This contraction can be partly attributed to TxA2-induced Ca2+ influx, which resulted in vascular contraction via Ca2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca2+ influx in vascular smooth muscle cells.Thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca2+, because removal of extracellular Ca2+ abolished the constriction. This constriction was partially inhibited by an L-type Ca2+ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583  had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca2+ ([Ca2+]i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.Application of U-46619, a thromboxane A2+ influx and contraction of smooth muscle cells.These data suggest a functional role of CNG channels in U-46619-induced Ca Functionally, TxA2 acts on the TP receptor (TxA2 receptor) to promote platelet aggregation 2-induced contraction of vascular smooth muscle. The first mechanism is the Ca2+ sensitization of contraction, which refers to a sensitized contractile response to a small rise in [Ca2+]i2+ sensitization can be attributed to a reduced activity of myosin light-chain phosphatase, followed by a greater degree of phosphorylation of myosin light chain 20, leading to the sensitized contractile response to Ca2+2-elicited increase in [Ca2+]i, which enhances the smooth muscle cell contraction via Ca2+-calmodulin-myosin light-chain kinase pathway. Relatively little is known about the detailed mechanism of how TxA2 elicits a [Ca2+]i rise. Tosun et al. found that both L-type and non-L-type Ca2+ influx channels could account for thromboxane A2 receptor-mediated contraction in rat aorta, but the identity of the non-L-type calcium channel is unclear (1998). Evidence also shows that the Ca2+ sensitization process also requires Ca2+ influx, because removal of extracellular Ca2+ abolished the Ca2+ sensitization Thromboxane A2+-permeable nonselective cation channels. Six CNG isoforms have been identified, these includes four A subunits and two B subunits. CNGA1–A3 subunits may form functional channels on their own, while B and A4 subunits serve modulatory functions. In native cells, CNG channels usually form heterotetrameric complexes consisting of A and B subunits Cyclic nucleotide-gated (CNG) channels are Ca2-induced Ca2+ influx and contraction in vascular smooth muscle cells. A stable TxA2 analogue U-46619 was used to induce contraction in the endothelium-denuded small mesenteric artery segments. This constriction was inhibited by L-cis-diltiazem and LY83583, two selective inhibitors for CNG channels, in dose-dependent manner. Immunoblot experiments found the expression of CNGA2 proteins in the primary cultured vascular smooth muscle cells. These data suggest a functional role of CNG channels in U-46619-induced Ca2+ influx smooth muscle contraction.In the present study, we tested the hypothesis that CNG channels may contribute to TxA2 is an unstable compound, we used a stable TxA2 analogue U-46619 (100 nM) to induce contraction in isolated rat small mesenteric artery segments , an L-type Ca2+ channel inhibitor nifedipine relaxed the vessels  to inhibit L-type Ca2+ channels -induced contraction  caused a concentration-dependent inhibition of U-46619-induced contraction. D-cis-diltiazem had similar inhibitory effect on the contraction, though its effect was less potent than that of L-cis-diltiazem  to inhibit L-type Ca2+ channels. This would allow us to better resolve the Ca2+ influx pathway that is independent of L-type Ca2+ channels. As shown in 2+ rise in these cells. L-cis-diltiazem (100 µM) inhibited this Ca2+ rise, supporting a role of CNG channels in this Ca2+ rise.U-46619-induced CaImmunoblots were used to examine the CNG channel isoforms that are expressed in vascular smooth muscle cells. 2 is implicated in many cardiovascular, renal and respiratory diseases 2-induced smooth muscle contraction, which leads to vascular constriction, ischemia, pulmonary hypertension and broncho-constriction 2-induced vascular smooth muscle cell contraction. Our results showed that, in the absence of extracellular Ca2+, U46619 failed to constrict the isolated small mesenteric artery segments. In addition, removal of extracellular Ca2+ relaxed the artery segments that were preconstricted with U-46619. Furthermore, inhibition of L-type Ca2+ channels by nifedipine reduced the contractile response to U-46619. These data agree with the previous reports by others 2+ influx is required for U46619-induced vascular constriction and that L-type Ca2+ channels play a role in this constriction.TxA2+ channels by nifedipine only caused a partial inhibition of U46619-induced constriction, we next explored the possible involvement of CNG channels in U46619-induced constriction. In these experiments, arterial segments were first treated with nifedipine to block L-type Ca2+ channels in order to better resolve the component that was independent of L-type Ca2+ channels. Under this condition, U-46619-induced contraction was found to be inhibited by L-cis-diltiazem in a dose-dependent manner. L-cis-diltiazem also inhibited U-46619-induced cytosolic Ca2+ rise in the primary cultured vascular smooth muscle cells. The inhibitory action of L-cis-diltiazem suggests the involvement of CNG channels in U-46619-induced Ca2+ rise in smooth muscle cells and subsequent vascular constriction. This hypothesis was supported by the LY83583 experiments, in which LY83583 also caused a dose-dependent inhibition of U-46619-induced vascular constriction. LY-83583 is a less selective agent and it inhibits both CNG channels and soluble guanylate cyclase at similar concentrations Because the inhibition of L-type Cacis-diltiazem even at mM concentration, olfactory-type CNGA2 is inhibited by D-cis-diltiazem though with much less potency compared to that of L-cis-diltiazem. In our experiments, high concentration of D-cis-diltiazem inhibited U-46619-induced vascular constriction with the dose-response curve mirrors that of olfactory-type CNGA2 reported by Frings et. al. CNG channels are widely expressed in different vascular tissues including cerebral and coronary arteries 2-induced vascular contraction was inhibited by two CNG channel inhibitors L-cis-dialtiazem and LY-83583. Our data suggest that CNG channels, olfactory-type CNGA2 in particular, contribute to TxA2-induced Ca2+ influx in vascular smooth muscle cells.In conclusion, TxAGuide for Animal Care and Use of Laboratory Animals published by the US National Institute of Health. The experiments were approved by the Department of Health of Hong Kong and carried out by investigators licensed (06-3indh/orhi/821pt.5) under section 7 of the Animals (Control of Experiments) Ordinance (Cap. 340).The animal study was conducted in conformity with the 2, 1.2 MgSO4, 25.2 NaHCO3, 1.2 KH2PO4, and 11.1 D-glucose. Second order small mesenteric artery ring segments of about 3 mm in length were isolated and cleared of adhering fatty tissues. Each segment was mounted in the tissue chamber of a Multi Myograph System  with two wires passing through the lumen. The tissue chambers contained KHS that was constantly bubbled with 95% O2 plus 5% CO2 and maintained at 37°C throughout the duration of the experiment. Endothelium was removed mechanically by gently rubbing the luminal surface with a piece of stainless steel wire. Each ring was stretched to an initial tension of 1 mN and the changes in tension were recorded in a myograph. The rings were precontracted twice in 60 mM K+ KHS and then contracted with 2 µM phenylephrine, followed by 1 µM acetylcholine to ascertain the completeness of endothelium removal. Only those with less then 5% relaxation were considered as endothelium denuded. The rings were then washed three times in normal KHS.Mesenteries with supplying blood vessels removed from adult Sprague Dawley rats (250 g–300 g) killed by carbon dioxide overdose were placed into Krebs-Henseleit solution (KHS) (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2+ influx is required for U46619-induced contraction, 100 nM U-46619 was added to initiate contraction in the rings. After that, the rings were washed three times with Ca2+-free KHS and incubated in 1 mM BAPTA before the rings were recontracted with the same concentration of U-46619. In some experiments, EGTA (3 mM) was added or extracellular Ca2+ was removed after the U-46619-induced tension became stable (n = 4).To study if Cacis-diltiazem on U-46619-induced contraction, the bathing solution was changed to 60 mM K+ solution to open the voltage-gated Ca2+ channels, followed by an addition of 1 µM nifedipine to block L-type voltage-gated Ca2+ channels. After the tone returned to basal level, which is an indication that most, if not all, of the voltage-gated channels were blocked, the vessels were recontracted with 100 nM U-46619. Cumulative doses (from 20 µM to 200 µM) of L-cis-diltiazem, a specific CNG channel blocker, were added to assess the role of CNG channels in the contraction. The concentration-dependent relaxant effect of D-cis-diltiazem was also examined for comparison. A control experiment running in parallel without the addition of diltiazem showed that the U-46619-induced contraction was sustained during the experimental time period.To assess the effect of L-2+-free PSS  for 25 min at 37°C under vigorous shaking. The tissues were then rinsed several times in PSS to remove the collagenase. Smooth muscle cells were dissociated from the tissues by pipetting up and down using a Pasteur pipet. The cells were seeded on coverslip and grown in 80% DMEM and 20% FBS with 1% antibiotic-antimicotic for a week before Ca2+-imaging experiments.The primary cultured rat aortic smooth muscle cells were isolated from SD rat aorta as described elsewhere 2, 10 glucose, 5 Hepes, pH 7.4. The experimental chambers were placed on the stage of an inverted microscope (Olympus IX81). Fluorescence was measured using the FV1000 laser scanning confocal imaging system. The excitation wavelength was at 488 nm and the fluorescence signals were collected using a 515 nm long pass emission filter. Data analysis was performed with FV1000 software. Changes in [Ca2+]i were displayed as a ratio of fluorescence relative to the fluorescence before the application of U-46619.Cultured cells were loaded with 10 µM Fluo-4/AM for 20 min in dark at 37°C in culture medium. Then the cells were briefly washed and bathed in a normal physiological saline solution (NPSS) that contained in mM: 140 NaCl, 5 KCl, 1CaClg for 5 min at 4°C. The supernatant was collected, and the protein concentration was quantified using Bradford reagent . Samples (50 g protein/lane) were subjected to SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membrane. Antibodies against CNGA1  or CNGA2  or CNGA3  were incubated with the membrane overnight at 4°C. The anti-CNGA2 polyclonal antibody was self-raised following a previous published protocol Thoracic aorta was isolated from Sprague Dawley rats (250–300 g) and placed in PBS immediately. The surrounding connective tissue was carefully trimmed off and endothelial cells removed by rubbing with a piece of cotton. The aorta was then homogenized in lysis buffer containing 50 mM Tris–base, 150 mM NaCl, 50 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, pH 7.5, with addition of the protease inhibitor cocktail tablets (Roche) and centrifuged at 10,000 cis-diltiazem were purchased from Sigma Aldrich, L-cis-diltiazem from Biomol International and U-46619 from Calbiochem. U-46619, BAPTA and nifedipine were dissolved in DMSO and others in distilled water.Chemicals. Acetylcholine, nifedipine, ethylene glycol tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) and D-


Sera were collected from patients with acute myelogenous leukaemia (AML) at various times during remission induced by chemotherapy, but after cessation of all immunosupressive treatment. These sera were tested, by a sensitive assay using radio-labelled antiglobulin binding, for the presence of antibodies which bound to the surface of autologous AML cells. The cell populations examined were chosen on the basis that they proliferated in short-term culture, did not bind anti-Ig reagents directly, and that more than 80% of the cells did not carry detectable Fc receptors. With 8/9 patients studied, no specific antibodies of the IgG or IgM class could be detected in serum samples taken during remission. IgG and IgM antibodies from the remission sera of one patient were found be bind to autologous leukaemic cells, but this was found to be due to the presence of rheumatoid factor (RF) and removal of the RF activity abolished this binding. This study has, like others, failed to detect autologous antibodies, in remission sera, that are directed against membrane components of AML cells.


Introduction of FPL (1 μm) resulted in a gradual development (>10 min) of diastolic electrical events and alternans in spontaneously beating hearts from which monophasic action potentials were recorded. In regularly paced hearts, they additionally led to non-sustained and sustained ventricular tachycardia (nsVT and sVT). Programmed electrical stimulation (PES) resulted in nsVT and sVT after 5–10 and >10 min perfusion, respectively. Pretreatments with nifedipine, diltiazem and cyclopiazonic acid abolished arrhythmogenic tendency induced by subsequent introduction of FPL, consistent with its dependence upon both extracellular Ca2+ entry and the degree of filling of the sarcoplasmic reticular Ca2+ store. Values for action potential duration at 90% repolarization when any of these agents were applied to FPL-treated hearts became indistinguishable from those shown by untreated control hearts, in contrast to earlier reports of their altering in long QT syndrome type 3 and hypokalaemic murine models for re-entrant arrhythmogenesis. These arrhythmic effects instead correlated with alterations in Ca2+ homeostasis at the single-cell level found in investigations of the effects of both FPL and the same agents in regularly stimulated fluo−3 loaded myocytes. These findings are compatible with a prolonged extracellular Ca2+ entry that potentially results in an intracellular Ca2+ overload and produces the cardiac arrhythmogenecity following addition of FPL.The experiments explored the extent to which alterations in L-type Ca The latter process requires a Ca2+-induced Ca2+ release (CICR) through sarcoplasmic reticular (SR) ryanodine receptor (RyR2) Ca2+ release channels  into the SR or extruded from the cell via the Na+–Ca2+ exchanger  in ventricular myocytes is initiated by extracellular Nachannels . The con2+ might eventually trigger mechanisms of this kind, through the use of the specific L-type calcium channel modulator FPL-64716 , housed at room temperature, fed sterile rodent chow with free access to water at all times and subjected to 12 h–12 h light–dark cycles were studied at ages between 5 and 10 months. They were killed by cervical dislocation  Act 1986).−1): NaCl, 119; Na2CO3, 25; KCl, 4; KH2PO4, 1.2; MgCl2, 1.0; CaCl2, 1.8; glucose, 11; and sodium pyruvate, 2.0. The buffer was made daily and equilibrated with a 95% O2–5% CO2 gas mixture  . Fluid in the water-jacket was circulated with a circulator  and warmed to 37°C by passing through a water-bath. The perfusate was then passed through 200 and 5 μm filters  before passing into the aorta of the heart. Hearts were thus perfused using buffer in which catecholamines were absent. They were then allowed to beat spontaneously until a steady state was reached, a time that permitted ample time for washout of any catecholamines that may have been present in the circulation at the time of killing.Hearts were dissected out and immediately immersed into ice-cold bicarbonate-buffered Krebs–Henseleit (KH) buffer  using square-wave stimuli of 2 ms duration . Monophasic action potentials (MAPs) were recorded from the epicardial basal surface of the left ventricle using a contact-type MAP electrode .Following the equilibration period, hearts were first studied in conditions of spontaneous activity, which typically took place at a heart rate of ∼6 Hz. They were then subjected to constant ventricular pacing at cycle lengths of 8 and 10 Hz. Finally, they were subjected to programmed electrical stimulation (PES) using a variation of existing clinical techniques  following previous MAP analysis protocols  and digitized at a frequency of 5 kHz using an analog-to-digital converter . Monophasic APs that were considered for analysis were all stable for more than 30 min, and the data were manually reviewed using Spike2 software . According to accepted criteria regarding MAP morphology, MAP traces selected should have a stable baseline with a rapid upstroke without inflection or negative spikes, consistent amplitude >1 mV, and rapid first phase repolarization lacking an early plateau. Action potential duration (APD) was analysed at 30, 50, 70 and 90% repolarization (APD2+ channel agonist FPL-64716 (FPL) was initially made up with dimethyl sulphoxide (DMSO) to make a stock concentration of 5 mm. Stock solutions of FPL were wrapped in foil because they are photosensitive and stored at −20°C. Cyclopiazonic acid (CPA) was dissolved in 96% ethanol to make an initial stock solution of 10 mm, which was stored at −20°C. Diltiazem was dissolved in distilled water to make a stock concentration of 1 mm and kept refrigerated at 4°C. Final drug solutions used were diluted with KH buffer made fresh before every experiment. The FPL was diluted to a working concentration of 1 μm. The CPA was diluted to an intermediary solution of 10 μm and then further diluted to a concentration of 150 nm. Nifedipine and diltiazem were both used at a final concentration of 100 nm.Pharmacological agents studied were all purchased from Sigma-Aldrich, Poole, UK. The CaArrhythmogenic phenotypes in the form of after-depolarizations, delayed after-depolarizations, alternans, and monomorphic and polymorphic ventricular tachycardia were examined both before and following introduction of FPL in both control conditions and following subsequent additions of nifedipine, diltiazem and CPA. After-depolarizations were defined as depolarizing electrical events that interrupted the restitution phases of action potentials, whereas premature activations took place following full action potential recovery. Spontaneous tachycardias prolonged over more than 30 s were defined as sustained, whereas those lasting less than 30 s were defined as non-sustained.s.e.m., and differences were considered significant at a level of P < 0.05.Statistical analysis of results obtained in the different pharmacological conditions was carried out using either paired or unpaired two-way ANOVA  for continuous data and Fisher's exact test for categorical data. Results are all expressed as means ±−1 with perfusion buffer containing (mm): NaCl, 120; MgSO4, 5; KCl, 5.4; sodium pyruvate, 5; Hepes, 10; glucose, 5; taurine, 20; nitrilotriacetic acid (NTA), 5 (Sigma-Aldrich); and adjusted using NaOH to pH 7.0 at 37°C. Subsequently, the heart was perfused for 25–28 min at 3 ml min−1 with digestion buffer containing (mm): NaCl, 120; MgSO4, 5; KCl, 5.4; sodium pyruvate, 5; Hepes, 10; glucose, 5; and taurine, 20, to which was added collagenase type II , hyaluronidase  and 200 μm Ca2+ and adjusted using NaOH to pH 6.8 at a temperature of 37°C. The pale and swollen heart was then removed and cut below the atria. The digested tissue was teased gently with fine forceps, and the dissociated cells were transferred to a conical tube containing 12.5 ml digestion buffer with 50 mg bovine serum albumin (BSA) to inactivate digestion. The resulting myocytes were subjected to further trituration using sterile plastic transfer pipettes, and allowed to sediment by gravity for 10 min.Single-cell experiments used mouse ventricular myocytes isolated by adapting an established enzymatic digestion described previously (g and the pellets resuspended twice in wash buffer containing (mm): NaCl, 119; KCl, 4.2; MgSO4, 0.94; KH2PO4, 1.2; Hepes, 20; glucose, 11.5; taurine, 20; and 1 mg ml−1 BSA, and bubbled with 95% O2–5% CO2, pH 7.4, to remove all traces of NTA, collagenase and hyaluronidase. Calcium chloride was then cautiously reintroduced by stepwise (0.2 mm) additions to reach an approximate concentration of 1.25 mm, by centrifuging the preparation at each Ca2+ reloading stage to remove dead or swollen cells. During this process, myocytes were examined periodically under the microscope to confirm a good yield of rod-shaped myocytes (better than 60%) before continuing the experiments. Cells were then transferred into a conical tube  and incubated at room temperature. The isolated myocytes were then placed in a control Hepes-buffered Krebs–Henseleit solution that was the same as the wash buffer mentioned previously without BSA and had a pH of 7.2, and transferred to a Perspex chamber 10 mm × 4 mm × 6.25 mm (length × width × depth). The myocytes spontaneously attached to the glass coverslip  forming the floor of the chamber. The myocytes were stimulated to contract using two in-built platinum field electrodes running the length of the chamber through which the periodic field stimulation was applied using a custom-built square-wave stimulator. This applied successive 40–60 V steps of duration 2.2 ms, immediately followed by a step of similar duration and amplitude but opposite polarity at a pacing frequency of 0.5 Hz. During this stimulation, cells were loaded with the acetoxymethyl (AM) ester of fluo-3  by incubating the cells in a bath of volume 500 μl containing KH buffer (with 1.25 mm Ca2+) with 2 μl of fluo−3 AM solution for 15 min at room temperature in the dark. The bath with the myocytes was then transferred onto the stage of a Zeiss LSM-510 laser scanning confocal microscope system  with a ×20 air objective  on a Zeiss Axiovert 100 M inverted microscope. Fluo−3 fluorescence emission was excited with a 488 nm argon laser and measured at 505–550 nm. Images were analysed using an in-house custom-written program. A series of 500 frames sampled at 98 ms per frame (128 × 128 pixels per frame) were used initially to monitor fluorescence changes over time. The appropriateness of the chosen frame capture rate was corroborated in some experiments by collecting data in the faster line-scan mode with a sampling rate of 960 μs per line. All traces had stable baselines, confirming consistent laser intensities and detector gains. The peak normalized fluorescence (F/F0) values following each response to each stimulus were calculated for each time series, and a mean was acquired for each series. Moreover, the mean peak F/F0 values as well as the baseline diastolic values were calculated. The results are expressed as mean values ±s.e.m. and compared using ANOVA (SPSS Inc.). Different solutions were perfused through the perfusion chamber as required. All confocal microscope studies were carried out at room temperature.The cell suspension was then centrifuged for 1.5 min at 100 m) increases extracellular Ca2+ entry through L-type Ca2+ channels, thereby enhancing cytosolic Ca2+ loading  into the perfusing KH buffer solution. The traces shown in each panel exemplify results from ∼2 min of recordings in each condition. Before introduction of FPL all hearts showed normal MAP waveforms whose time courses resembled those reported on earlier occasions (Previous work has demonstrated that FPL (1 μ loading . It theractivity A and duractivity B before ccasions Aa and BaP= 0.009) incidence of arrhythmic events compared with control results observed before addition of FPL ; of these, two hearts showed EADs that intercepted the recovery phase of approximately two and six out of 20 successive APs. Moreover, four hearts showed DADs, giving an overall statistically significant incidence of arrhythmic phenomena (P= 0.006) compared with control hearts  than corresponding incidences obtained before addition of FPL.In paced hearts, with 1 min of exposure to FPL there were no arrhythmic episodes Bb apart n= 8; P < 0.001); and (3) all eight hearts showed episodes of sVT with further perfusion with FPL for more than 10 min (P < 0.0001).Programmed electrical stimulation procedures then provided quantifiable tests for arrhythmic substrate as employed on previous experimental and clinical occasions n= 8; A; (2) se2+ channel blocker nifedipine (100 nm), the benzothiazepine Ca2+ blocker diltiazem (100 nm) and the effects of the SERCA inhibitor CPA (150 nm) before restoring FPL to the buffer, in both regularly paced hearts .None of the hearts on pretreatment with nifedipine  and hypokalaemic murine cardiac models and have been implicated in the re-entrant substrate responsible for their abnormal electrical activity .x= 30, 50, 70 and 90% recovery  from left ventricular MAPs obtained from hearts paced at 8 Hz (90 was 48 ± 3.5 ms (n= 6 hearts), in agreement with previous reports , at different perfusion times  identical to those adopted above upon values of APDx. Application of two-way ANOVA to each of the above groups demonstrated that there were no significant differences between treated or untreated groups with the exception of APD30 values obtained between 5–10 min. Therefore, FPL did not significantly alter action potential time course despite the occurrence of EADs and alternans but not sVT at times <10 min and the appearance of all three phenomena at times >10 min. This progressive development of arrhythmogenesis is therefore more likely to arise from progressive increases in intracellular Ca2+ resulting from increased net Ca2+ influx brought about by FPL. The findings complement recent reports in a murine RyR2 model whose arrhythmogenic properties were attributable to alterations in RyR2 , 30 ± 1.5  and 35 ± 1.8 ms (n= 3 hearts); the corresponding value for the longer of each pair was 45 ± 0.7 (n= 1 heart), 46 ± 0.3 (n= 4 hearts) and 65 ± 2.1 ms (n= 3 hearts), respectively , diltiazem (n= 5 hearts) and CPA (n= 9 hearts) nor a further addition of FPL resulted in any significant change in APDx as indicated by ANOVA , whose timing is indicated by the dots below the F/F0 traces in F/F0 traces showed stable baselines and reproducible peak responses to regular stimulation with minimal evidence of fluorophor bleaching over the adopted sampling periods as assessed by readings obtained at the beginning and end of the adopted, 50 s, sampling periods (P≫ 5%).The experiments that followed correlated these arrhythmic effects with changes in the amplitude and pattern of Ca2+ signals observed immediately following commencement of stimulation consisted of series of responses with consistent amplitudes, each directly following the individual stimuli with a peak F/F0 of 1.53 ± 0.02 . The myocytes did not show spontaneous ectopic peaks in the intervals between stimuli, nor did they show any subsidiary events during the recovery phases of the evoked Ca2+ transients  that showed subsidiary events  with subsidiary events of amplitude 1.27 ± 0.02 , from 2.11 ± 0.04 (n= 6 cells), 1.77 ± 0.02 (n= 3 cells) and 3.20 ± 0.04 , 1.30 ± 0.01 (n= 3 cells) and 1.80 ± 0.06 (n= 12 cells) , 1.31 ± 0.01 (n= 4 cells) and 1.57 ± 0.05  was similar to those observed with FPL. Nevertheless they occurred significantly less frequently, in 5 ± 0.20 out of 23 evoked transients  of diastolic electrical events and alternans in non-paced hearts. In paced hearts, FPL led to an additional appearance of arrhythmic, nsVT and sVT phenomena. Finally, hearts were subjected to PES. Results from this have been shown to correlate with arrhythmogenic tendency in genetically modified hearts modelling Brugada syndrome  reactivation during prolonged AP plateaus (2+, leading to cytosolic Ca2+ waves and oscillations and increased NCX activity (In contrast, diastolic events of the kind observed here have previously been attributed to a range of Caplateaus  and DADs2+ that might result from digitalis toxicity and catecholaminergic polymorphic ventricular tachycardia (The alternans that involved APD parallel similar phenomena in clinical situations, in which such beat-to-beat fluctuations in the electrocardiographic T-wave are thought to be a strong diagnostic precursor of ventricular tachycardias and ventricular fibrillation, potentially leading to sudden cardiac death (2+ channel blockers, with diltiazem additionally inhibiting calcium leak through RyR2s. Previous experiments using PES protocols showed that both these agents block triggered VT induced by either caffeine or isoproterenol (2+ uptake (2+ sensitivity of the contractile apparatus (2+ currents (2+ transients and force–frequency relationships (The subsequent experiments in intact hearts accordingly proceeded to explore the effects of pretreatments using three different pharmacological agents, the diydropyridine nifedipine, the benzothiazipine diltiazem and the indole tetramic acid CPA on arrhythmogenic tendency induced by subsequent introduction of FPL (oterenol . In cont2+ entry; FPL-induced arrhythmogenesis was abolished in hearts subjected to pretreatment with either nifedipine or diltiazem. The effects were also dependent upon the degree of filling of the SR Ca2+ store, and accordingly abolished by CPA. Furthermore, none of these agents, whether in the presence or absence of FPL, altered APD values, in contrast with earlier reports of the actions of such agents in LQT3 (These experiments demonstrated that these effects of FPL could be attributed to its enhancing extracellular Ca2+ homeostasis at the single-cell level. These experiments investigated the effects of FPL, as well as nifedipine, diltiazem and CPA, upon regularly stimulated fluo−3 loaded myocytes subjected to similar pharmacological manoeuvres to those used in the whole hearts. Thus, FPL alone resulted in an immediate increase in peak evoked F/F0 as well as an onset of diastolic Ca2+ transients in isolated myocytes subjected to regular stimulation. This change was dependent upon a Ca2+ entry enhanced by FPL. Thus, both nifedipine and diltiazem reduced such transients, whose amplitude was then not restored by a subsequent administration of FPL; in such conditions, diastolic Ca2+ transients were absent. Similarly, myocytes pretreated with CPA had reduced peak F/F0 transients. These were not restored by a subsequent addition of FPL. Nevertheless, addition of FPL resulted in an appearance of diastolic transients whose amplitude was substantially lower than diastolic transients observed with FPL alone. This is as expected for an action of FPL in activating cardiac RyR2 either by Ca2+ entry or through a direct effect reported earlier (In contrast, these arrhythmic effects correlated with alterations in Ca2+ entry in this arrhythmogenecity that is attributable to a gradually developing intracellular calcium overload.The present findings are thus compatible with a major role for extracellular Ca


Serum albumin is a key component in mammalian sperm capacitation, a functional maturation process by which sperm become competent to fertilize oocytes. Capacitation is accompanied by several cellular and molecular changes including an increased tyrosine phosphorylation of sperm proteins and a development of hyperactivated sperm motility. Both of these processes require extracellular calcium, but how calcium enters sperm during capacitation is not well understood.BSA-induced changes in intracellular calcium concentration were studied using Fluo-4 and Fura-2 calcium imaging with wild-type and Catsper1 knockout mouse sperm.We found that the fast phase of the BSA-induced rises in intracellular calcium concentration was absent in the Catsper1 knockout sperm and could be restored by an EGFP-CATSPER1 fusion protein. The calcium concentration increases were independent of G-proteins and phospholipase C but could be partially inhibited when intracellular pH was clamped. The changes started in the principal piece and propagated toward the sperm head.We conclude that the initial phase of the increases in intracellular calcium concentration induced by BSA requires the CATSPER channel, but not the voltage-gated calcium channel. Our findings identify the molecular conduit responsible for the calcium entry required for the sperm motility changes that occur during capacitation. This process can also be mimicked in vitro by incubating sperm in defined capacitation media. Several commonly used components are essential for successful in vitro capacitation in sperm from many mammalian species. Among them are bovine serum albumin (BSA), Ca2+ and bicarbonate (HCO3-) [i) and Ca2+ concentration ([Ca2+]i), membrane hyperpolarization, and hyperactivated motility i and intracellular [pH]i are believed to play central roles in both sperm capacitation and the acrosome reaction (AR) i changes are presented as ΔF/F0 ratios after background subtraction, where ΔF was the change in fluorescence signal intensity and F0 was the baseline as calculated by averaging the 10 frames before stimulus application. In sperm loaded with Fura-2, [Ca2+]i changes are presented as the ratio of fluorescence from excitation at 340 nm to that at 380 nm (F340/F380) after background subtraction. All the imaging experiments were done at room temperature. Cells with uneven dye loading were excluded from the analysis. Motile sperm (~60% of the population) that had at least two points attached to the coverslip were used for analysis. Cells with peak changes of >50% in ΔF/F0 (for Fluo-4) or >0.1 in F340/380 (for Fura-2) after application of stimuli were counted as responsive. To detect the Ca2+ responses at "clamped" membrane potential (Vm), K+ ionophore valinomycin (1 μM) was added to the imaging buffer, with additional K+ as indicated to replace an equimolar amount of Na+. EK was calculated with the assumption of a 120 mM intracellular K+ concentration i in the sperm head , 98% of wild-type sperm (107/109) showed initial responses in the head within 20 s and 17% (10 of 58) had a 2nd response more than 2 min later  increased [Ca2+]i simultaneously in the principal piece, mid-piece and sperm head  can lead to CATSPER-dependent Ca2+ influx likely via pertussis toxin (PTX)-sensitive G proteins and a phospholipase C-dependent signaling pathway that can be inhibited by neomycin i changes in the absence and presence of PTX  can lead to a CATSPER-dependent rise in [Ca2+]i [2+]i rises and the extent to which CATSPER channels contribute to the increases. To do this, we compared the [Ca2+]i responses to bath application of 20 mM NH4Cl, which leads to intracellular alkalization and induces a slow [Ca2+]i rises in sperm [2+]i rises in wild-type, but not in Catsper1 null sperm  and BSA i responses to bath application of ATP, which was reported to increase [Ca2+]i in bovine and mouse sperm by activating a P2 purinergic receptor[2+]i in the wild-type sperm head in a concentration-dependent manner  rais receptor,49. Simir Figure . Unlike m Figure . The ATPm Figure , indicat2+]i increases in mouse sperm. Stimuli known to induce a CATSPER-dependent Ca2+ entry now include some of the most important mediators in sperm physiology: cyclic nucleotides, zona pellucida and serum albumin. There are clearly also CATSPER-independent Ca2+ entry paths that are responsible for the [Ca2+]i increases induced by stimuli such as extracellular ATP, as we showed in this study, and mechanical force . Differences in signal transduction pathways that link various stimuli to CATSPER channels also exist. For example, the Ca2+ entry induced by the zona pellucid is dependent on G-proteins and phospholipase C i rises were reduced when pHi was "clamped" , can lead to a CATSPER-dependent Ca2+ influx i rises are not dependent on a membrane voltage change since they persist when the voltage is "clamped" with K+ ionophore. Second, inactivating the T-type CaV channel, that is a proposed candidate for the Ca2+ entry [2+]i rises. This result is consistent with our recent finding that mature sperm do not possess detectable functional CaV channels i increases are absent in Catsper1 knockout sperm; second, such responses can be restored by an EGFP-CATSPER1 fusion protein; and finally, these [Ca2+]i rises start in the principal piece of sperm where CATSPER proteins and current through CATSPER channels are localized. The mechanisms by which BSA is coupled to the CATSPER channel are largely unknown. They are unlikely through a voltage change or a cholesterol efflux. An intracellular alkalization appears to be involved (Figure i change-independent component that remains to be uncovered.In summary, we determined the molecular identity of an ion channel responsible for the fast Cad Figure  but ther2+ requirements for the two aspects of sperm capacitation: one mediating the change of motility via the CATSPER channel and another mediating the increase of tyrosine phosphorylation via a source yet to be determined. One possible source for the latter is the second phase of [Ca2+]i rises that are also present in the Catsper1 mutant sperm.Together with previous findings that the capacitation status and changes in the pattern of tyrosine phosphorylation during capacitation do not require CATSPER while the hyperactivated motility does ,20,24,28The authors declare that they have no competing interests.JX performed all the experiments and did data analysis. JX and DR designed the experiments and wrote the paper. Both authors read and approved the final manuscript.


Oxidative metabolism, resulting in the formation of hydroxylated polybrominated diphenyl ether (PBDE) metabolites, may enhance the neurotoxic potential of brominated flame retardants.2+ concentration ([Ca2+]i) and vesicular catecholamine release in PC12 cells.Our objective was to investigate the effects of a hydroxylated metabolite of 2,2′,4,4′-tetra-bromodiphenyl ether  on changes in the intracellular Ca2+]i using amperometry and imaging of the fluorescent Ca2+-sensitive dye Fura-2, respectively.We measured vesicular catecholamine release and [Ca2+]i, which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 μM). An additional late increase in [Ca2+]i was often observed at ≥1 μM 6-OH-BDE-47. The initial transient increase was absent in cells exposed to the parent compound BDE-47, whereas the late increase was observed only at 20 μM. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and thapsigargin to empty intracellular Ca2+ stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca2+, whereas the late increase originates primarily from mitochondria.Acute exposure of PC12 cells to 6-OH-BDE-47 (5 μM) induced vesicular catecholamine release. Catecholamine release coincided with a transient increase in i cannot be ruled out.The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca This correlation between increased [Ca2+]i and the occurrence of exocytosis has been widely studied in neurons , in the environment, human food chain, and human tissues raise concern about possible neurotoxic effects. High concentrations of PBDEs and the structurally related polychlorinated biphenyls (PCBs) increase intracellular Cain vitro only at concentrations ranging from 3 to 20 μM  is the predominant PBDE congener . Neonatain vitro endocrine studies  on interactions with the estrogen and thyroid hormone receptor systems indicate that hydroxylated metabolites of PBDEs are more potent than the parent compounds  at the Wallenberg Laboratory of Stockholm University as described by 2+ imaging experiments, we subcultured undifferentiated PC12 cells in poly-l-lysine–coated glass-bottom dishes  as described previously (Rat pheochromocytoma (PC12) cells  obtainedeviously . For ampWe used cell density as an indicator of cell viability. After 20 min of exposure to dimethylsulfoxide (DMSO) or 20 μM 6-OH-BDE-47, cells were cultured in fresh cell culture medium for another 24 hr. After replacing the culture medium, which washes away most dead, detached cells, and trypan blue inclusion, which stains the remaining dead cells, we determined the proportion of the surface of the cell culture dish occupied by living PC12 cells in triplicate for three dishes per experimental condition.+-evoked and spontaneous vesicular catecholamine release from dexamethasone-differentiated PC12 cells using carbon fiber microelectrodes were made as described previously  to determine their responsiveness. Cells were allowed to recover for 2 min before a 15-min exposure to BDE-47 or 6-OH-BDE-47 to investigate acute effects on vesicular catecholamine release. Recordings were performed at room temperature. To ensure exclusion of nonresponsive or extraordinary cells, we determined basal release frequency for 22 cells. Cells that showed a basal release frequency larger than the average + 2 standard deviations were considered to have an extraordinary high release frequency. Based on these findings, we excluded cells with a basal release frequency > 5/min. Similarly, cells with an evoked release frequency < 16/min were excluded. We used the resulting 20 cells for further data analysis.Amperometric recordings of Keviously . Followi2+]i using the Ca2+-responsive fluorescent ratio dye Fura-2 as described previously  for 20 min at room temperature; this was followed by 15 min de-esterification in external saline. The cells were then placed on the stage of an Axiovert 35M inverted microscope  equipped with a TILL Photonics Polychrome IV . Fluorescence evoked by 340 and 380 nm excitation wavelengths (F340 and F380) was recorded every 12 sec at 510 nm with an Image SensiCam digital camera (TILL Photonics GmBH). The digital camera and polychromator were controlled by imaging software , which was also used for data collection and processing. We further analyzed changes in the F340/F380 ratio, reflecting changes in [Ca2+]i, using custom-made Excel macros . After 5 min baseline recording, cells were exposed to 0.2–20 μM BDE-47 or 6-OH-BDE-47. Maximum and minimum ratios were determined after 25 min recording (20 min exposure) by addition of ionomycin (5 μM) and ethylenediamine tetraacetic acid  as a control for experimental conditions.We measured changes in i (Exposure of PC12 cells to a high concentration (20 μM) of the brominated flame retardant BDE-47 was previously shown to induce vesicular catecholamine release, coinciding with a gradual increase in [Ca2+]i . To inve2+]i , 20 min +-containing saline to determine their responsiveness. Responsive cells displayed depolarization-evoked release (at least 16 released vesicles/min), after which the release frequency returned to baseline values. During the first 2.5 min of a subsequent exposure to 5 μM 6-OH-BDE-47 (n = 9), the release frequency was enhanced from 1.0 ± 0.3 to 13 ± 5.3 events/min (p < 0.05). This enhancement did not occur in cells exposed to DMSO only . After the burst of exocytotic activity, the release frequency in 6-OH-BDE-47–exposed cells declined to a value not significantly different from basal release.To investigate whether exposure to 6-OH-BDE-47 has functional consequences for neuronal communication, we measured vesicular catecholamine release . First, 2+]i. Exposure of PC12 cells to 6-OH-BDE-47 (≥ 1 μM) resulted in a dose-dependent increase in [Ca2+]i in PC12 cells , and an even larger late increase (3.2 ± 0.4) compared to the normalized baseline. At this high concentration, non-specific effects are likely to occur. Therefore, we investigated the concentration dependence of the amplitude of the two types of increases in [Ca2+]i within the range of 1 μM (the lowest concentration where the effects occur) to 5 μM. ANOVA analysis indicated no relationship between the applied 6-OH-BDE-47 concentration and the amplitude of the initial Ca2+ transient -pretreated PC12 cells to 5 μM 6-OH-BDE-47 under physiologic Ca2+ conditions. The occurrence and amplitude of the initial transient increase were markedly reduced under these conditions (data not shown), suggesting that store-operated Ca2+ entry (SOCE) largely accounts for the influx of extracellular Ca2+.To investigate whether the influx of extra-cellular Ca2+]i in PC12 cells, although the hydroxylated metabolite does so at much lower concentrations. The initial transient and the late increase in [Ca2+]i are due to release of Ca2+ from endoplasmic and mitochondrial Ca2+ stores, respectively; extracellular Ca2+ also plays a role in the observed initial increase in [Ca2+]i during exposure to 6-OH-BDE-47. Interestingly, the initial increase in [Ca2+]i is temporally linked with vesicular catecholamine release, raising concern about effects of BDE exposure on neurotransmission.The results of the present study demonstrate that both the abundant PBDE congener BDE-47 and its hydroxylated metabolite 6-OH-BDE-47 increase [Ca2+]i is mainly caused by release of Ca2+ from intracellular stores, which are involved in controlling intracellular Ca2+ homeostasis and neurotransmitter release  and the 6-OH-BDE-47–induced burst of exocytotic activity strongly suggests a causal relationship. Because the observed late increase in [Ca2+]i (by Ca2+ release from mitochondria) has a smaller effect on [Ca2+]i than the emptying of the ER and subsequent SOCE (2+]i and neurotransmitter release is likely to be of less toxicologic concern.The effects of 6-OH-BDE-47 on exocytosis and [Caexposure . The strent SOCE  at conce2+]i primarily originates from the ER. Another brominated flame retardant (tetrabromobisphenol A) has recently been shown to be a potent inhibitor of the SERCA Ca2+ pump (2+]i increase under Ca2+-free conditions (without TG pre-treatment) indicates that both an intracellular and extracellular Ca2+ component contribute to this transient increase. The extracellular Ca2+ component could be caused by a direct effect of 6-OH-BDE-47. However, the TG experiments suggest it is more likely that SOCE, through SOCE channels, in response to 6-OH-BDE-47–induced emptying of the ER, accounts for the involvement of extra-cellular Ca2+. SOCE is commonly observed in PC12 cells after depletion of Ca2+ stores  PCBs increase [Ca2+]i in cultured neural cells and brain preparations - and ryanodine receptors, and disruption of plasma, mitochondrial, and endoplasmic membranes have all been proposed as possible mechanisms. Furthermore, the commercial PBDE mixture DE-71, which contains 31.8% BDE-47, has also been shown to disrupt microsomal Ca2+ homeostasis  could be responsible for the observed effects on [Ca2+]i. In rat hippocampal neurons as well as in human SH-SY5Y cells and neutrophil granulocytes, BDE-47 has been shown to induce ROS formation at exposure concentrations of 41, 4, and 6 μM, respectively  oxidase; release of arachidonic acid; and disturbed and increased [Ca2+]i  PBDE-induced disruption of Caectively . However [Ca2+]i . BecauseDetermination of the relative potency of 6-OH-BDE-47 and BDE-47 based on half maximal effective concentrations requires full dose–response curves with similar slopes and efficacy, which is not realistic due to the occurrence of nonspecific effects at high concentrations of 6-OH-BDE-47 and the relatively low potency of the parent compound and consequent solubility problems. Nonetheless, comparison of lowest observed effect concentrations clearly reveals that 6-OH-BDE-47 has a potency at least one order of magnitude higher than the parent compound BDE-47.in vivo values are still orders of magnitude lower than those that exert effects in the present in vitro study. However, particular concern about neurotoxicity arises from the fact that comparable or even higher levels were observed for several other hydroxylated PBDE metabolites, for which even fewer toxicity data are available than for 6-OH-BDE-47.In the 1990s, an association between delayed human neurodevelopment and prenatal or neonatal exposure to PCBs was reported in cohort studies, which were corroborated by experiments demonstrating developmental neu-rotoxicity of PCBs , a cumulative neurotoxic effect (on [Ca2+]i) of both groups of compounds can not be ruled out. Further research should determine whether combined exposure to PBDEs and ortho-PCBs is of neurotoxicologic relevance in humans.In summary, exposure of PC12 cells to ≥1 μM 6-OH-BDE-47 increases exocytosis and [Cabehavior  and synabehavior . Human ebehavior . In this


Acute myeloid leukemia (AML) is a heterogenous disorder that results from a block in the differentiation of hematopoietic progenitor cells along with uncontrolled proliferation. In approximately 60% of cases, specific recurrent chromosomal aberrations can be identified by modern cytogenetic techniques. This cytogenetic information is the single most important tool to classify patients at their initial diagnosis into three prognostic categories: favorable, intermediate, and poor risk. Currently, favorable risk AML patients are usually treated with contemporary chemotherapy while poor risk AML patients receive allogeneic stem cell transplantation if suitable stem cell donors exist. The largest subgroup of AML patients (~40%) have no identifiable cytogenetic abnormalities and are classified as intermediate risk. The optimal therapeutic strategies for these patients are still largely unclear. Recently, it is becoming increasingly evident that it is possible to identify a subgroup of poorer risk patients among those with normal cytogenic AML (NC-AML). Molecular risk stratification for NC-AML patients may be possible due to mutations of NPM1, FLT3, MLL, and CEBPα as well as alterations in expression levels of BAALC, MN1, ERG, and AF1q. Further prospective studies are needed to confirm if poorer risk NC-AML patients have improved clinical outcomes after more aggressive therapy. Acute Myeloid Leukemia (AML) is a broad range of disorders that are all characterized by an arrest of maturation along with uncontrollable proliferation of hematopoietic progenitor cells. The French-American-British classification is still widely used in clinical setting that groups AML into 8 subgroups (M0-M7) based on its degree of differentiation and morphology. Due to the heterogenous nature of AML even within specific FAB subtypes, there is a highly variable prognosis among AML patients. The overall 5-year survival rate for AML is still less than 50% in adults and significantly lower in the elderly . The medNM_002520). Both mutations lead to aberrant cytoplasmic localization of NPM1 as shown after immunostaining with anti-NPM1 monoclonal antibodies. This is caused by open reading frameshift mutations that lead to either the disruption of the NPM1 nucleolar-localization signal or the generation of a leucine-rich nuclear export motif. In all mutated cases, the resulting frameshift led to a product five amino acids longer with the new C-terminal tail CFSQVSLRK, peculiar to the NPM1-mutated product [Mutations of NPM1 have recently been described as one of the most frequent genetic lesions in AML, occurring in 50–60% of adult AML with normal karyotype . Additio product .Recent studies in cell lines and knockout mice have shown that NPM1 is involved in the control of genomic stability and contributes to growth-suppressing pathways through its interaction with ARF. Therefore, the loss of NPM1 expression can contribute to tumorgenesis . SeveralNPM1 gene mutations appear to occur more frequently in adult female AML patients -16 and aDetection of NPM1 gene mutations may be useful in the dissection of the heterogeneous group of AML patients with normal karyotype into prognostically different subgroups . FurtherFLT3 is a tyrosine kinase that is primarily expressed on hematopoietic progenitor cells and functions in the proliferation and differentiation of these cells. FLT3 is the most commonly mutated gene in AML with the mutation occurring in approximately 30–40% of AML patients . The mosParticularly in AML patients with normal cytogenetics, FLT3-ITD status is important in assessing the prognosis of patients. Several studies have demonstrated that FLT3-ITD in NC-AML patients correlates with an adverse prognosis for both DFS and OS -25. Not In addition to mutation of FLT3 and the decreased expression of the wild-type allele, over-expression of FLT3 in the absence of mutation has also been observed in AML patients. Over-expression of FLT3 even in the absence of FLT3-ITD is also an unfavorable prognostic factor for OS . As FLT3The association of FLT3 mutations have also been evaluated in patients with favorable cytogenetics , inv16, t). FLT3 mutations were noted in 15 of 17 patients studied. Of these patients, 41% of those with t were associated with FLT3 mutations and only 9% of cases with inv16. Those with PML-RARα had decreased to no CD11c or HLA-DR expression. However, this study did not illustrate significant correlations with outcomes and FLT3 mutational status .As FLT3 mutations lead to constitutively activated signaling, much work has been performed to develop small molecule FLT3 inhibitors. Unfortunately FLT3 inhibitors have thus far shown disappointing results as remission induction has been short lived. Nevertheless, there is optimism that FLT3 inhibitors may be more efficacious when used in combination therapies . BesidesMLL is frequently rearranged in AML and ALL and has been found in combination with greater than 30 different genes. The most frequent rearrangements in the current published series were unbalanced translocations leading to loss of chromosomal material. Overall, loss of 5q and/or 7q chromosomal material seemed the more common event, and losses of 5q, 7q, and 17p in combination were observed in many cases. Overrepresented chromosomal material from 8q, 11q23, 21q, and 22q was found recurrently and in several cases this was due to the amplification of the MLL (located at 11q23) and AML1/RUNX1 (located at 22q22) genes . MLL encIn addition to rearrangements, the MLL gene can also undergo partial tandem duplications of exons 5–11 or exons 5–12 and produce an elongated protein. This abnormal protein, which contains a DNA-binding and transcriptional repression domain, can suppress the expression of the wild-type allele by an unknown mechanism. Interestingly, the silencing of wild-type MLL in blasts positive for MLL-PTD was reversed by DNA methyltransferase and histone deacetylase inhibitors . These fIn one large study of 247 young adult patients with AML, MLL-PTD was found in 7.7% of patients. In this study, MLL-PTD was an adverse prognostic indicator as the median remission duration was 19 months in the absence of MLL-PTD and 7.75 months in its presence . In geneCEBPα is an essential transcription factor for granulocytic differentiation as demonstrated by CEBPα-null mice that lack mature granulocytes ,41. StudSuprisingly, as CEBPα is required for differentiation, mutation of CEBPα is correlated with beneficial effects on remission, CR duration , event-fWhile NPM1, FLT3, MLL-PTD, CEPBα are all mutations noted in NC-AML, additional genetic abnormalities are noted in the form of over-expression. More detailed discussion of BAALC, MN1, ERG-1, and AF1q give insight to over-expression of genes in NC-AML.BAALC has also been found to be an important adverse prognostic factor in NC-AML. Though little is known about the biological function of BAALC, it is highly expressed in hematopoietic precursor cells as well as leukemic blasts and is down-regulated during differentiation. BAALC has been postulated to function in the cytoskeleton network due to its cellular location ,45. SeveMN1 is an oncoprotein that has been found to function as a transcription coactivator. In AML it has been found as part of the translocation t which leads to the MN1-TEL fusion gene . In animERG is a member of the ETS family of transcription factors. High ERG expression is associated with the upregulation of many genes which are involved in cell proliferation, differentiation, and apoptosis . The ERGAs the ERG gene is located on chromosome 21, it has been speculated that ERG expression may play a role in the pathogenesis of acute leukemia in patients with Down's syndrome. Patients with Down's syndrome are known to have a higher incidence of acute leukemia . The higTwo small studies conducted by Tse et al suggest that elevated AF1q expression is associated with poor outcomes both in pediatric AML and adult myelodysplastic syndrome (MDS) ,56. In tlow) had better OS (p = 0.026) and CR rate with initial induction chemotherapy (p = 0.06) compared to high AF1q expressing patients (AF1qhigh). The AF1qhigh patients had a significantly greater incidence of concurrent FLT3-ITD. A subgroup of the AF1qhigh patients who received allogeneic stem cell transplantation (SCT) had a significant better relapse-free survival compared to patients who received chemotherapy/autologous SCT (p = 0.04). This suggests that high AF1q expression is a poor prognostic marker for adult NC-AML patients and may help direct post-induction treatment strategies.Recently, Strunk et al examined AF1q expression in 290 adult NC-AML patients (aged < 60). They found NC-AML patients with low AF1q expression  can provide an early indication of potential relapse in AML post-treatment. There has been some initial evaluation of the utility of FLT3-ITD, CEBPα, ERG, NPM1, and MLL in MRD whereas AF1q, MN-1, and BAALC have not. The impact of FLT3 was analyzed in 11 patients. All of the six patients with positive quantitative real-time polymerase chain reaction (RQ-PCR) post-treatment eventually relapsed . Real-tiThough NC-AML comprises the single largest subgroup of AML, these patients pose considerable challenges in diagnosis, risk stratification, and post-treatment monitoring for minimal residual disease. Gene expression and profiling studies have shown that NC-AML is very heterogeneous at the molecular level. Multiple studies have shown that NC-AML patients usually exhibit two or more genetic aberrations. While assessment of these molecular prognostic markers is not widely available to the community physician outside of a clinical trial, future studies will help to further validate the prognostic importance of these altered genetic abnormalities in systemic multivariate analysis in the NC-AML patients. Once the prognostic importance of these genetic abnormalities is clear, it may be possible to appropriately tailor the aggressiveness of therapy to NC-AML patients. Further, these studies also have the potential to identify novel therapeutic targets that may be used to design targeted therapies. In the future, specific genetic abnormalities may be profiled in AML patients in a similar manner to the immunophenotyping that is currently done by flow cytometry to obtain information on accurate diagnosis, prognosis, and disease monitoring.The authors declare that they have no competing interests.All authors participated in the drafting of the manuscript. TG edited the manuscript and WT and TG read and approved the final manuscript.


Calliphora salivary glands by the parallel activation of the InsP3/Ca2+ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca2+ signaling and InsP3-induced Ca2+ release from the endoplasmic reticulum (ER).Serotonin induces fluid secretion from 2+]i changes into a sustained increase. Intraluminal Ca2+ measurements in the ER of β-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP3-induced Ca2+ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP3 receptor Ca2+ channel for InsP3. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP3-induced Ca2+ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP3/Ca2+ signaling pathway for 5-HT, because IBMX potentiated Ca2+-dependent Cl- transport activated by a threshold 5-HT concentration.Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca3-induced Ca2+ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP3/Ca2+ signaling pathways enhances transepithelial electrolyte transport.This report shows, for the first time for an insect system, that cAMP can potentiate InsP InsP3-induced Ca2+ release with or without Ca2+ entry from the extracellular space generates temporally and spatially coordinated Ca2+ signals leading, in many cells, to intracellular Ca2+ oscillations and waves i . Treatment with cAMP or IBMX had the same effect as forskolin at all tested preparations . Forskolin did not affect the sustained Ca2+ elevation produced by 30 nM 5-HT  induced intracellular Cags. 2+]i . As show-HT Fig. , a conce2+ increase produced by forskolin at low 5-HT concentrations was attributable to Ca2+ influx from the extracellular space, we stimulated glands with a sub-threshold concentration of 5-HT . As seen in Fig. 2+ oscillations (in Ca2+-containing PS), but application of 100 μM forskolin stimulated a transient Ca2+ elevation even in the absence of extracellular Ca2+. Taken together, these results suggested that cAMP did not induce Ca2+ influx but rather augmented Ca2+ release from the ER produced by low 5-HT concentrations.To determine whether the extra Caepletion ) and app2+ from the ER: the InsP3R and the ryanodine receptor Ca2+ channel (RyR). Blowfly salivary glands, however, seem to lack RyR L) L) ,35,36. F2+ release from the ER, whereas the Ca2+-ionophore ionomycin led to a dramatic loss in intraluminal Ca2+  -induced Ca2+ release gave a mean half maximal cAMP concentration (EC50) of 2.5 μM  Fig. , squares μM Fig. .3-induced Ca2+ release was not simply the result of the addition of fresh InsP3(+cAMP)-containing ICM, we superfused several preparations with InsP3(no cAMP)-containing ICM twice. A second InsP3 application never increased Ca2+ release induced by a prior InsP3 application , but rather its rate as measured by the decline in the Mag-fura-2 fluorescence ratio per minute. The rates were obtained from regression lines fitted to the fluorescence traces over a one minute period before and after application of the cAMP analog  by 5-HT concentrations that were just sufficient to stimulate fluid secretion and (2) by saturating 5-HT concentrations. Because cAMP also stimulates transepithelial K+ transport by activating an apical vacuolar-type H+-ATPase that energizes K+ transport . Sequence information for Calliphora InsP3R is still lacking but the dipteran fly Calliphora is closely related to Drosophila. Thus, whether fly InsP3 receptor Ca2+ channels can be phosphorylated, or whether the InsP3R in Calliphora differs from that in Drosophila with respect to consensus sites for PKA-mediated phosphorylation remains unknown. Therefore, we cannot yet explain the molecular basis of the cAMP-induced and PKA-mediated sensitization of Ca2+ release in this species. DmInsP3R seems to have consensus sequences for phosphorylation by PKC and CaM-kinase II i changes, and this potentiation also results from enhanced Ca2+ release attributable to cAMP-dependent and PKA-mediated potentiation of InsP3-induced Ca2+ release from the ER inositol release from isolated glands .Calliphora salivary glands indicate that cAMP also augments the Ca2+-dependent transepithelial Cl- transport induced by low 5-HT concentrations, an observation suggesting that the cAMP-dependent enhanced Ca2+ release additionally affects fluid secretion. This notion is supported by experiments dating back more than 30 years. In the early 1970s, Berridge i was measured as described previously [2.[Caeviously . In briet-test, and P values < 0.05 were considered significant.Signal processing and curve fitting were performed by using GraphPad Prism 4 . Data are expressed as means ± S.D. Statistical comparisons were made by a Student's paired RS carried out all experiments and drafted the manuscript. BW and OB participated in the conception of the project and the design of the experiments, and they helped to write the manuscript. All authors approved the final manuscript.


Nevertheless, the specific role of calpains in Ca2+ signalling during the onset of dystrophies remains unclear. We investigated Ca2+ handling in skeletal cells from calpain 3-deficient mice. [Ca2+]i responses to caffeine, a ryanodine receptor (RyR) agonist, were decreased in −/− myotubes and absent in −/− myoblasts. The −/− myotubes displayed smaller amplitudes of the Ca2+ transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca2+ channels (LCC) suppressed the caffeine-induced [Ca2+]i responses in −/− myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR) Ca2+ release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca2+ signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.Mutations of Ca It was(LGMD2A) , a disea(LGMD2A) . To bett(LGMD2A) . The pat(LGMD2A)  suggestiThe identification from skeletal muscle of a 94 kDa protein that possesses thiol-dependent proteolytic activity specifically directed against the skeletal muscle ryanodine receptor (RyR) has several implications for the pathogenesis of LGMD2A . In part2+-release channel, is the key protein responsible for the extremely rapid movement of Ca2+ (millions of ions per second) from the internal stores named the sarcoplasmic reticulum (SR) to the cytosol during the process of excitation-contraction coupling. The latter is described by a plasma membrane depolarisation coming from motor neurones and activating dihydropyridine receptors or L-type Ca2+ channels (LCCs) mechanically coupled to the skeletal RyR isoform 1. In turn, Ca2+ release from RyR causes the contractile filaments to slide along one another, thus triggering the twitch of the muscle cell. Then Ca2+ has to be removed from the cytosol by extrusion through the Na+/Ca2+ exchanger and by reuptake into the SR by the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump. Hence Ca2+ cycling is a fine-tuned process that requires strong control of its homeostasis. Indeed, RyR, also referred to as Ca2+]i, which controls the excitation-contraction coupling in muscle. We measured [Ca2+]i in primary cultures of normal and capn3-deficient mice skeletal muscle cells by single cell fast fluorescence microspectrofluorometry. In vivo, satellite cells are activated into myoblasts, proliferate and fuse to form myotubes to repair damaged muscle fibres. By comparing myoblasts and myotubes in culture, we aimed to investigate the different steps involved in the process of regeneration. Our pharmacological approach enabled us to decipher the mechanisms leading to an impairment of Ca2+ release in skeletal muscle when calpain 3 is absent, as it might be the case in LGMD2A patients. To test the hypothesis of RyR modulation by calpain 3, we investigated the effect of caffeine, a potent activator of RyR, and cyclopiazonic acid (CPA), an inhibitor of the SERCA pump, on i) were performed as described i as determined by preliminary fura-2 measurements). [Ca2+]i levels in single cells were measured using the FFP photometer system  based on an inverted microscope (Axiovert-100) equipped with epifluorescence. Band pass filters (340/10 nm and 380/10 nm) were alternately positioned with a filter wheel, and the cells were excited through an oil-immersion objective . With fluorescence values corrected for background and dark current, [Ca2+]i was calculated from the ratio between 340 and 380 nm recordings. For the in vitro calibration of [Ca2+]i measurements based on the procedure described by Grynkiewicz et al. i levels before and after the application of test substances, while bar diagrams and numerical data are given as mean ± S.E.M. and represent the peak amplitude of the [Ca2+]i increase. Depending on the data, the results were analysed using ANOVA or Mann-Whitney Rank Sum test. Unless otherwise stated, differences were considered statistically significant if  P < 0.05.The results are expressed as mean ± S.D or means ± 2, 20 mM HEPES (pH 7.4), 200 μM phenylmethylsulfonylfluoride (PMSF), 1 mM diisopropylfluorophosphate (DFP). Protein samples were denatured 30 min at room temperature and subjected to SDS-PAGE on 4%–15% acrylamide gradient gels and electro-transferred to Immobilon membranes for 3 hours at 0.8 A. The membrane was blocked with 4% nonfat dry milk in PBS, 0.1% Tween 20 (PBS-T) for 30 minutes at room temperature and then incubated overnight at 4°C with a polyclonal rabbit antibody against RyR diluted 1/10 000 i, that peaked after 3 minutes of caffeine application and decayed slowly, even before the caffeine was washed out (+/Ca2+ exchanger at the plasma membrane level [2+]i after the application of caffeine. No [Ca2+]i response to caffeine was observed for repeated applications (data not shown) even when the caffeine was quickly taken off. By contrast, +/+ myotubes exhibited a constant, repeated and reproducible increase in [Ca2+]i after brief (30s) and repeated applications of caffeine (2+]i response is plotted in 2+]i-induced response to caffeine in a huge majority of cells (2+]i response to repeated caffeine applications (3 min each) was observed in −/− myotubes  mice . Restingshed out  presumabne level . The numcaffeine . The meaof cells . In contmyotubes . In addi n = 12; .2+-ATPase i of much lower amplitude  Ca+-ATPase , 22 and e stores . In +/+ y higher  than they higher . The difcapn3-deficient cells (−/−), the [Ca2+]i responses to caffeine and CPA were quite different  mice. Indeed, the removal of extracellular Ca2+ did not significantly affect the [Ca2+]i responses induced by 20 mM caffeine on +/+ myotubes (results not shown). However, a major reduction in the [Ca2+]i response to caffeine was observed in −/− myotubes, in the absence of extracellular Ca2+ (results not shown). The i response induced by caffeine in +/+ myotubes , omega GVIA-N-type, omega MVIIC/MVIIA-a P/Q type, omega-Aga-IVA (a P/Q-type blocker), and SNX-482 (a R-type blocker). Interestingly, only nicardipine at 800 nM significantly blocked the Ca2+ response induced by caffeine in −/− myotubes  suggesting that the response could not be directly mediated by caffeine-sensitive channels. The next experimental design aimed to dissect the functional interaction between RyR and Ca Figures . In shar Figures . It is n Figures , the bas2+ entry , the bas2+-dependent cysteine proteases . Moreover, protein degradation was enhanced in mdx muscle i rise in myoblasts and myotubes from normal and capn3 −/− mice. Notably, it is possible that calpain 3 acts as a feedback regulator for calcium homeostasis in skeletal muscle cells by exerting an action on RyR. The latter, also known as the Ca2+ release channel of the SR, is a key protein involved in excitation-contraction coupling. Its activity is regulated by a 94 kDa thiol-protease of the junctional SR membranes which specifically cleaves one site on RyR. This cleavage results in enhanced Ca2+ efflux from SR vesicles i transients than in normal cells, which could indicate a lower SR Ca2+ loading state in the KO skeletal cells, a decreased number of RyR at the SR membrane surface, or a decreased sensitivity of these receptors but independently of any cleavage by calpain 3.The effectiveness of caffeine in normal myotubes in comparison to myoblasts indicates a maturation of proper RyR signalling in culture during the fusion process. Importantly, both myoblasts and myotubes from 2+ stores [2+]i under all conditions tested, it appeared that those responses were weaker in capn3 −/− myotubes in comparison to wild-type myotubes, reinforcing the hypothesis that SR Ca2+ loading decreased in capn3 −/− myotubes or indicating that SR Ca2+-ATPases were less expressed or less sensitive to the blocker in KO myotubes. The difference in CPA responses between wild-type myoblasts and myotubes is most likely due to a change in the size of the SR that correlates with a change in the size of the cells during fusion in culture, myotubes having a larger area. While CPA, a compound that depletes internal Ca+ stores , evoked 2+ from the SR into the cytosol, independently of any Ca2+ influx through the plasma membrane. The fact that low extracellular Ca2+ and blockers of LCCs abolished caffeine-induced [Ca2+]i increases in −/− myotubes suggests that RyR opening and SR Ca2+ release lead to a Ca2+ influx through LCCs solely in the KO myotubes. Since cytosolic Ca2+ is known to negatively regulate these channels (inactivation) and that the release from the SR is not sufficient to evoke a depolarization enabling the opening of LCCs [2+]i increases in −/− myotubes.Caffeine is thought to directly activate RyR at the SR membrane, leading to the opening of this channel and the release of Ca of LCCs , it is m2+ entry in order to induce the proper opening of the receptor. Finally, another possibility would be that the lack of calpain 3 leads to a decrease of RyR sensitivity to caffeine, probably involving a regulation of the post-translational maturation of the receptor, and thus independently of any functional cleavage of RyR. It is noteworthy that RyR contains many endogenous cysteines in the cytoplasmic domain of the protein. Hence the binding of caffeine to its site on the cytosolic face of RyR would require an activation of RyR by extracellular Ca2+ entry), but (iv) no increase of the basal intracellular Ca2+ concentration. As the major system of Ca2+ extrusion from the cytosol is the Na+/Ca2+ exchanger, it is very tempting to speculate that the activity of the exchangers could be increased (higher protein expression and/or higher rate of ion flow) in KO myocytes to maintain the level of Ca2+ in the cells. However, this will be the subject for another specific investigation.Taken together, the results obtained in −/− myotubes indicate: (i) a decrease of the SR load, (ii) an alteration of RyR signalling, (iii) an increase of LCC activity  an increase of caspase 3 activity, (ii) a deregulation of the I{kappa}B{alpha}/NF-{kappa}B pathway leading to apoptosis , 16,  uncoupling in normal and capn3 −/− skeletal muscle fibres [2+ signalling. Of interest, recent work from our group showed that not calpain 3, but e fibres , suggest


Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na+/H+ ionophore monensin, K+/H+ ionophore nigericin and the H+ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca2+ from intracellular stores upon ATP addition.We have previously reported that a Teiid lizard red blood cells (RBCs) such as Tropidurus torquatus store Ca2+ in endoplasmic reticulum (ER) pool but unlike in the referred Teiidae, these cells do not store calcium in monensin-nigericin sensitive pools. Moreover, mitochondria from T. torquatus RBCs accumulate Ca2+. Addition of ATP to a calcium-free medium does not increase the [Ca2+]c levels, however in a calcium medium we observe an increase in cytosolic calcium. This is an indication that purinergic receptors in these cells are P2X-like.Here we report, that RBCs from the tropidurid lizard T. torquatus RBCs present different mechanisms from Teiid lizard red blood cells (RBCs), for controlling its intracellular calcium levels. At T. torquatus the ion is only stored at endoplasmic reticulum and mitochondria. Moreover activation of purinergic receptor, P2X type, was able to induce an influx of calcium from extracelullar medium. These studies contribute to the understanding of the evolution of calcium homeostasis and signaling in nucleated RBCs. The first assembles the snakes, amphisbaenas, and a diverse array of lizard families, among them the Teiidae, while the last comprise a less diverse assort of lizard families which includes the Tropiduridae. We have previously shown that RBCs from the teiid lizards  pathway -20. HereTropidurus torquatus with Fluo-3 AM, we characterized the intracellular pools involved in calcium homeostasis within these cells. We found [Ca2+]c in the RBCs of Tropidurus torquatus around 51,2 ± 1,7 nM (n = 18). In order to verify if T. torquatus RBCs contain intracellular Ca2+ stores compartments, the cells were treated with the tumor promoter thapsigargin, known to specifically inhibit the Ca2+ ATPase from the sarco-endoplasmic reticulum (SERCA) [2+ ([Ca2+]c) of 18.1 ± 2.1 nM (n = 6) when the cells were incubated in Ca2+ medium (1 mM). Addition of thapsigargin in Ca2+-free medium (supplemented with 15 mM EGTA) resulted in an increase of fluorescence in RBCs of T. torquatus loaded with Fluo-3 AM. Subsequent addition of ionomycin (10 μM) resulted in further increased fluorescence  . Fig 1A ence Fig  indicatiease Fig . The inc+/H+)  (data not shown).Differently from Teiidae family, RBCs from Tropiduridae lizards are not sensitive to Monensine (ionophore Na/H+) Fig  nor NigeAmeiva ameiva and Tupinambis merianae lizards. In these cells, we have characterized the G-protein coupled P2Y type receptor as ATP mobilized calcium from internal pools in a calcium-free medium. We also reported that the second messenger InsP3 could be the signal for calcium mobilization from internal pools within these cells. In Tropidurus torquatus, the addition of ATP (50 μM) promotes a sustained increase of [Ca2+]c of 26.4 ± 3.4 nM (n = 10) when the cells are incubated with Fluo-3 AM in Ca2+ medium (CaCl2 1 mM) . To observe the presence of a P2Y purinoceptors we treated the RBCs with a specific antagonist to this type of receptor. We have shown that addition of α2MetATP (50 μM) did not induce an increase of [Ca2+]c in RBCs from T. torquatus loaded with Fluo-3 AM  no effect was observed in fluorescence with addition of ATP (50 μM), although further addition of ionomycin (10 μM) promotes Ca2+ release . These results indicate that the increase of [Ca2+]c, promoted by purinergic receptors agonists, is from extracellular medium by activation of a Ca2+-channel in the plasma membrane, probably a P2X type.ATP triggers calcium release in the RBCs of  mM) Fig . To veri3 AM Fig . When thease Fig . When weease Fig , for 30 2+ storing in RBCs from T. torquatus we loaded the erythrocytes with mitochondria indicator Mito Fluor Green (5 μM)    Fig  (n = 3)  μM) Fig  (n = 3). μM) Fig  denotes /ml) Fig  (n = 3),2+ homeostasis in T. torquatus RBCs. These cells were incubated with the citosolic Ca2+ indicator Fluo 4 AM and with mitochondrial Ca2+ indicator Rhod-2 AM. The addition of ATP (50 μM), as shown previously in Fig 2+ by activation of a membrane purinoceptor P2X type that promotes an influx of the ion. Here we show the increase of mitochondria [Ca2+]mt after an increase of [Ca2+]c induced by ATP (50 μM) in T. torquaturs RBCs loaded with both Fluo 4 AM and Rhod-2 AM (in the Ca2+ medium)  Fig  (n = 4).]mt. Fig  (n = 4).]mt. Fig  (n = 4) ]mt. Fig  (n = 4).n of ATP 0 μM, as  ATP Fig  or ionom ATP Fig  in a Ca2ADPH Fig  (n = 3).T. torquatus ER can sequester Ca2+ and that blockage of the pump results in a rapid release of this ion to the cytosol. The fact of the increase of [Ca2+]c, induced by thapsigargin, in T. torquatus was lower than the values found for RBCs in the previously studied teiid lizards suggests that the capacity of the ER to sequester Ca2+ is higher in the Teiidae, or the affinity of SERCA pump to thapsigargin is lower in T. torquatus. Differences on SERCA pump sensitivity to thapsigargin have been reported in different models. For example, micromolar concentrations of thapsigargin inhibit SERCAs in non-mammalian vertebrate cells of turtles c promoted by mobilization of Ca2+ from ER or by influx of this ion by activation of purinergic receptors in the plasma membrane.The mitochondria, in most mammalian cells loaded with fluorescent indicators, appear disperse in the cytosol ,33. Howeted RBCs . This inl models -37. The l (1993) . The autAgama sp (Iguania) (type P1 and P2)  = Kd (F-Fmin)/(Fmax-F) where the Kd utilized for Fluo 3 was 390 μM, F is the measured fluorescence intensity in the condition of the experiment, Fmax the fluorescence in the presence of digitonin and Fmin the fluorescence in the presence of 8 mM of EGTA. Unless otherwise specified all experiments were performed at 37°C. All the experimental conditions and the range of drug concentration were previously standardized by teiid lizards as described by Beraldo et al. 2001 and 2002 [After the passage in the cellulose column, the cells were washed 3 times in buffer A  are resuspended in buffer A where Mitofluor Green (5 μM) and Rhod-2 AM (5 μM) are added. The RBCs are incubated at 37°C by 30 min. After incubation, the RBCs are washed three times in buffer A to remove the extracellular dye. The cells are observed in confocal microscopy LSM-510 Zeiss using wavelengths: Mitofluor Green EX = 490/EM = 516 and Rhod-AM EX = 545/EM = 560–615 nm. Samples were observed using slides that were previously incubated for 1 hour with polylisine in PBS 2 mg/ml and washed after incubation.In these experiments we used the mitochondrial probe Mitofluor Green and the mitochondrial CaLizard RBCs are ressuspended in 1 ml of buffer A where 10 μM Fluo-4 AM and 40 μM of probenecid were added. RBCs are incubated for 60 minutes at 37°C with Fluo-4 AM. After that, 5 μM of Rhod-2 AM were added and the RBCs were incubated for 30 minutes at 37°C. Finally, RBCs were washed three times with buffer A to remove the extracellular dyes. The experiments were performed in confocal microscopy in plates previously incubated with polylisine using the wavelengths (EX = 488/EM = 550) Fluo 4 AM and (EX = 545/EM = 560–615 nm) for Rhod-2 AM.2+ elicit an increase of activation of mitochondrial dehidrogenases, which can be detected by measuring the level of NADPH fluorescence [4, 5.5 mM D-glucose, 1 mM CaCl2 and 50 mM MOPS, pH 7.2) at room temperature by centrifugation at 1500 g for 5 min and resuspended (at 106 cells/ml-1) in the same buffer. After incubation with 5 μM de Rhod-2 AM the cells were washed three times with buffer A to remove the extracellular dyes. The experiments were performed in spectrofluorimeter Shimadzu using the wavelengths EX = 540/EM = 570 for Rhod 2 AM and EX = 350/EM = 460 nm for NADPH auto fluorescence.The changes in mitochondrial Carescence . After tFHB and CRSG designed the experiments. FHB carried out the experiments with RBCs. FHB and CRSG wrote the manuscript and designed the figures. All the authors read and approved the manuscript.


Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer.First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+)-free medium. The potential of the most promising treatments  to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats.All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7%) and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%); incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos  were transferred into 19 recipient females, 4 animals became pregnant. All of the fetuses developed from oocytes activated by electroporation followed by cycloheximide and cytochalasin B incubation; no fetal development was detected as a result of thimerosal/DTT activation. Although heartbeats were detected in two of the cloned fetuses, no term development occurred.Electroporation proved to be the most effective method for the activation of cat oocytes reconstructed by nuclear transfer. The combined thimerosal/DTT treatment followed by cycloheximide and cytochalasin B incubation triggered development effectively to the blastocyst stage; whether it is a viable option to stimulate term development of cloned cat embryos needs further investigations. Changes in the [Ca2+]i can also be induced artificially and as a result parthenogenetic oocyte activation can take place. Although mammalian parthenogenetic embryos never develop to term, a great number of invertebrate and vertebrate animal species are able to reproduce via parthenogenesis [Ovulated mammalian oocytes are arrested at the metaphase stage of their second meiotic division . Normall2+]i must be able to trigger the numerous biological events that are associated with fertilization i rise is induced to stimulate development of the reconstructed oocyte i changes in cat oocytes induced by different stimuli; 2) develop new activation methods that could be applied after nuclear transfer to produce cleavage-stage embryos; and 3) assess the feasibility of nuclear transfer to produce cloned cats affected with the lysosomal storage diseases MPS or AMD.Activation of oocytes of many domestic species including the domestic cat has been described and used in reproductive research . Cats arAll chemicals were purchased from Sigma-Aldrich Chemical Company  unless otherwise indicated. Experiments were conducted according to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) at the Cincinnati Zoo & Botanical Garden.Cat ovaries were collected after routine ovariectomy. Cumulus-oocytes complexes (COCs) were collected by slicing in Hepes-buffered Tyrode's Lactate (TL-Hepes) medium and matured in groups of 10 in 100-μl drops of Feline Optimized Culture Medium  supplem2+ indicator dye fura-2 by incubation in the presence of 2 μM fura-2 AM (acetoxymethyl ester form) and 0.02% detergent pluronic F-127  for 45 min. The oocytes were then rinsed and transferred to a chamber with a glass coverslip as the bottom. They were exposed to various treatments and changes in the [Ca2+]i were recorded using InCyt Im2, a dual-wavelength fluorescence imaging system . Fluorescence was recorded by calculating the ratio of fura-2 fluorescence at 510 nm excited by UV light alternatively at 340 and 380 nm. The Ca2+ concentration was calculated by comparing the ratio of fluorescence at 340 and 380 nm against a standard curve of known Ca2+ concentration prepared with fura-2 potassium salt (Invitrogen). During measurements, the baseline [Ca2+]i was detected first and then different stimuli were applied. For each treatment, the fluorescence measurement was performed for at least 10 min and then it was repeated several times using different oocytes.Mature oocytes were loaded with the Ca2+]i of the oocytes. Then, based on the results of these measurements and on data published in other species, a number of treatments were identified that were deemed to have the potential to trigger parthenogenetic development of the oocytes. The oocytes were treated using the selected methods, then a portion of them were incubated in the presence of 10 μg/ml cycloheximide (a protein synthesis blocker) and 7.5 μg/ml cytochalasin B (an inhibitor of actin polymerization) for 4 h prior to culture; the rest of the oocytes were cultured without inhibitor supplementation. In previous studies the use of such inhibitors had a positive effect on embryo development following oocyte activation  and placed on the stage of an inverted microscope. After a 5-min equilibration period the steady-state Ca2+ levels were measured, then two DC pulses of various characteristics , 1 sec apart, were applied to the oocytes [2+]i were monitored. To stimulate development two DC pulses of either 1.0 kV/cm for 20 μsec or 1.2 kV/cm for 60 μsec were applied.Individual oocytes were transferred to a chamber with two stainless steel electrodes where the bottom of the chamber had been replaced with a glass coverslip. The chamber was filled with electroporation medium  was added to the medium to obtain 7% (v/v) final ethanol concentration [2+]i were recorded by the fluorescence imaging system. In order to trigger parthenogenetic development, oocytes were exposed to 7% (v/v) ethanol for 5 min.Oocytes were placed into the Cantration . ChangesEach oocyte was transferred into TL-Hepes and ionomycin dissolved in dimethyl sulfoxide (DMSO) was added to the medium at a final concentration of 5 or 50 μM . The sam2+ release proteins thimerosal is known to elevate [Ca2+]i in oocytes i of cat oocytes. The application of DC pulses of larger voltage or longer duration led to more robust changes in cytoplasmic Ca2+ levels and increasing these parameters above a certain threshold (e.g. raising voltage above ~1.8 kV/cm) caused elevated intracellular Ca2+ levels that did not return to baseline possibly indicating permanent membrane damage (data not shown). A typical change induced by two pulses of 1 kV/cm, 20 μsec each is shown in Figure 2+]i in the oocytes was approximately 200 nM. The [Ca2+]i elevation started 3 ± 0.4 sec after applying the electrical shock and peaked at a concentration of 485 ± 28 nM after which it decreased gradually. The transient had an average duration of 73 ± 15 sec and the [Ca2+]i reached near baseline levels by the end of the measurements (n = 15). Electroporation caused a single [Ca2+]i rise in all oocytes tested  could elicit a rapid Ca2+ response in all  oocytes tested. Figure 2+]i change induced by 50 μM ionomycin. The [Ca2+]i rise in these cases started within a few seconds after adding ionomycin and peaked at a concentration of 336 ± 12 nM. In all cases the [Ca2+]i elevation had an extended duration that lasted as long as the oocyte was exposed to the ionophore since no [Ca2+]i return to basal levels was observed during the measurement period. The Ca2+ rise triggered by 50 μM ionomycin started within a shorter period of time and had higher amplitude compared to those stimulated by lower ionomycin concentration (data not shown). DMSO alone did not elicit changes in the [Ca2+]i (data not shown).Incubation of mature cat oocytes in 7% ethanol induced a single [Cas Figure . The ele2+]i elevation in 15 out of 18 oocytes; the signal took place as an oscillation in the intracytoplasmic Ca2+ level in 11 oocytes. On average, the initial [Ca2+]i rise started 356 ± 51 sec after the addition of thimerosal, with an average peak concentration of 496 ± 75 nM and a mean duration of 85 ± 8 sec. Subsequent rises in the [Ca2+]i were generated at intervals of 351 ± 30 sec; the number of Ca2+ spikes induced in the oocytes was between 2 and 5. In all oocytes the initial transient had the highest peak, each subsequent transient tended to have a lower average amplitude . As a result of 20 mM SrCl2 stimulation, the baseline [Ca2+]i increased to 431 ± 52 nM within 85 ± 10 sec. The average duration of the elevated [Ca2+]i was 335 ± 28 sec .Addition of SrClc Figure . The low2+]i rise caused by Na+-free-medium is displayed in Figure +-free-medium induced a gradual [Ca2+]i elevation in 15 out of 28 oocytes. The rise started 135 ± 24 sec after addition of the Na+-free-medium and peaked at a concentration of 639 ± 79 nM. The average duration of the [Ca2+]i rise induced by the Na+-free-medium was 1,470 ± 242 sec.The profile of the [Ca2+]i in mature cat oocytes. Next, in preliminary experiments the different stimuli were evaluated for their ability to induce parthenogenetic development and based on the outcome of these experiments four different treatments were finally selected and compared. The treatments selected for further evaluation were the following: 1) electroporation using two DC pulses of 1.0 kV/cm for 20 μsec each; 2) 15 min incubation in 200 μM thimerosal followed by a culture in 8 mM DTT for 30 min; 3) incubation in the presence of 20 mM SrCl2 in Ca2+/Mg2+-free medium for 4 h, and 4) a treatment in Na+-free medium for 45 min. Each treatment was applied with or without subsequent incubation in the presence of 10 μg/ml cycloheximide and 7.5 μg/ml cytochalasin B for 4 h.The fluorescence recordings indicated that all the artificial stimuli tested were able to generate an elevation in the i elevations in occytes of many species. According to the literature, the parameters of ionomycin treatment used to activate oocytes of various species vary significantly: concentrations ranging from 5 μM to 5 mM i changes caused irreversible damages and were lethal to the oocytes.Ethanol was reported to activate oocytes of various species. In Xenopus it was demonstrated to stimulate the production of inositol 1,4,5-trisphosphate . During activation, MPF activity has to drop in order to allow release from the metaphase II arrest [2+ elevation-inducing stimulus tested and the improvement was significant in several cases. Although the incubation did not increase the total cell number of the developing blastocysts, the results indicate that these inhibitors are beneficial to increase the efficiency of oocyte activation in the cat.The results obtained also showed that incubation of cat oocytes in cycloheximide and cytochalasin B after an induced CaI arrest . CycloheI arrest ,48. In oThe objective of the oocyte activation experiments was to develop potential methods to be used during nuclear transfer for the generation of useful disease models. In the domestic cat, somatic cell nuclear transfer not only provides the opportunity to generate genetically identical animals for research purposes, it also has the potential to facilitate preservation of rare and valuable felid populations, including cat research models and possibly endangered felid species . In addiFor nuclear transfer, fibroblast cells from two animals affected by different metabolic defects were collected and used as nuclear donors. The defects included AMD and MPS I, two lysosomal storage diseases that have been extensively characterized in cats. They are homologous to human inborn errors, involve single gene mutations and inherited as autosomal recessive traits ,50. Thes2+ releasing proteins in cat oocytes.Transfer of cloned embryos in which development was stimulated with electroporation followed by cycloheximide and cytochalasin B incubation resulted in 5 females having distinct implantation sites and 4 females with established pregnancies . Despite a total of 8 implantation sites, 5 fetuses and detectable heartbeats in two fetuses, no term development occurred; all fetuses were eventually reabsorbed by day 49 of pregnancy. Our previous research has demonstrated the developmental competence to term of MPS-affected and MPS-carrier IVF embryos following transfer into our primary recipient type . Pregnan2+ release in cat oocytes for the stimulation of embryo development. In addition, the results support previous findings that electroporation is an effective way for the activation of oocytes reconstructed by nuclear transfer. Based on in vitro data, the combined thimerosal/DTT activation may also be suitable for cat oocyte activation but this needs further verification from additional studies.This study described a number of novel methods to induce CaThe authors declare that they have no competing interests.CW carried out in vitro oocyte maturation, fluorescence measurements, parthenogenetic oocyte activation, performed statistical analysis and drafted the manuscript. WFS conceived of the study, participated in its design and performed oocyte collections and embryo transfers. JRH participated in oocyte collections and embryo transfers. KL performed nuclear transfer. ZM participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.