorylation, disrupted interaction with regulatory proteins and ions, or altered RyR2 domain interactions will be examined, and potential strategies for therapeutic intervention will be considered.Both physically and functionally, the RyR2 Ca22.12+ store (2+ channel (LCC) and the Na+/Ca2+ exchange protein (NCX). Although in cardiac muscle there is no allosteric contact between these proteins they are co-ordinately regulated via the changing concentrations of Ca2+, Na+ and K+ within the dyadic cleft .Of vital importance to the reliability of ECC is the myocyte intracellular architecture . The pre2+ store . Traversic cleft  Fig. 1)2+ store ic cleft  dependinic cleft , with faic cleft . Althoug2.22+ concentrations, both dyadic, luminal and within the transmembrane pore. These changes are critical to RyR2 regulation and numerous studies have modelled the precise modal gating of the RyR by Ca2+. These aspects are beyond the scope of this review, but have been given comprehensive treatment by others , but unlike single channel studies only gives a snapshot of the steady state  phenomenon was first proposed as an ECC mechanism in skinned muscle fibres  entering through one LCC (the ICa current) to activate a cluster of opposing RyR2 molecules to discharge SR store Ca2+  although 12% of spark events emanated from a single RyR2 channel opening , innate regulation of the individual RyR and LCC channels and the numbers activated  is 10–100  pumps and their regulatory protein phospholamban (PLB) situated on the longitudinal SR. SERCA resequesters the released Ca2+ into the SR Ca2+ store. The raised Ca2+ in the dyadic cleft has a retrograde inhibitory action on LCC and also activates the NCX, which effectively drives Ca2+ out of the cell   , and a sproposed . This phntration  and spar(2+ load . Phosphoload (2+  — see Se2.2.42+ (2+-dependent deactivation (2+ (∼ 600 µM) and each molecule can bind about 40 Ca2+ ions, which cause a major conformational change in the protein , triadin and junctin, directly regulate the RyR2 channel sensitivity to luminal Ca2+ , as parttivation . CSQ has protein . Since Cntration . In addiproposed . The twoproposed  and juncproposed  and form2+ rises .2+ store was regulated by the amount of CSQ expressed , immediately beneath a RyR2 cluster during Ca2+ release from the SR, which complemented a Ca2+ spark in the dyadic space . This NCX activity leads to a depolarisation which invokes the transient inward current — Iti   .2.4.1The flight-or-fight response, driven by adrenergic stimulation of cardiac β-adrenoreceptors raises intra-myocyte cAMP, which causes PKA activation. PKA can phosphorylate and co-ordinately regulate  all of the major components of the ECC cycle see . This ha2+ sparks  phosphorylation of LCC enhancing ICa  an increased sensitivity of phosphorylated RyR2 to activator Ca2+ concentrations (2+ concentrations (2+ store using thapsigargin (SERCA inhibitor) have shown Ca2+ release to be governed by ICa (2+. Others have suggested that the size of the Ca2+ store governs increased Ca2+ release. PLB knock-out or PLB-DM (double-mutant) knock-in mice with S16A/T17A substitutions, in which the SR Ca2+ store would remain constant, have shown that in permeabilised myocytes (no ICa), isoprenaline did not alter Ca2+ sparks  phcing ICa ) thus intrations , or (iv)trations . Experimd by ICa  such tha+ sparks . A seconrylation .2+ block (2+ concentrations (∼ 1 mM), while ATP no longer modified channel gating though Po was increased  Ca2+  Ca2+ .2.4.2δC isoform in heart  is acti2+ loads  and is aads  and Ca2+ entry via LCC  and S2815 (the phosphorylation site for CAMKII ) were ph not PKA . Two stuactivity . Mice ex via LCC . Both thaddition . Using aised ECC .2+ inhibition in single channel studies, however, these showed that Po was decreased overall  for the inner helix of each monomer but a poor fit for the outer helices  all forramerise  and the ramerise . Deletioramerise . Sequencramerise . Models ramerise , 6  during the open to closed conductive transition  and RyR23.12+ sensitivity and ryanodine binding of SR vesicles and inhibited interaction in overlay assays with domains 799–1172 and 3010–3225  or whether there is any co-operativity once one subunit is modified. In this respect it has been calculated that the stoichiometry for RyR2 and FKBP12.6 is > 1:3 . It has 42+ diastolic leak causing DADs is a feature of fatal arrhythmia in HF and CPVT. Patients with the latter condition carry point mutations in the RyR2 gene. In CPVT arrhythmias are triggered by exercise or emotional stress implicating adrenergic drive  and statistics suggest that 50% will eventually die from SCD associated with an arrhythmic episode, usually a ventricular tachycardia (VT) leading to ventricular fibrillation , which inhibits PP1 activity, is decreased in human HF  of FKBP12/12.6 from normal channels results in RyR dysfunction including increased  studies  and incrmyocytes . The relred mice . Disconcred mice . In the + sparks . The rea3H]FK506 to [3H]ryanodine was measured in microsomal fractions prepared from a 4 week paced dog model of HF   but anot4.1.2.2.1For RyR2 to lose FKBP12.6 from its binding site the binding affinity must either change or the concentration of FKBP12.6 must fall and there is evidence for both.Po of S2808D RyR2 and ‘stabilise’ channel activity , as seen in a detubulated myocyte model (+ and Na+ channels and the Na+/K+ ATPase) that interact functionally through the Ca2+, K+ and Na+ ionic changes in the dyadic cleft will also be compromised by such structural changes.There is sufficient evidence, particularly from experimental models to suggest that RyR2 function in HF is perturbed and that diastolic leak is a feature of HF even if there is no agreement on the precise mechanism of dysfunction. These changes in RyR2 function in HF take place against a greatly modified cellular landscape, which could also contribute to the propensity for arrhythmia. The close spatial relationship between T-tubule and junctional SR — the dyadic cleft, which is so important to the electrophysiological homeostasis of the heart beat can become disrupted in HF . Co-locate model . The ratte model  and a fute model . The int+ channels occurs early in the development of hypertrophy  normally stabilises the resting Em but its activity is reduced by Ca2+ . Some of these RyR2 mutations have been identified in patient groups screened for Long QT syndrome (Casq2 gene (CPVT2) which is linked to chromosome1p13–p21 but these patients have an autosomal recessive phenotype  in Europe and the USA annually . The num4.2.2The RyR2 point mutations are traditionally grouped into three regions of the RyR molecule N-terminus, central domain and C-terminus clusters which are largely homologous to similar groups of point mutations in the RyR1 gene see . See FigThe first RyR point mutation was detected in the skeletal muscle RyR1 isoform a decade earlier than the RyR2 counterparts and this was the R615C mutation causing porcine stress syndrome . Many mum-creosol and a reduced sensitivity to inhibiting concentrations of Ca2+ and calmodulin (in vitro contracture test (IVCT) a diagnostic test for MH although genetic screening is now also available . Those associated with the MH plus CCD, in addition, showed underlying SR Ca2+ store depletion and CCD mutations had SR Ca2+ store depletion but also a decreased response to activation — EC uncoupling   and the naltered . The R44naltered . The R17naltered . Epicardnaltered . This wonaltered .2+ transients. ECG registered complex ventricular arrhythmias when isoproterenol (with or without caffeine) was administered  could evoke DADs .4.2.32+ signalling, translated through RyR2 modulation and possibly manifest through SR store Ca2+ concentrations. Simulations of CPVT1 and CPVT2 have tested some of these proposed mechanisms, which might underlie abnormal Ca2+ homeostasis. These models were able to predict DADs (in response to β-adrenergic stimulation) induced by impaired luminal Ca2+ sensing in CPVT2 (2+ release (SOICR) in CPVT1 (The sub-cellular manifestation of increased adrenergic drive is PKA phosphorylation of key ECC proteins (including RyR2), which cause changes in the RyR2 microdomain environment as outlined above in in CPVT2  and alsoin CPVT1 . Impairein CPVT1 . Thus so4.2.3.1Several observations have led to the hypothesis that the clusters of RyR1/2 point mutations may be coincident with one or more domain interactions. In CPVT, mutations fall into the N-terminus or central domain clusters, which make up the proposed interacting zipper domains  or into 4.2.3.1.12+ spark frequency but DP4 (R2458C) had no such effect (2+ and increased ryanodine binding but DPc10M (R2474S) had no effect (The synthetic peptide DP4 (2442–2477 of RyR1) was able to disrupt the zipper domain interaction, whereas DP4 with the R2458C MH mutation had no effect . When inh effect . The DP4h effect . NMR anah effect . It was h effect  containih effect  that theo effect . The imp4.2.3.1.22+ when expressed in CHO cells. However, when challenged with caffeine all of the mutant constructs showed an increased sensitivity (The C-terminus cluster of CPVT mutations can be sub-divided into two regions 3534–4610 (the I-domain ) and 449sitivity . Noise asitivity .The I-domain/C-terminus interaction was explored in RyR1 using the DP15 (4821–4841) peptide  correspo4.2.3.22+ in the cis chamber but PKA phosphorylation increased the Po of all channels and some sub-conductance states were seen. The mutants were also more sensitive to activating Ca2+ concentrations  and P23r R4497C  and inhir V4653F ). The mur V4653F . The ‘stechanism .4.2.3.32+ release) hypothesis postulates that the aberrant feature of CPVT mutations is an enhanced sensitivity to luminal Ca2+ concentration  when store load was varied with extracellular Ca2+ and the cells expressing the mutants all exhibited a reduced Ca2+ store content compared to WT . Singlee others . The muted to WT . The latuse DADs . There w4.2.3.42+ via the CSQ/triadin/junctin complex. The CSQ mutant, with a D307H substitution, was characterised by adenoviral expression in rat myocytes and showed a reduced Ca2+ storage capacity and reduced spontaneous Ca2+ sparks  in some way   . N2386I mutation . Mouse Rcaffeine .4.2.42+ spark activity and response to caffeine and increased sensitivity to luminal Ca2+ in another , which cannot be met by the available healthy heart tissue. Without means of healing or replacing tissue damaged or lost, for example following a myocardial infarction (as might one day be possible with stem cell replacement) the best options currently available are to sustain the functional capacity of surviving tissue for as long as possible. The cells in this tissue, including the cardiomyocyte, are not passive bystanders. They bring to bear a veritable arsenal of genetic pathways intended for development, cell maintenance or short-term physiological flexibility but not ‘designed’ to mend a ‘broken’ heart . This stUntreated, the mortality for CPVT is high (∼ 30% by the age of 30) with sudden death the first symptom in some instances . The gen2+ transients (Currently available therapeutic treatments for HF ameliorated symptoms and slow progression. The recommended treatment for chronic HF are angiotensin converting enzyme (ACE) inhibitor, β-blocker and diuretic often combined, dependent upon severity of symptoms . β-blockansients .5.15.1.1LVADs are used to support failing hearts awaiting donor transplantation. Their short-term use has been shown to reverse some of the changes in protein expression associated with end-stage HF and to aid recovery from the negative force frequency relationship by improving myocyte function . In part5.1.22+-sensitizers also act to improve contractility, ideally without increasing oxygen demand and altering diastolic function  with metopropol, in a canine model of congestive HF, demonstrated improved haemodynamic parameters and restoration of SERCA and NCX expression to normal levels as a result of treatment  or FK506  in stimulated myocytes  suggests that RyR2 dysfunction is similarly compromised by dysregulation as also suggested in end-stage HF. The demise of RyR2 in both HF and CPVT have been considered jointly in this review, not least because RyR2 dysfunction, resulting in the diastolic leak and arrhythmia (DADs) in both conditions provide important clues to the underlying regulation and channel gating of RyR2 and its relationship to its microdomain environment. The understanding of these processes will provide the basis for therapeutic strategies.2+ this might result in a molecular reorganisation of the regulatory I-domain, which radiates to the pore to control channel gating function. When the channel is activated by Cal gating . In turnl gating . These cl gating . Perturba camera ) perhapsrevealed . As discIs RyR2 function and the inherent instability of CICR the ‘Achilles' heel’ of ECC? Indeed, in HF, because it is such a pivotal protein in ECC, acting as a regulator and coincidence detector of cellular events, RyR2 is buffeted by every type of stressful change encountered by the myocyte and in microcosm may merely reflect the myocyte's malaise. When faced with a chaotically regulated environment or wounded by a point mutation this magnificently robust protein may finally run out of possibilities to autoregulate coherently, when assaulted by phosphorylation. This might explain why broad spectrum strategies to rescue the myocyte such as β-blockade are successful in HF because they ‘treat’ ECC as a whole and partially successful in CPVT where phosphorylation maybe the trigger to arrhythmia.Despite the intense spotlight on hyperphosphorylation and loss of FKBP12.6, this has not led to any real understanding of the relationship between RyR1/2 and FKBP12/12.6 or phosphorylation, in the context of the normal flight or fight response or the regulation of domain interactions in normal RyR2. In fact the molecular mechanism by which phosphorylation changes RyR2 function is uncertain and the experimental finding contradictory see . The reaThe correction of RyR2 dysfunction in both point mutation carriers and HF patients and the development of novel therapeutic strategies is currently the focus of a much effort, particularly methods to prevent the onset of fatal arrhythmia. The efficacy of current and new therapeutic approaches is, in turn, providing valuable information on the underlying molecular mechanisms regulating RyR2 channel gating and function. In the final analysis, the pathological changes in RyR2 which cause spontaneous diastolic leak and arrhythmia must be understood from a perspective which correlates the dynamics of RyR2 channel gating with intramolecular movement, and this presents an intriguing challenge.S/P and RyRS/S genotypes demonstrated that gene dosage of the mutation increased the propensity to arrhythmia in myocytes and Langendorff heart preparations, particularly on the addition of isoproterenol. The physiological characteristics of CPVT were similar to the two models described in Section 4.2.2 describing C-terminal and N-terminal CPVT mutations . Physiological consequences of the P2328S mutation in the ryanodine receptor (RyR2) gene in genetically modified murine hearts. Acta Physiol (Oxf) 194, 123–140).A third knock-in mouse model of CPVT has been reported for the central domain P2328S mutation. Comparison of WT, RyR