Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes.There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing K2+ uptake. Moreover, pretreatment of cells with the mitochondrial Ca2+ uptake blocker, Ru-360 (10 μM), markedly suppressed the effect of acetate on contraction.Acute exposure of myocytes to 10 mM sodium acetate caused a marked, but transient, decrease in systolic sarcomere shortening (1.49 ± 0.20% vs. 5.58 ± 0.49% in control), accompanied by a significant increase in diastolic sarcomere length (1.81 ± 0.01 μm vs. 1.77 ± 0.01 μm in control), with a near linear dose response in the 1–10 mM range. Unlike palmitate, acetate caused no change in action potential duration; however, acetate markedly increased mitochondrial Ca2+ uptake in isolated mitochondria. Here we show that this effect of acetate appears to extend to isolated cardiac myocytes where it transiently modulates cell contraction.Lehninger and others have previously demonstrated that the anions of weak aliphatic acids such as acetate stimulate Ca Lipids, carbohydrates, ketone bodies, and amino acids can all support some degree of ATP synthesis in the heart. The loss of metabolic flexibility in the diseased heart may lead to abnormal contractile function. For example, we recently demonstrated that mice overexpressing fatty acid transport protein (FATP4) in the heart have impaired diastolic function . Similarotential .In a continued effort to understand the relationship of cardiac metabolism and cell function, we set out to test the effects of acetate on contraction. Several studies have examined the effect of acetate on cardiac contraction with equivocal results. In isolated cells, acetate tends to increase cell shortening after 10 minutes of exposure . In isol2 output in humans [2+ and osmotic swelling [Although it is typically found in low concentrations (~0.2 mM) in non-ruminant mammals , acetaten humans . Acetaten humans  and the n humans . Metaboln humans . In somen humans , althougn humans . Acetateswelling -15.2+ uptake with Ru-360, indicating that acetate causes effects on cardiac contraction by increasing Ca2+ uptake into the mitochondria.In this context, we investigated the effects of acetate on cardiac contractility in isolated cardiac myocytes. We tested the effects of acetate throughout a 10 minute exposure. The results demonstrate that acetate inhibits systolic function and increases cell relaxation within 2 minutes of exposure. The decrease in systolic function is transient, however, and contraction amplitude is restored within 10 minutes. These effects are independent of changes in action potential duration; however, the effects of acetate were inhibited by blockade of mitochondrial CaGuide for the Care and Use of Laboratory Animals . Protocols were approved by the USUHS Institutional Animal Care and Use Committee.All animals used in this study were male, aged 2–4 months, C57Bl6/J. All procedures complied with the standards for the care and use of animal subjects as stated in the 2PO4, 0.16; glucose, 10; MgCl2, 0.5; CaCl2, 1.8; HEPES, 5.0; NaHCO3, 3.0; pH 7.35 - 7.4.Normal Tyrode Soution (NT): NaCl, 137; KCl, 5.4; NaH2PO4, 1.2; glucose, 11.6; MgCl2, 3.7; HEPES, 20; L-glutamine, 2.0; NaHCO3, 4.4; KH2PO4, 1.5; 1X essential vitamins; 1X amino acids; pH 7.3-7.4.Wittenberg Isolation Medium (WIM): NaCl, 116; KCl, 5.3; NaH2+-free WIM solution for 5 minutes followed by perfusion with a digestion solution containing 100 μM CaCl2 and 1 mg/mL collagenase . Left ventricular cells were gently dispersed by manual trituration using a pasteur pipette in WIM solution supplemented with bovine serum albumin (1 mg/mL) and 500 μM CaCl2. Cells were washed twice with WIM solution and twice with HEPES-buffered M199 solution and stored at room temperature. Cells were used for experiments within 12 hours of isolation in all cases.Isolation of ventricular myocytes was performed as described previously ,2,16. BrUnloaded sarcomere shortening was measured in freshly isolated ventricular myocytes, as described previously ,2. Brief2 to quench cytoplasmic fluorescence as has been previously reported [2 and transferred to the microscope stage. Cell images were obtained every 10 seconds for 10 minutes. Data were plotted as background-subtracted Rhod-2-AM fluorescence normalized to mean signal during the first 6 images recorded prior to addition of acetate.Freshly isolated ventricular myocytes were plated on laminin-coated (100 μg/mL) Mat-Tek dishes for fluorescence imaging. Cells were loaded with Rhod-2-AM (5 μM) for 30 minutes in normal Tyrode solution containing probenecid (500 μM) to inhibit dye export and 200 μM MnClreported ,18. Afte90) was determined at 90% repolarization and referenced to the peak of the action potential.Action potentials were measured in freshly isolated ventricular myocytes using whole cell current clamp. Briefly, following acquisition of the whole cell mode, cell holding potential was adjusted to −70 mV using current injection. Action potentials were evoked by suprathreshold stimuli  delivered at 1 Hz. Action potentials were recorded continuously during 5 minute exposure to acetate followed by 5 minutes washout. Average traces constructed from 25 consecutive action potentials during control, acetate exposure (2 minutes) and washout (5 minutes) were analyzed. Action potential duration  results are presented as mean ± SEM (standard error of the mean). Statistical analysis was performed with built-in functions of Excel or with the Sigma XL software add-in. Statistical tests and p-values are denoted in the figure legend and text where appropriate.50 = 5.6 mM and Hill coefficient of 1.4. There was no apparent concentration dependence for the effect on diastolic sarcomere length.To test whether the short chain fatty acid, acetate, exerts negative inotropic effects, we continuously monitored average sarcomere length in isolated mouse cardiomyocytes acutely exposed to Tyrode solution containing 10 mM sodium acetate. Three major consequences of acetate exposure were observed. Figure + currents encoded by Kv2.1 and Kv1.5, with no effect on IK1 [90)  was unaffected by acetate , suggesting that a different molecular mechanism underlies the inotropic effects of acetate.We previously demonstrated that acute exposure to long chain fatty acids shortens the action potential duration principally by increasing outward voltage-dependent K) Figure . In cont2+ uptake in isolated liver and heart mitochondria [2+ uptake, leaving less Ca2+ available to activate the myofilaments. To test this hypothesis, we first monitored mitochondrial Ca2+ during exposure to acetate using the fluorescent Ca2+ indicator, Rhod-2 AM (5 μM) in the presence of 200 μM MnCl2 to quench cytosolic fluorescence [2+ uptake inhibitor, Ru-360 (n = 9 and 8 respectively). This concentration of Ru-360 was chosen as it has previously been shown to have no effect on transmembrane Ca2+ fluxes other than mitochondrial uptake [2+ uptake.It has been shown previously that acetate increases Cachondria -15. In trescence ,18. Figul uptake ,20. The 2+ is responsible for the reduction of myocyte fractional shortening, we reasoned that inhibiting mitochondrial Ca2+ uptake would reduce or abolish the effect of acetate on myocyte contraction. To test this hypothesis, we incubated cells for 30 minutes with 10 μM Ru-360, and then measured the effects of acetate on sarcomere shortening. Figure 2+ uptake, leading to reduced availability of Ca2+ for myofilament activation.If the acetate-dependent increase in mitochondrial Ca2+ uptake appears to be the seminal event leading to reduced inotropy. However, in the sustained presence of acetate, fractional shortening recovers to control levels by 10 minutes. This suggests that during the early phase of acetate exposure, cytosolic Ca2+ released from the SR bypasses the myofilaments and enters the mitochondria. We hypothesized that the recovery of contraction during the latter phase of acetate exposure reflects refilling of the SR to replace the Ca2+ lost to the mitochondria. Previous studies have utilized brief application of 1 μM thapsigargin to partially inhibit SERCA activity [2+.The acetate-induced stimulation of mitochondrial Caactivity . We therThere have been previous studies that have examined the effects of acetate on cardiovascular function and energetics in different contexts. Consistent with the results of the present study, acetate is reported to exhibit negative inotropic effects which appear to be transient ,5. It sh2+ accumulation. Moreover, the effect of acetate was markedly attenuated by pretreatment of myocytes with Ru-360, an inhibitor of mitochondrial Ca2+ uptake. Ru-360 is commonly used to inhibit mitochondrial Ca2+ uptake in a number of studies due to its ability to permeate cell membranes and it has been previously shown to have little if any effect on other transmembrane Ca2+ transport processes, including SR Ca2+ uptake and release, L-type Ca2+ channel and sodium calcium exchange function [2+ and that this decreases, at least transiently, the amount of Ca2+ available for contraction of unloaded cardiomyocytes.The present data provide evidence for a calcium-dependent mechanism linking acetate exposure with contractile function. Unlike the long chain fatty acid palmitate , the shofunction . Our dat2+ is sequestered in the mitochondria in the presence of acetate, additional Ca2+ enters the cell and refills the SR Ca2+ stores. We found that the acute decrease in contraction amplitude and increase in diastolic sarcomere length were unaffected by pretreatment with thapsigargin to attenuate SERCA activity. Interestingly, however, the recovery of depressed myocyte contractility during sustained acetate exposure and the abrupt increase in contraction amplitude due to acetate removal were no longer observed when cells were pretreated with thapsigargin. One possible explanation of this phenomenon is that inhibition of SR Ca2+ store replenishment prevents the restoration of fractional shortening.Interestingly, the effects of acetate on cell contraction were transient, with complete recovery of fractional shortening within 10 minutes of continuous exposure. This result could imply that as Ca2+ uptake and swelling [2+ uptake into the mitochondrial matrix. Similar observations demonstrate that acetate or phosphate increase the rate of mitochondrial Ca2+ uptake in a concentration dependent manner [2+ uptake in heart mitochondria [2+ entry into the mitochondria. In addition to the mitochondrial Ca2+ uniporter [2+/H+ exchanger (Letm1) [2+. Nevertheless, here we present pharmacological evidence showing that acetate is apparently coupled with an increase in mitochondrial Ca2+ in isolated cardiac myocytes and it is plausible to assume that this decreases the amount of Ca2+ available for contraction.Mitochondrial uptake of acetate has been shown to be coupled with increases in Caswelling . It was t manner  and thatchondria . The datniporter , the mitniporter , or Ca2+ (Letm1)  could pl2+, the mitochondria remain swollen and the increased diastolic sarcomere length is maintained. Given the structural constraints, it seems possible that swelling is physically limited possibly explaining the absence of a clear concentration dependence.In isolated mitochondria, acetate also caused osmotic swelling . Given t2+. However, this is very unlikely since substrate availability governs only the pathways used to generate ATP, while workload or demand is the principal determinant of respiration [2+ uptake. Rather, we consider the possibility that mitochondrial uptake of the acetate anion is electrically balanced by the uptake of Ca2+ in line with the conclusion of others based on experiments with isolated mitochondria [The observed delay from acetate application to initial effects on contraction is similar to the time course for conversion of acetate to acetyl CoA observed by Randle . Acetatepiration ,28. In uchondria -15.2+ content [2+ and that pretreatment of cells with Ru-360 markedly attenuates the effects of acetate argues that an acute change in mitochondrial Ca2+ uptake, rather than cytoplasmic acidification, is the predominant mechanism underlying the effects of acetate on contraction. Moreover, it is tempting to predict that other anions of weak organic acids  may cause similar changes.Short chain fatty acids like acetate and butyrate may also cause changes in intracellular pH with effects on contraction and SR Ca content ,30. The 2+ uptake has been proposed as an important player in regulating cardiac energetics, reactive oxygen species generation and supply–demand matching [2+ overload is also associated with the activation of cell death pathways [2+ loading is required in order to achieve proper regulation of metabolism, but avoid overloading and cell death. In ischemia-reperfusion experiments, excessive Ca2+ uptake is associated with a poor outcome, and treatment with Ru-360 to inhibit mitochondrial Ca2+ uptake is beneficial [2+ resulting from increases in cytosolic Na+ concentrations and increased mitochondrial sodium-calcium exchange activity and blocking mitochondrial Ca2+ export  is beneficial [In the heart, mitochondrial Camatching ,32; howepathways . Thus a neficial . Acetateneficial -37. Convneficial . The dat2+ uptake. This finding is consistent with the results of Lehninger and others using isolated liver and heart mitochondria, where it has been shown that acetate causes an increase in mitochondrial Ca2+ uptake and osmotic swelling [2+ homeostasis in the intact heart in vivo.In summary, we have shown that acetate causes an acute but transient reduction in contractile function in isolated cardiac myocytes. Mechanistically, the transient negative inotropic effect appears to result from an acetate-dependent increase in mitochondrial Caswelling -15. Here


We use fluorimetry-based fluorescence resonance energy transfer (FRET) to monitor changes in STIM1 oligomerization in COS7 cells transfected with STIM1 constructs containing selected truncations, deletions, and point mutations, and labeled with donor and acceptor fluorescent proteins at either the luminal  or the cytoplasmic  ends. Our results with sequential truncations of STIM1 from the C-terminus support previous evidence that the CRAC activation domain  is an oligomer-promoting segment of STIM1, and they show that truncation just after CAD/SOAR (1–448) causes significantly elevated basal cytoplasmic Ca2+ and spontaneous STIM1 clustering. We find that a 14 amino acid sequence just C-terminal of CAD/SOAR (449–462) prevents spontaneous clustering and activation of STIM1 in COS7 cells. In response to store depletion, C-terminally labeled STIM1 without CAD/SOAR clusters together with CAD/SOAR-containing STIM1 constructs. However, these donor-acceptor pairs do not undergo a stimulated increase in FRET, exhibiting instead a decrease in FRET consistent with a stimulated conformational extension in full length STIM1. We find that the 14 amino acid sequence plays a regulatory role in this process. Overall, our FRET results provide evidence in live cells that Ca2+ store depletion stimulates a conformational extension in the cytoplasmic segment of STIM1 that accompanies its oligomerization.Oligomerization of the Ca Our results provide direct evidence for an activating conformational extension that occurs in wt STIM1, as well as in STIM1 truncated at residue 462 (STIM1(1–462)) in the cytoplasmic segment. In contrast, STIM1 truncated just after the CAD/SOAR region (STIM1(1–448)) is not similarly regulated, exhibiting spontaneous clustering and Orai1 activation as measured by elevated cytoplasmic CaThe primers used to generate DNA constructs are listed in 5 cells per 35 mm dish) were transiently transfected with the indicated constructs (0.5 µg of DNA per 35 mm dish) using Lipofectamine 2000 for 24 h as described previously [2, 0.7 mM Mg(SO4)2, 10 mM glucose, 10 mM Hepes, pH 7.4, using a Zeiss LSM 710 scanning confocal microscope and a x63/1.4 oil immersion objective. Fluorimetry-based measurements of FRET were performed at 37 °C as previously described [COS-7 cells . COS-7 cells previously transfected with R-GECO1, together with untagged Orai1 and STIM1 plasmid DNA, were harvested and suspended in modified Krebs-Ringer buffer. Cells (~106/ml) were stirred in an acrylic cuvette at 37°C in Ca2+-free Krebs-Ringer buffer supplemented with 100 µM EGTA, and the time course of R-GECO1 fluorescence  was monitored.Cytoplasmic CaCalculations were performed with MS Excel and p values were calculated with two-tailed and unpaired T-test distribution functions. FRET data points were normalized based on the same day control experiments with time “0” taken one minute before activation of store depletion with ATP/TG treatment. Steady-state values of stimulated FRET responses were taken 5 min after initiation of store depletion. Positive changes in FRET correspond to increases in acceptor/donor fluorescence and negative changes in FRET correspond to decreases in this ratio.2+ store depletion, similarly to wt STIM1-AcGFP. However, STIM1(1–448)-AcGFP at low expression levels forms spontaneous clusters in unstimulated COS7 cells in the absence of Orai1, and larger clusters that are observed after stimulation by ATP + TG are similarly located. A similar result was recently described with a related construct [Our initial experiments evaluated the spatial distributions of C-terminally truncated forms of human STIM1 transfected into COS7 cells, which express little endogenous STIM1 and no Orai1 . We componstruct . In cont2+ entry. We tested this by coexpressing YFP-STIM1 constructs together with untagged Orai1 and the cytoplasmic Ca2+ reporter, R-GECO1. In these experiments, COS7 cells were harvested in modified Krebs-Ringer buffer without Ca2+, then 2 mM Ca2+ was added, followed by 200 nM thapsigargin. As summarized in 2+ spike that declines over 5 min to an elevated basal Ca2+ level compared to YFP-STIM1(1–462) or YFP-STIM1(wt) co-expressed with Orai1, which exhibit intermediate basal Ca2+ levels relative to YFP-STIM1(1–343) or endogenous STIM1 together with Orai1 , and STIM1(1–462), co-expressed with Orai1. For these stimulated responses, the sustained Ca2+ level with YFP-STIM1(1–448) is intermediate between YFP-STIM1 (wt) and YFP-STIM1(1–462). As expected, YFP-STIM1(1–343) co-expressed with Orai1 exhibits a transient stimulated response that is not significantly different from that for endogenous STIM1 with Orai1 (Spontaneous formation of clusters by STIM1(1–448) suggests that this truncated construct might spontaneously activate Orai1 to enable Cath Orai1 . After tth Orai1 . Togetheth Orai1 . Togethe2+-free buffer. Addition of millimolar Ca2+ results in an increase in donor fluorescence (green) and a temporally corresponding decrease in the ratio of sensitized acceptor fluorescence to donor fluorescence (red). These changes are consistent with a decrease in FRET due to increasing separation of STIM1 proteins that occurs with reversal of oligomerization as Ca2+ refills the initially depleted ER stores. Subsequent stimulation by ATP + TG to fully deplete Ca2+ stores causes a time-dependent increase in FRET corresponding to oligomerization as previously described [2+ to the buffer and subsequent stimulation  can be explained by the absence of CAD/SOAR in these constructs.We next compared the stimulated changes in FRET for both N-terminally and C-terminally labeled STIM1 proteins with the truncations described above  to the F+ uptake . Little These results establish our capacity to measure stimulated oligomerization of C-terminally labeled STIM1 and to detect differences in this oligomerization that depend on the length of the cytoplasmic segment. This capacity is important for the subsequent experiments which show that these C-terminally labeled STIM1 constructs can also sense the conformational extension of the cytoplasmic segment occurs upon store depletion.2+-free medium; the clusters disassemble after addition of Ca2+ and form again upon stimulation by ATP + TG -mApple, indicating an increase in the average nanoscale distance between the asymmetrically placed donor and acceptor labels that occurs upon store depletion. This FRET decrease is shown in We next investigated the roles of CAD/SOAR 342–448;  and CC1αATP + TG . These oIn the same experiment we also evaluated the stimulated change in FRET between wt STIM1-AcGFP and STIM1 (del344–448)-mApple, in which CAD/SOAR is deleted but CC1α3 is retained. This pair also exhibits a stimulated decrease in FRET -mApple as a reference, and we compared sequential truncations of donor-labeled STIM1 . As summPrevious studies showed that the CRAC-activating mutation L251S causes a conformational change in the cytoplasmic segment of STIM1 to a more extended structure , 10. We 2+ from ER stores. We further identify a 14 amino acid sequence (449–462) adjacent to the C-terminal end of CAD/SOAR that is important for maintaining STIM1 in an inactive conformation in the absence of store depletion. Our initial experiments showed that truncation of the cytoplasmic segment of STIM1 just after CAD/SOAR (STIM1(1–448)) results in spontaneous, microscopically visible clustering of this construct and elevated basal cytosolic Ca2+ levels in resting COS7 cells. C-terminal addition of the 14 amino acids just after CAD/SOAR (STIM1(1–462)) is sufficient to prevent spontaneous clustering in resting cells and elevated basal cytosolic Ca2+, such that basal Ca2+ with STIM1(1–462) is similar to that with wt STIM1 -mApple in which the CC1α3 and CAD/SOAR sequences are deleted, and we observed a decrease in FRET caused by store depletion, despite microscopically visible co-clustering of these asymmetric constructs under these conditions . We foun2+ depletion from ER stores (Our results are consistent with a model in which the basic sequence in CAD/SOAR 382–386) that is critical for activation of SOCE 2–386 tha,17 is seUsing synthetic transmembrane STIM1 constructs, Fahrner et al. recently provided evidence that CC1α1 (238–271) association with CC3 (400–450) contributes to the inactive state of STIM1 in the absence of store depletion . They prIn previous models, the C-terminal polybasic sequence is commonly depicted in contact with the ER membrane in unstimulated cells ,32,33. HOur results further indicate a critical role for the 14 amino acid sequence (449–462) just C-terminal to CAD/SOAR in mediating the inactive conformation of STIM1 prior to stimulation, and thereby preventing spontaneous coupling to Orai1. The absence of this sequence in the CAD/SOAR-deleted acceptor construct (STIM(del302–462)-mApple) substantially reduces the magnitude of the stimulated decrease in FRET from wt STIM1-AcGFP . This reC. elegans CC1α3/SOAR [2+ binding to an inactivation domain (475–483) [2+ that is observed with STIM1(1–448) (trans with CC1α3 (308–337) in the unstimulated STIM1 dimer, this is not likely to participate in the regulation of a conformational extension. This same (449–462) sequence in the donor is clearly important for maintaining it in an inactive state prior to stimulated store depletion, even when the acceptor construct lacks CC1α3  –36. Howe1(1–448) . Elimina1(1–448) , and its1(1–448) . Althougks CC1α3 . Further2+ stores. Our measurements of increased FRET between both C- and N-terminally labeled symmetric STIM1 constructs are consistent with oligomerization that accompanies this stimulation, and a conformational extension is revealed by the stimulated decrease in FRET measured between a STIM1 that is donor-labeled at its C-terminus and CAD/SOAR-deleted STIM1 that is acceptor-labeled at its C-terminus. Furthermore, our results identify a 14 amino acid sequence just C-terminal to CAD/SOAR that is necessary for maintaining STIM1 in an inactive conformation until store depletion activates both conformation extension and oligomerization leading to exposure of CAD/SOAR for functional coupling to Orai1.In summary, our results with live cells substantially extend previous evidence, both with isolated fragments in solution ,10 and wsupplemental


Therefore, we aimed to study the effect of 1,2-DCB on the Ca2+-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca2+ concentration.1,2-Dichlorobenzene  is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca2+-ionophore. At higher concentrations (0.25–0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca2+ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca2+ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca2+ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-32P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C.Concentrations of 0.05–0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca2P · Ca2 state to E2 and Pi, which explains the uncoupling of the ATP hydrolysis from the Ca2+ transport.The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism – E The Ca2+ ionophore A23187 , EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetra acetic acid), Mops (4-morpholinepropanesulfonic acid), liquid scintillation cocktail (Sigma-Fluor S-4023), ATP disodium salt, LaCl3, K-oxalate, benzene, 1,2-DCB was obtained from Sigma, Co. . CaCl2 and other chemicals ware purchased from J.T. Baker (México). The Ca2+ ionophore A23187 was dissolved in ethanol. Benzene and 1,2-DCB were dissolved in methanol. Ethanol and methanol never reached a concentration higher than 1 % (v/v) in the reaction media after the addition of 1,2-DCB or A23187.glucose as a marker. This approach was used to test the Ca2+ high-affinity conformational state of SERCA (E1Ca2 stage). The effect of 1,2-DCB on E1Ca2 state was evaluated at 25 °C for 5 min in a Ca2+-saturating medium containing 20 mM Mops, pH 7.0, 5 mM MgCl2, 80 mM KCl, 0.1 mM EGTA, 0.105 mM [45Ca]CaCl2 (~5000 cpm/nmol) (10 μM free Ca2+), 1 mM [3H]glucose , and 0.2 mg/ml of SR protein. Subsequently, we added different concentrations of 1,2-DCB. In order to test the effect of 1,2-DCB on E2 stage we used a Ca2+-free media containing 20 mM Mops, pH 7.0, 5 mM MgCl2, 80 mM KCl, 0.1 mM EGTA, 1 mM [3H]glucose , and 0.2 mg/ml of SR protein, and different concentrations of 1,2-DCB. The incubation was performed at 25 °C for 5 min. Later, the reaction for E2 stage was supplemented by adding a buffer containing 20 mM MOPS, pH 7.0, 5 mM MgCl2, 80 mM KCl, 0.1 mM EGTA, 3.15 mM [45Ca]CaCl2 (~5000 cpm/nmol) and 1 mM [3H]glucose  with a final free-Ca2+ concentration of 10 μM. Following, the assay was prolonged for 1 min at 25 °C. Samples of 1 ml (0.2 mg of protein) were filtered under vacuum in 0.45 μm nitrocellulose filters . Any further washing to determine 45Ca and 3H associated with the filters and the amount of radioactivity in the filters was determined by scintillation spectroscopy. Specific binding of Ca2+ to the SERCA was calculated by subtracting unbound Ca2+ retained by the filter.The binding of Ca32P]ATP as a substrate and starting from different incubation conditions: E1Ca2 -DCB, E2 -DCB and E2[γ-32P]ATP-DCB in similar manner as we described in previous work ATP . When the enzyme (0.2 mg/ml) was in nominally Ca2+-free medium (E2 -DCB stage), the phosphorylating medium contained CaCl2 to give 10 μM free Ca2+ after mixing. In order to determine if 1,2-DCB was directly affecting the E2ATP in a Ca2+ free medium, the formation of the EP stage was started when Ca2+ (to give 10 μM free) was added into the reaction medium. Phosphorylation time was 2 s when the enzyme turnover was studied at 0 °C, and 5 s when studied at 25 °C. The reaction was started under continuous vortexing and stopped by adding 1 ml of ice-cold 250 mM perchloric acid and 2 mM sodium phosphate. Stopped reactions were incubated on ice for 5 min before filtration through 0.45 μm nitrocellulose filters . SR vesicles retained by the filters were washed 5 times with ice-cold 250 mM perchloric acid and 2 mM sodium phosphate. Finally, the amount of radioactivity in the filters was determined by scintillation spectroscopy. These results are expressed as nmol EP/milligram of protein.Maximal levels of EP (phosphorylated enzyme intermediate) were determined using [γ-ous work , 26. Thep < 0.05 determined by t-student’s paired t-test.Statistical and kinetic analysis was performed using Sigma Plot 11 software. Unless otherwise stated, the experiment values are represented as the mean of at least three independent experiments, each performed in triplicate +/− average SE, with statistical significance of 2+-ATPase assay where SERCA hydrolyzed ATP to ADP and Pi as described before. In this experiment we incubated SR vesicles with A23187, a Ca2+ ionophore, which is used to generate leaky vesicles (absence of gradient) in order to avoid saturation of SR vesicles with Ca2+, which would otherwise stop Ca2+-ATPase activity. The dose-response curve revealed a biphasic effect of 1,2-DCB on the ATP hydrolytic activity of SERCA: while lower concentrations of the compound highly stimulated ATP hydrolysis, 1,2-DCB concentrations higher than 0.3 mM inhibited SERCA hydrolytic activity . To determine the importance of the Cl atoms on the effect of the compound on SERCA hydrolytic activity, we used the benzene molecule, which is the same ring base structure, but depleted of Cl atoms . In order to avoid Ca2+ leakage caused by the Ca2+ ionophore (A23187), we used K-oxalate in both Ca2+-transport and hydrolytic activity experiments in order to make them comparable to Ca2+ transport. The dose-response curves showed that 1,2-DCB did not stimulate Ca2+ uptake mediated by SERCA, but in contrast, Ca2+ uptake was inhibited by DCB concentrations above 0.5 mM until achieving full inhibition at 1 mM  therefore these experimental conditions did not allow us to compare the Ca2+ uptake assay with the previously conducted experiments of hydrolytic activity. The approach employed for Ca2+ uptake measurement is less sensitive than the one using Ca2+ ionophore and reduces enzyme activity by ~1 fold, thus showing less effect of 1,2-DCB , which indicates that the enzyme activity ratio was uncoupled, as opposed to normal physiological conditions during which the enzyme maintains a coupled ratio of 2Ca2+ for each mol of ATP hydrolyzed to ADP and Pi , thus demonstrating that 1,2-DCB did not disrupt membrane vesicles. In addition, we performed a positive control with a Ca2+ ionophore (A23187) knowing that it produces leaky vesicles and therefore does not allow Ca2+ accumulation . This demonstrates that, at least in short time exposure, DCB does not disrupt the SR membrane integrity, which indicates that the effect of 1,2-DCB on SERCA hydrolytic activity was due to an effect on SERCA functionality.1,2-DCB is a typical hydrophobic molecule and as such, it has affinity for hydrophobic environments such as biological membranes. For this reason, we used our model of study to investigate the possibility of 1,2-DCB targeting the SR membranes. First, we conducted a set of experiments using a high-dose of 1,2-DCB in reactions having 10-fold different amounts of SR vesicles . In these experiments we tested hydrolysis of ATP mediated by SERCA and we found that the inhibitory effect of 1,2-DCB was significantly reduced with high amounts (5–10 folds higher) of SR vesicles , which strongly suggested that 1,2-DCB molecules were being diluted into the SR membranes Fig. . These r1P stage of SERCA , the EP levels were drastically reduced: E1Ca2 with 0.05 mM of 1,2-DCB barely reached 13.04 % of accumulated EP, with 0.1 and 0.2 mM of 1,2-DCB further reducing EP levels to 6.08 and 5.65 %, respectively and 0.5 mM of the compound completely abolishing EP accumulation. In the case of E2, the EP levels were similarly affected: 0.05 mM of 1,2-DCB decreased EP to 17.5 %, with 0.1 mM further reducing it to 3.0 %, and the remaining concentrations showed no EP at all , Ca2+ reacts with oxalate to form calcium oxalate thus precipitating inside of the intact vesicle as crystals [2+-ionophore acts as a freely mobile carrier that transports Ca2+ across the membrane, and it is possible that it is acting simultaneously with the 1,2-DCB, stimulating the pump due to increased membrane permeability and absence of gradient [2+-ionophore than when measured with K-oxalate, where the vesicle interior is not gradient-free. We also found that the Ca2+/Pi ratio was partially uncoupled at 1,2-DCB concentrations between 0.05 and 0.5 mM, and fully uncoupled at 1 mM. This phenomenon of uncoupled Ca2+-ATPase with a lower Ca2+/Pi ratio has been previously described in terms of the increased thermogenic activity that occurs when using intact vesicles [2+ saturation, ~82 % of the hydrolytic activity is uncoupled from the Ca2+ transport, thus decreasing the Ca2+/Pi ratio.We also addressed the possibility of 1,2-DCB affecting the activity of binding/translocation of Cacrystals , which agradient , 56, 57.vesicles –15, wherPrevious reports showed that non-polar organic solvents facilitate the incorporation of hydrophobic compounds to cell membranes, and that enzymatic stimulation or inhibition is related to both solvent and membrane protein quantity , 49. In 2+ uptake assay employed in this study measured accumulated Ca2+ inside the SR vesicles [2+, this may be reflected as an inhibition on the accumulated Ca2+ due to a loss of Ca2+ caused by vesicle damage. Molecules such as 1,2-DCB can target SR membranes affecting the filling time of SR vesicles by the SERCA pump. For example, hexachlorocyclohexane, a hydrophobic compound, was shown to affect the activity of membrane proteins by disrupting the lipid bilayer [2+ using the active transport of SERCA, followed by the addition of a high concentration of DCB (1 mM) 5 s before stopping the reaction. In agreement with our previous findings, 1,2-DCB was unable to decrease the levels of accumulated Ca2+ when compared to controls (without DCB)  alone or with 1 mM 1,2-DCB, which did not allow accumulation of Ca2+ inside the SR vesicles . At 0.05 mM DCB, ~30–40 % of accumulated EP can be observed at both temperatures, indicating that ATP could be protecting the enzyme from inhibition. However, higher DCB concentrations (0.1 mM and subsequent) caused a drastic decrease in EP levels (Fig. 1P to E2P transition include detergents and the microviscosity of the lipid environment of the ATPase [1P to E2P isomerization is rapidly affected at high KCl concentrations [In order to get more insights into the mechanism by which 1,2-DCB caused uncoupling of the SERCA activity, we studied the EE1 stage , 63. Ourterature , 64, 65.ntration , 67 Our position , in othe at 0 °C . Howevere ATPase . Temperatrations . Temperatrations .2+ transport and EP stage formation due to uncoupled enzyme activity, even without affecting the Ca2+ bound to the enzyme. Finally, to the best of our knowledge, this is the first study designed to study the effect of 1,2-DCB, a chemical that is widely found in the environment, on the functionality of SERCA proteins, and thus the data presented here not only provide insights into the molecular mechanisms of the enzyme’s catalytic cycle, but on the possible toxic effects caused by 1,2-DCB.Our data demonstrates that 1,2-DCB affects the ATP hydrolytic activity of SERCA, causing both stimulation and inhibition of ATP hydrolysis. This dual effect caused by 1,2-DCB fits it into a common pattern displayed by several other hydrophobic compounds. We also found that 1,2-DCB partially inhibited Ca


Ionic solutions with lower K+/Ca2+ ratios induced an increased sap flow rate in stems of Olea europaea L. and Laurus nobilis L. The different ascent rates of ionic solutions depending on K+/Ca2+ ratios at a fixed total concentration increases our understanding of ion-responsiveness in plants from a physicochemical standpoint. Based on these results, effective structural changes in the pit membrane were observed using varying ionic ratios of K+/Ca2+. The formation of electrostatically induced hydrodynamic layers and the ion-responsiveness of hydrogel structures based on Hofmeister series increase our understanding of the mechanism of ion-mediated sap flow control in plants.Sap water is distributed and utilized through xylem conduits, which are vascular networks of inert pipes important for plant survival. Interestingly, plants can actively regulate water transport using ion-mediated responses and adapt to environmental changes. However, ionic effects on active water transport in vascular plants remain unclear. In this report, the interactive ionic effects on sap transport were systematically investigated for the first time by visualizing the uptake process of ionic solutions of different ion compositions (K Plants transport sap water through specific porous structures and xylem conduits, which are composed of dead cells. Since the geometrical features of xylem conduits are believed to determine hydraulic conductance, xylem water transport is considered a passive process. Nonetheless, plants can pull water above 100 m in height without any mechanical pumps. They have also survived over hundreds or thousands of years by regulating water transport against unfavorable situations such as severe freezing weather and prolonged drought. Interestingly, water transport in plants can be actively modulated through the xylem (a system of inert pipes).Flow modulation responds to the small molecules and ion concentration in, and pH levels of, the sap water, and the ion-mediated response in vascular plants is considered a major factor in sap flow regulation. The flow rate of tap water is known to be faster than distilled water in plant stems +Due to interest in active flow regulation, previous studies have investigated the effect of ions on sap flow control in terms of a specific cation, K+, Ca2+, Mg2+, and Na+ for optimizing water transport under different environmental conditions + to Ca2+. These two ions are the major cations in xylem sap water + facilitates water flow by controlling evaporation rates from stomata via guard cell regulation 2+ by modulating cell wall structure, stomatal aperture and aquaporin stimulation Interestingly, ion-mediated sap flow regulation is a unique process that occurs in dead cells in situ visualization of water transport in plants Synchrotron X-ray and neutron imaging methods allow us to directly monitor ion-responsiveness in the stems of vascular plants in a noninvasive and nondestructive manner. Unlike perfusing solutions, these imaging techniques can be employed to characterize sap flow without exerting an artificial positive pressure, which could induce morphological changes in the plant nanostructure + and Ca2+ on sap flow in olive and laurel stems were examined using synchrotron X-ray and neutron imaging methods. Our experimental results on the interactive ionic effects increase our understanding of structural changes in pit membranes and the physicochemical mechanism of active sap flow regulation in xylem conduits.In this report, to characterize ion-mediated sap flow regulation in vascular plants from a physicochemical standpoint, the interactive ionic effects of KOlea europaea L.) and laurel (Laurel nobilis L.) as test samples because they are known to be highly sensitive to ions in sap water +/Ca2+ ratios, and the uptake process was monitored using synchrotron X-ray and neutron imaging techniques. During the ascent of ionic solutions, the temperature and humidity of environmental air were maintained at 19±1°C and 95±1% relative humidity (RH). During the experiments, the photosynthetic active radiation was 200 µmol m−2 s−1.We selected olive , KEK (High Energy Accelerator Organization), Tsukuba, Japan. Using a white (full-energy spectrum) X-ray beam with an energy of 70 keV, X-ray images with a field of view (FOV) of 1.5×1.5 mm were obtained with a spatial resolution of ∼10 µm. X-ray images were recorded consecutively at 3-min intervals. To minimize radiation damages, a mechanical shutter and attenuating plates were employed to expose the sample plants to attenuated X-ray beam only when we captured X-ray images. Iopamidol solution was mixed with the ionic solutions as an X-ray contrast agent to clearly visualize the uptake process of ionic solutions in plant stems.6LiF/ZnS:Ag scintillator  and a CCD camera attached with a zoom lens . The thermal neutron flux was 5.07×106 neutrons cm−2 s−1 and the effective beam size was approximately 350×450 mm. The FOV of neutron radiography was 11.3×11.7 cm and each image was expressed at 1340×1300 pixels with a spatial resolution of 50 µm. Neutron images were recorded consecutively for several hours and the total image acquisition time for one image was 41.5 s, which consisted of 40-s exposure and 1.5-s readout. Deuterium oxide (D2O), which has physiological properties similar to those of H2O in plants, was employed as a contrast agent in neutron imaging experiments 2O, we observed no wilting and no morphological changes in the test plants. This implies that D2O application as a contrast agent does not perceptible damages to the test plants.The neutron imaging experiments were conducted at the neutron radiography facility (NRF) of HANARO (30 MW), Korea Atomic Energy Research Institute (KAERI), Korea. A thermal neutron beam with a mean energy of 14 meV was collimated and detected using a http://www.xraylab.com). Flat-field correction was adopted to eliminate the CCD dark current noises and the heterogeneity of the beam profile. The corrected image  was selected to quantitatively analyze the X-ray images. The intensity values in the ROI were then averaged and the results in the consecutive images were compared. To show the relative variations in ionic water uptake effectively, normalized intensity  but different K imaging . The ima+/Ca2+ ratios was compared using intensity-based analysis of X-ray images. Normalized intensity .In 2O containing varying K+/Ca2+ ratios in excised laurel stems at 0, 15, 30, and 60 h after dipping in ionic D2O. The degree of neutron transmission was also color scaled as shown in the scale bar on the right of 2O solution uptake and replacing H2O present in the stems. Assuming the stem samples showed the same initial water potential, the neutron images clearly demonstrated that when the K+/Ca2+ ratio was 0.01, the ionic D2O uptake was much faster than at higher K+/Ca2+ ratios of 1 or 100 for a longer time scale in the excised laurel stems. The modulation of water uptake according to K+/Ca2+ observed by neutron imaging for an extended time period (65 h) was similar to the results obtained by synchrotron X-ray imaging for short time durations (50 min). These results suggest that the interactive effect of different K+/Ca2+ ratios at a fixed concentration (50 mM) has a considerable influence on the water uptake process, irrespective of plant species or time scale.To further investigate the uptake of ionic solutions over longer time scales, neutron imaging techniques were employed with laurel stems. Laurel responded to ions more slowly than olive. 2O thickness  was similar to the results obtained from synchrotron X-ray imaging for a short time . The interactive effect of different ionic compositions (K+/Ca2+) at a fixed concentration (50 mM) strongly affected the water uptake process irrespective of plant species and time scale.Temporal characteristic variations in ionic Dolutions . The ion+/Ca2+ ratios were investigated in laurel stems with and without leaves. 2O thickness (2O uptake (see 2O was more significant and faster at the lowest ionic ratio (K+/Ca2+  = 0.01). The uptake rate of ionic solution was in the order of the K+/Ca2+ ratio of 0.01>1>100 for both systems with and without plant leaves. The varying cationic ratios synergistically regulated sap water transport in plants, irrespective of the existence of leaves.To determine whether ion-mediated water flow modulation was independent of that driven by transpiration in leaves, the effects of different Ktake see . On aver+/Ca2+ ratios were then compared quantitatively  +/Ca2+ ratios corresponded to the ratio of porosity to tortuosity (+/Ca2+  = 0.01, compared with that at K+/Ca2+  = 1. Conversely, the ratio +/Ca2+  = 100, compared with that at K+/Ca2+  = 1. These results suggested that the effective porosity of pit membranes decreased sap flow as the K+/Ca2+ ratio increased. Since the water flow through pit membrane plays a major role in determining the overall water transport through xylem conduits The diffusion through pit membranes differed from that through homogeneous solution because it was influenced by the structural features of pores, such as size, shape, and connectivity. The To characterize the mechanism of interactive ionic effects on effective structural changes of pit membranes, we should consider the chemical compositions of pit membranes and the ion-structure interaction. Pit membranes are composed of coextensive cellulose-hemicellulose networks forming a fiber composite material as a primary cell wall structure Taking into account the controversy over the existence of pectins in pit membranes of plants +/Ca2+ ratio decreases (ecreases . This me+ causes shrinking of the hydrogel, while salting-in Ca2+ induces swelling. Thus, a lower K+/Ca2+ ratio is expected to induce swelling in the hydrogel structure, whereas a higher K+/Ca2+ ratio causes relatively less swelling or shrinking (+/Ca2+ ratios. The effects of structural variation in a hydrated gel matrix on the ion-responsive flow regulation in vascular plants can be used to characterize ionic effects.Second, by assuming that pit membranes are composed of a pectin-based hydrogel, the swelling and shrinking phenomena of hydrogels hrinking . Shrinki+/Ca2+ ratio, while a decrease in the K+/Ca2+ ratio accelerated the ascent of ionic solution. This interactive ionic effect is a common type of systematic flow regulation in vascular plants, irrespective of the plant species. The active ion-mediated flow regulation in the stem of a vascular plant may result from structural changes in pit membranes. The effective structural changes mediated by ions can be explained by the formation of electrostatically induced hydrodynamic layers and the ion-responsiveness of hydrogel structure based on Hofmeister series. The interactive ionic effects from a physicochemical standpoint describe the role of ion-responsiveness during water flow regulation in vascular plants.In conclusion, we examined the ion-responsiveness of water transport in olive and laurel stems to increase our understanding of the mechanism of active water flow regulation in two representative vascular plants. Different ion types significantly affected water transport in xylem conduits. Sap flow rate decreased with an increasing K


Cultured neonatal Sprague Dawley rat cardiomyocytes were used to compare the effects of normoxia, total saponins of Panax ginseng (TSPG), ginsenoside Rg1 (Rg1) and SM treatments in rat myocardial cells following I/R. For each of these treatment groups, the mRNA and protein levels of PLB and the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated, in addition to the cytoplasmic Ca2+ concentration [Ca2+]i and the rate of apoptosis. The results indicated that I/R markedly decreased phosphorylated PLB and SERCA expression and that SM was able to mitigate this effect, while TPSG and Rg1 were not. Furthermore, SM appeared to prevent aberrant apoptosis and restore the depleted [Ca2+]i resulting from I/R. The protective efficacy of SM against heart disease following I/R may, therefore, be due in part to its effect on intracellular Ca2+ homeostasis. SM may exert its protective effects by relieving PLB inhibition, and the pharmacodynamic actions of SM appear to be significantly more effective than those of its bioreactive components, TPSG and Rgl.The aim of the present study was to determine whether the myocardial protective function of Shenmai injection (SM) during ischemia/reperfusion (I/R) is attributable to its regulation of intracellular calcium (Ca In its lum (SR) . The pholum (SR) . We ther2+ channels (2+ overload (Panax ginseng (TSPG), i.e. the total quantity of ginsenosides extracted from ginseng, can increase cardiac SR Ca2+ levels, reduce mitochondrial Ca2+ levels and increase mitochondrial calcium pump activity (2+ influx in neurocytes following hypoxia-reoxygenation (2+]i via the regulation of PLB phosphorylation. An aim of the present study was to elucidate the mechanism underlying PLB involvement in each of these processes.Shenmai injection (SM), derived from the shendong drink described in the Zheng Yin Mai Zhi  by Qin Changyu of the Ming dynasty , has beechannels ,19 and rchannels –24. Prevoverload . Treatmeactivity ,23. Furtgenation . It rema2+]i through the relief of PLB inhibition. In addition, a further aim was to determine whether the pharmacodynamic activity of SM injection is superior to that of the pure ginseng saponins compounds.This study aimed to determine whether the myocardial protection from SM injection during I/R is associated with the maintenance of the i measurements were conducted using the Ca2+-sensitive ﬂuorescent probe ﬂuo-4 AM. Cardiomyocytes were incubated in six-well plates with 5 µM ﬂuo-4 AM for 30 min at 37°C. The cells were washed with HBSS three times. Fluorescence levels were measured using ﬂow cytometry, with excitation at 484 nm and emission at 516 nm.After 1 h of reoxygenation, i (P<0.01); however, this increase was suppressed by the SM (P<0.01) and TSPG (P<0.05) treatments. The I/R + Rg1 group exhibited a modest but insignificant decrease in [Ca2+]i compared with the I/R group (P>0.05). The I/R + SM group showed the greatest decrease in [Ca2+]i following I/R treatment (P<0.01) (As shown in (P<0.01) .2+ transport within heart myocardial cells. In accordance with a previous study, the apoptosis rate and [Ca2+]i of cardiomyocytes increased significantly following I/R, indicating that I/R injury successfully occurred (In the present study, the mechanism underlying the myocardial protective effect of SM following I/R was investigated. It was demonstrated that exposure to I/R resulted in significant alterations in a variety of functional indices for cardiac function, including a disruption in Caoccurred .In order to further clarify the effects of SM on myocardial cells, three different drug treatments were employed to determine their comparative efficacies in abating I/R-related complications. Ginsenoside Rg1 is a major bioactive component of TSPG, while TSPG is the primary bioactive component of SM. It has been established that 1 ml SM contains 65±6.68 µg Rg1 and 293.38±40.54 µg TSPG ,31, 5 ml2+-ATPase is responsible for restoring the SR Ca2+ load per excitation-contraction cycle. A reduction in SERCA content is associated with reduced SR Ca2+ loading and elevated [Ca2+]i (2+ uptake, as observed in line-scan and frequency-dependent experiments (2+]i overload in cardiomyocytes following I/R treatment. The present study has demonstrated that SM and TSPG are able to increase SERCA expression and that this improves intracellular Ca2+ cycling. Notably, the numerous changes observed following I/R treatment were not reversed by Rg1 treatment, which is inconsistent with the results of previous studies (The SR Ca [Ca2+]i ,34. The eriments . It is t studies ,26. This2+ homeostasis, specifically the relief of PLB inhibition. Notably, the present results indicate that the pharmacodynamic activity of TPSG is significantly superior to that of Rg1; however, the effects of SM are significantly superior to those of TSPG and Rg1.In conclusion, the myocardial protective effects of SM following I/R treatment may be partially attributable to the effects of SM on intracellular Ca


Exceptional scientific work inquires into new areas or utilizes techniques circumventing existing limitations to study. Alternatively, it may provide insights extending or complementing existing understanding, or generate hypotheses amenable to or worthy of further testing. Adeniran et al.  completeKCNH2 and KCNJ2 and therefore in repolarizing IKr  and cytoplasmic volumes, SR-Ca2+ leak and pump currents, Ca2+ current from dyadic to bulk cytoplasmic space, and background, plateau and Na+/Ca2+ exchange membrane Ca2+ current. The resulting Ca2+-troponin binding coupled this to a myofilament mechanics model chosen for its realistic basis in the cross-bridge cycling model of cardiac muscle contraction (Rice et al., 2+ and sarcomere length-Ca2+ relations. The simulations compared results from inclusion or exclusion of stretch-activated current, ISAC.At the cellular level, an established electrophysiological single cell model recapitulated human ventricular myocyte electrical and membrane channel properties as well as transmural action potential timecourse heterogeneities across the ventricular wall (Ten Tusscher et al., These cellular level findings were built into predictions for mechanical activity in two and three-dimensional human ventricular tissue models. The modeling of cardiac tissue mechanics permitted a non-linear elasticity (Marsden and Hughes, ISAC in normal as well as SQT hearts. In addition, at the theoretical level, the present modeling inevitably entails assumptions of particular implicit physical mechanisms operating within the reconstructions of physiological behavior. It did yield predictions matching previous experimental force-frequency data over frequencies including that at which the simulations were conducted. As outlined in the limitations section of the article, further validation including improved and more physiologically detailed representations of Ca2+ dynamics (Iribe et al., P, its derivative, dP/dt, as well as the timecourse and amplitude of chamber contraction and relaxation.The action potential shortening associated with SQT did appear to reduce ventricular mechanical function. Furthermore, this was rectified by an inclusion of stretch-activated channels. This prediction justifies future experimental testing as to whether such channels indeed exert functional effects on cardiac electro-mechanical coupling in SQTS1 and SQTS3. The experimental testing might then explore involvements of 2+ homeostatic and biomechanical changes with realistic descriptions of cardiac anatomy and mechanical properties. At the broader level we look forward to applications and developments of such approaches at the whole organ level in the analysis of other ion channel exemplars of abnormal cardiac physiology hitherto mainly studied at the cellular or tissue levels. These might include models for the long QT, Brugada and catecholaminergic polymorphic ventricular tachycardia, syndromes (Thomas et al., Nevertheless, the approach adopted by the authors draws attention to the potential value for modeling that integrates cellular electrophysiological, CaThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With 60 ± 5 %, children with AMKL had a lower 5-year overall survival . Yet, we achieved an improved 5-year OS in AML-BFM 04 compared to AML-BFM 98 . Allogeneic hematopoietic stem cell transplantation in first remission did not provide a significant survival benefit . Cytogenetic data were available for n = 78 patients. AMKL patients with gain of chromosome 21 had a superior 5-year OS , whereas translocation t was associated with an inferior 5-year event-free survival . However, multivariate analysis showed that treatment response (bone marrow morphology on day 15 and 28) was the only independent prognostic marker . Interestingly, GATA1-mutations were detected in six patients (11 %) without previously known trisomy 21. Thus, AMKL (excluding DS) remains an AML subgroup with inferior outcome. Nevertheless, with intensive therapy regimens, a steep increase in the survival rates was achieved.Despite recent advances in the treatment of children with acute megakaryoblastic leukemia (AMKL) using intensified treatment protocols, clear prognostic indicators, and treatment recommendations for this acute myeloid leukemia (AML) subgroup are yet to be defined. Here, we report the outcome of 97 pediatric patients with de novo AMKL (excluding Down syndrome [DS]) enrolled in the prospective multicenter studies AML-BFM 98 and AML-BFM 04 (1998-2014). AMKL occurred in 7.4 % of pediatric AML cases, at younger age (median 1.44 years) and with lower white blood cell count (mean 16.5 × 10The online version of this article (doi:10.1007/s00277-015-2383-2) contains supplementary material, which is available to authorized users. Acute megakaryoblastic leukemia (AMKL), M7 according to the French-American-British (FAB) classification , is a spAMKL occurs predominantly in childhood and particularly in children with Down syndrome (DS) –5, accout, which creates the RBM15-MKL1  fusion protein, is unique to pediatric AMKL [t (n = 5) as a good prognostic marker [Similarly, the prognostic impact of cytogenetically defined subgroups could not be clearly defined, mostly due to the small size of the study cohorts. A limited number of specific genetic abnormalities characterizes non-DS-AMKL. Translocation ric AMKL , 10. Whic marker , other sc marker . Also, tThe AML-BFM 98 and AML-BFM 04 studies were randomized, controlled phase III studies running in 75 centers in Germany, Austria, Switzerland, and the Czech Republic. The AML-BFM 98 study opened in July 1, 1998 and closed in June 30, 2003. Between July 1, 2003 and April 30, 2004, the AML-BFM 98 Interim Study, continuing the best arm of the AML-BFM 98 trial, recruited further patients. The AML-BFM 04 study opened in April 2004 and closed in February 2014. The final protocols were approved by the protocol review committee of the German Cancer Aid (DKH) and by the ethics committee of the University of Münster.Simultaneously, Germany, Austria, and Switzerland (BFM-Core group) participated in the prospective study on allogeneic HSCT vs. chemotherapy for high-risk (HR) childhood AML in first complete remission (AML CR1 HLA id) on behalf of the European Bone Marrow Transplantation (EBMT) Pediatric Working Party and the International BFM Study Group (I-BFMSG), which was approved by the ethics committee of the University of Tübingen. HR patients and family members were required to undergo HLA typing after the assignment to the risk group. All HR patients with a matched sibling donor were eligible for allogeneic HSCT in first complete remission.n = 37) and AML-BFM 04 (n = 60), including patients younger than 18 years with de novo AMKL. Patients with DS, myelosarcoma, secondary AML, or pretreatment of more than 2 weeks were excluded.The French-American-British (FAB) classification was used for the initial diagnosis of AML . The diaChildren with non-DS-AMKL were treated according to regimes AML-BFM 98 and AML-BFM 04, being similar for most parts Fig. .Fig. 1TrIn AML-BFM 98, patients received chemotherapy with an induction of cytarabine (Ara-C), idarubicin, and etoposide (AIE) and thereafter HAM , followed by a randomized consolidation therapy .In AML-BFM 04, patients received a randomized induction therapy of cytarabine (Ara-C), liposomal daunorubicin (L-DNR), and etoposide (ADxE) or AIE . After tAllogeneic HSCT should be performed in CR1 after consolidation according to the AML CR1 HLA id protocol. Children who were not transplanted in CR1 received one course of HAE as an intensification therapy (high-dose cytarabine and etoposide) and 1-year maintenance therapy . After 2006, the indication for allogeneic HSCT was restricted to patients with BM blasts >5 % after second induction. Allogeneic HSCT was performed in 23 SCT centers as reported previously . The recn = 78) of the included patients (n = 97) were available. Complete karyotypes were described according to the International System of Human Cytogenetic Nomenclature [GATA1 was prospectively performed as previously described [Cytogenetic analyses were carried out and centrally reviewed in the AML-BFM reference laboratory in Giessen (AML-BFM 98) and Hannover (AML-BFM 04), Germany, as previously described . Comprehnclature . In the escribed . For 15 Complete remission (CR) was defined by fulfillment of the Cancer and Leukemia Group B (CALGB) criteria , early dU test for continuous variables. The effect of HSCT on survival was tested using the Mantel-Byar method for comparisons of patients treated or not treated with HSCT. For graphic presentation, patients without HSCT and EFS below the median time to transplantation (0.4 years) were excluded. Follow-up was as of June 2014. Computations were performed using SAS .The Kaplan-Meier method was used to estimate survival rates . DifferePFisher’s < 0.0001). The mean white blood cell (WBC) count of children with de novo AMKL (16.5 × 109/L) was lower than of children with other AML subtypes (58.5 × 109/L). Additionally, initial CNS involvement occurred more frequently among other AML subtypes (12.4 %) than among AMKL patients (2.1 %). Patients with AMKL frequently presented with a longer disease history. Only 41.8 % of AMKL patients had an anamnesis less than 3 weeks, as compared to 57.2 % of children with other AML subtypes.Of 1316 pediatric patients with AML enrolled in the population-based studies AML-BFM 98 and AML-BFM 04, 97 (7.4 %) presented with de novo non-DS-AMKL. Patient characteristics are summarized in Table 9/L vs. 14.5 × 109/L (P = 0.002).Comparing AML-BFM 98 and AML-BFM 04, the study population did not significantly differ. We only noted statistical significance in the mean WBC of 19.7 × 10Plog rank = 0.079). The complete remission (CR) rate of non-DS-AMKL patients was comparable to other children with HR-AML (defined by the reported criteria of the AML-BFM study group [n = 32) relapsed after first CR, and 4.1 % (n = 4) died in first CR. Thirteen patients (13.4 %) were partial- and non-responders  to induction therapy and 3.1 % (n = 3) suffered from early death (ED). The cumulative incidence of relapse (CIR) was 45 ± 5 % compared to 39 ± 1 % (PGray = 0.07) for patients with other AML subtypes.With a 5-year OS of 60 ± 5 % Fig.  and 5-yedy group , 23). EiComparing AML-BFM 98 to AML-BFM 04 Fig. , the 5-yPlog rank = 0.84; 74 ± 11 % vs. 68 ± 8 %, Plog rank = 0.57), only 15.3 % of AMKL patients in AML-BFM 04 (ADxE and AIE) displayed a poor treatment response, as assessed by bone marrow morphology on day 15 and day 28 of therapy (>5 % blast cells). This is a clear improvement to AML-BFM 98 with 33.3 % (Table Plog rank = 0.021) and 5-year EFS of 56 ± 6 % (Plog rank = 0.0001)  in the induction of AML-BFM 04 (randomization of ADxE vs. AIE) resulted in no significant improvement regarding 5-year EFS and 5-year OS  Fig. . The intPMantel-Byar = 0.95; 5-year EFS 44 ± 17 % vs. 45 ± 11 %, PMantel-Byar = 0.6) and AML-BFM 04  were excluded. In AML-BFM 98, allogeneic HSCT in CR1 was performed on 9 (27 %) non-DS-AMKL patients, whereas 24 patients (73 %) were treated by chemotherapy alone. In AML-BFM 04, the percentages were 19 and 81 %. The outcome was not significantly different in patients undergoing allogeneic HSCT rather than chemotherapy alone in AML-BFM 98  was found in 8 patients  children with non-DS-AMKL Tables  and 2. OPlog rank = 0.034; Supplementary Fig. Other recurrent aberrations were mainly numerical changes, such as gain of chromosome 8 or 21. Gain of chromosome 21 was associated with better survival rates  or had a history of transient abnormal myelopoiesis (TAM) during the neonatal period. Six patients (11.3 %) were positive for a GATA1-mutation, and a gain of chromosome 21 in the leukemic blasts was detected , gain of chromosome 8 (+ 8), gain of chromosome 21 (+ 21), monosomy 7 and der(3)) as risk factors, only poor treatment response  was of independent prognostic significance , which was statistically significant in the univariate analysis, multivariate analysis indicated a trend towards poor prognosis  was extremely poor in the early AML-BFM 87 trial , which could be significantly improved in the following trials AML-BFM 93 and 98  [n = 28, 2-year EFS/OS 14/14 %; O’Brien 2013: AML02 (n = 26), 3-year EFS/OS 49/54 %; POG 9421 (n = 49), 5-year EFS/OS 35/36 %; Barnard 2007: CCG 2891 (n = 53), 5-year EFS/OS 23/28 %) [Our study with 97 patients demonstrates that AMKL is nowadays not necessarily associated with a poor outcome, as we and others previously reported  after allogeneic HSCT compared to 0 % after chemotherapy alone, stating that allogeneic HSCT during remission offers the best chance of cure . The EurThe response to induction therapy is of independent  and strong prognostic relevance. Patients with more than 5 % bone marrow blasts on day 15 or 28 of therapy have a poor 5-year OS of only 35 ± 11 %. Still, the number of non-responders could be reduced in AML-BFM 04 (8 %), compared to AML-BFM 98 (17.1 %). PCR- or flow cytometry-based residual disease monitoring may help to distinguish between increased numbers of normal myeloblasts in the regenerating bone marrow after chemotherapy and persistence of malignant blasts to more accurately determine the treatment response in the future –28.t to be a good prognostic factor. Contrarily, we demonstrated for 8 patients (10.1 % of our cohort) a significantly lower 5-year EFS compared to patients without this translocation. Though this may be attributable to the small number of patients and the high NR rate, our report puts a clear role of t as a good prognostic indicator into question. Interestingly, neither monosomy 7/7q-, complex karyotype nor 11q23 aberrations indicated a poor prognosis. A good prognostic factor in our study was gain of chromosome 21, which we found in 21.9 % of the patients . Based on the GATA1-status, which is strongly associated with DS-AMKL, four of these patients were diagnosed with trisomy 21-mosaicism that was previously unknown. Two patients did not have constitutional trisomy 21, suggesting that trisomy 21 was acquired as an early step during leukemogenesis. All six patients are in continuous remission after treatment, indicating a similar biology and treatment response of AMKL with acquired or constitutional (DS-AMKL) trisomy 21 in conjunction with GATA1s. Therefore, it is crucial to perform GATA1 mutation analysis on each patient with pediatric AMKL and it may not be advisable that children with GATA1-mutations are stratified to the high-risk arm of AML protocols like other AMKL patients. All children with GATA1s-mutation showed CD7 surface marker expression, which could serve as a marker to predict the cases with GATA1s-mutation [Our cytogenetic analyses mark a difference to earlier reports. Carroll et al.  and Ducht1;2) and CBFA2T3-GLIS2-fusion (inv(16)) needs to be addressed [Thus, the focus on cytogenetic analyses remains of high prognostic importance. Especially, the role of the newly defined recurrent cytogenetically cryptic NUP98-JARID1A fusion Fig. S1(PDF 233 kb)Fig. S2(PDF 186 kb)Table S1(PDF 18 kb)


Variant patterns of peritoneal folds could be formed due to the complex nature of the embryology of the peritoneum and the gastrointestinal tract. When uncommon peritoneal folds are formed, they could influence aberrant formation of surrounding gastrointestinal structures and create spaces that may harbour peritoneal fluids in cases of infection or malignant tumor. One of such variant peritoneal folds is the cystoduodenal ligament which is a doubled peritoneal membrane attaching the gallbladder to the duodenum. Yet no study was found that had reported the frequency of occurrence of the cystoduodenal ligament. The current study determined the prevalence of the cystoduodenal ligament in forty adult cadavers. The ligament was reported in 35% of cases. The ligament was further classified as types I and II. Type I cystoduodenal ligament was attached partially to the gallbladder  while type II was attached to the entire extent of the gallbladder. Type I occurrence was found in 44% and type II was found in 56% of the occasions of cystoduodenal ligament formation. It is concluded that the cystoduodenal ligament could be commonly found, it possesses important vascular structures, and it could affect the shape of the gallbladder. Surgeons, radiologists, and anatomists should be kept abreast of these findings. It is important to take cognizance of the possibilities of encountering variant peritoneal folds when assessing the abdominal region, either radiologically, surgically, or in cadaveric demonstrations. Such peritoneal formations may potentiate the kinking of intestinal structures, leading to intestinal necrosis or increased tendency of intestinal herniation . They coThe study was conducted on forty cadavers  in the Department of Anatomy, Bowen University, Osun State, Nigeria. This study was conducted between February 2011 and October 2014. The use of cadavers was in line with the Nigeria Anatomical Act. Departmental approval was sought from the head of department of the involved institution. The ages of the cadavers were not retrievable but they were presumably adults.The abdominal cavity was carefully dissected, and then the supracolic region was painstakingly exposed. The visceral surface of the liver was upwardly reflected to subject the cystoduodenal ligament to qualitative and quantitative examination. Qualitative analysis was done by physical visual examination while quantitative measurements were performed with the digital vernier caliper.The presence of the cystoduodenal ligaments was confirmed when ligaments were seen being superiorly attached to the inferior border of the gallbladder, either completely or partially; and they are distally attached to the duodenum. The cystoduodenal ligament was carefully dissected to record the possible existence of the cystic artery, vein, or duct. The upper, middle, and lower width of the cystoduodenal ligament were measured.The cystoduodenal ligament was differentiated from peritoneal adhesion by tracing the two membranes that formed it superiorly and inferiorly around the gallbladder and duodenum. The continuity of the two membranes of the cystoduodenal ligament with visceral peritoneum of the gallbladder and the duodenum is a confirmation that they are not mere peritoneal adhesions.This current study reports 35% (fourteen out of forty) occurrence of the cystoduodenal ligament Figures  and 2. TThe current study is the first to demonstrate the prevalence of the cystoduodenal ligament. The study demonstrated that the cystoduodenal ligament is more prevalent 35%) than it was ocassionally expected 5% than i, 3–9. SuIt was ensured in the current work that the cystoduodenal ligament was differentiated from peritoneal adhesion which usually occurs due to peritonitis. The formation of the cystoduodenal ligament would be clinically important if it disrupts the function(s) of the surrounding structures . The conThe cystoduodenal ligament was associated with anomalous gallbladder in 5% of all cases studied and only type I formation was involved. This supports earlier finding that peritoneal folds could disrupt gallbladder structure and emptying , while aCongenital bands have been reported to cause contriction or obstruction of duodenum or jejunum , 16; howIt should be noted that, commonly, variant peritoneal fold formations result from abnormal gastrointestinal rotation and they sometimes occur alongside gastrointestinal anomalies. In the case reported by Ashaolu et al. , 4, the The mode of origin of variant peritoneal folds has been attributed to errors in development, previous inflammation (peritonitis), and mechanical traction by the gut . It has The cystoduodenal ligament formation may potentiate the kinking of intestinal structures, leading to intestinal necrosis or increased tendency of intestinal herniation , 4, 5. TIt is therefore important for surgeons, anatomists, and radiologists to be aware of the dominance of the ligament.


In this review, we describe the properties of airway store-operated channels and their role in regulating airway epithelial cell function.The epithelial cells of the lung are at the interface of a host and its environment and are therefore directly exposed to the inhaled air-borne particles. Rather than serving as a simple physical barrier, airway epithelia detect allergens and other irritants and then help organize the subsequent immune response through release of a plethora of secreted signals. Many of these signals are generated in response to opening of store-operated Ca2+ and ATP together to control life and death’.This article is part of the themed issue ‘Evolution brings Ca In the UK, the proportion of children with asthma and allergies, in general, is higher than most other European countries, imposing a serious clinical and economic burden on the National Health Service. There is no cure for the disease, and therapies are directed at controlling the severity of the symptoms. There has been little change in treatment for several years with corticosteroids being prescribed for prolonged management and long-lasting β2 agonists for more immediate relief through bronchodilation. However, lung function in 30–40% of patients shows no signs of clinical improvement with these treatments , and chrA characteristic of chronic asthma is substantial remodelling of the airway wall . ChangesThe epithelial cells of the lung are at the interface of a host and its environment and are therefore directly exposed to inhaled air-borne particles. Although long considered to have a passive role in the airway remodelling process, growing evidence suggests that airway epithelial cells may make a more active contribution by reacting directly to allergens and triggering and then helping orchestrate the subsequent immune response .2, thymic stromal lymphopoietin, regulated on activation, normal t expressed and secreted (RANTES) and various interleukins (ILs), including IL-1, -8 and -33 [Lung epithelial cells release a plethora of signals that recruit and activate immune cells that shape the subsequent inflammatory response. Epithelia-derived stimulants include ATP, uric acid, lysophosphatidic acid, granulocyte macrophage-colony stimulating factor, chemokine (C–C motif) ligand 2/20 (CCL2/CCL20) chemokine ligands, prostaglandin E and -33 ,7. These2+ is a critical intracellular signal in airway epithelia and this trigger Ca2+ is mainly derived from store-operated Ca2+ channels in the plasma membrane.An important question concerns the mechanisms of activation of airway epithelial cells. Which intracellular second messenger pathways lead to the synthesis and secretion of the epithelia-derived signalling molecules? Identification of the underlying pathways may open up new potential therapies for manipulating epithelial cell activity and thereby impact on the remodelling process itself. As with many other cell types, it turns out that cytoplasmic Ca2.2+ can activate a range of temporally distinct responses in airway epithelial cells .Airway mucus production is dependent on another form of secretion, namely that of Clthmatics . Studiesthmatics , the lat +100 mV . At phys2+. Flagellin, a component of bacterial flagella, can bind to the glycolipid receptor asialoGM1, leading to interaction with Toll-like receptor 2 [2+ release and activation of downstream Ca2+-dependent responses such as Ca2+-dependent Cl− secretion.In cystic fibrosis, bacterial pathogens contribute to mucus hypersecretion through mobilization of intracellular Caceptor 2 . This re2+ in airway epithelial cells is to regulate ciliary beat frequency, an important step in clearance of mucus. ATP is an effective stimulus of ciliary beating while inducing numerous cytoplasmic Ca2+ oscillations [2+ [2+ near the base of the cilia [2+ oscillations induced by the stimulants acetylcholine or ATP, with high frequencies of Ca2+ signal inducing a steady elevated beat frequency [2+ and, when activated, increase cytoplasmic Ca2+. This rise in Ca2+ was shown to increase ciliary beat frequency, providing a neat feedback loop whereby the cilia, having detected harmful particles, mechanically move the compounds out of the lung [Another important role for cytoplasmic Callations . Simultaions [2+  or Ca2+ he cilia  showed trequency . Interesrequency . These cthe lung .2+ can also spread through airway epithelial cells as an intercellular Ca2+ wave following mechanical stimulation of a single cell. The Ca2+ wave can be propagated by inositol1,4,5-triphosphate (InsP3) [2+ wave is significant, spreading to 40 cells or more following mechanical stimulation of just one cell [2+-dependent manner and which acts through G protein-coupled receptors that link to phospholipase C and thus produce InsP3. Possibilities include ATP or leukotriene C4, which acts on cysteinyl leukotriene type I receptors to drive intercellular Ca2+ waves in mast cells [Ca (InsP3) , diffusi (InsP3) . The spaone cell . An auxist cells .2+ can exert long-term effects on epithelial cell function through regulation of gene expression. Damage to airway epithelia such as mechanical or viral insults leads to remodelling and subsequent thickening of the airways and a major role for EGF in regulating the process has been established [In addition to the relatively rapid onset responses described above, cytoplasmic Caablished . Insults2+ signals often regulate gene expression through activation of Ca2+-dependent transcription factors such as CREB and members of the NFAT family. NFAT proteins are heavily phosphorylated at rest and confined to the cytoplasm [2+ rise can activate the protein phosphatase calcineurin, the target for immunosuppressants cyclosporin A and tacrolimus, which dephosphorylates NFAT. Dephosphorylation leads to exposure of a nuclear localization sequence, resulting in nuclear import as part of a transport complex with importin β [2+ entry activates nuclear CREB through a series of phosphoryation reactions [2+ microdomains near open CaV1 (L type) channels activate βCa2+/calmodulin-dependent protein kinase II (CaMKII) which is held close to the channel pore. Active βCaMKII then phosphorylates γCaMKII on threonine 287. Once phosphorylated, γCaMKII acts as a shuttle, transporting Ca2+–CaM into the nucleus. Nuclear Ca2+–CaM then activates Ca2+/calmodulin-dependent protein kinase kinase that in turn activates CaMKIV, resulting in CREB phosphorylation and transcription of CRE-regulated genes [Caytoplasm . A cytopportin β . By conteactions . Ca2+ mied genes .3.2+ concentration in most mammalian cells is typically 50–100 nM. However, high concentrations of free Ca2+ (hundreds of micromolar to millimolar) are stored in intracellular organelles such as the endoplasmic reticulum and Golgi apparatus  and Golgi-located secretory pathway Ca2+ATPase (SPCA) pumps. Mitochondria, by virtue of the large negative potential across the inner mitochondrial membrane, are able to take up large amounts of cytoplasmic Ca2+ rapidly though the mitochondrial uniporter channel and then release the Ca2+ more slowly through Na+–Ca2+ exchange [2+ after stimulation [Resting free cytoplasmic Caiewed in ,26). Theexchange ,28. In amulation .2+ can be rapidly released by agonists that couple to phospholipase C and thereby increase InsP3 levels. Treatment with a maximally effective concentration of the SERCA pump inhibitor thapsigargin, which gradually depletes stores of Ca2+, resulted in substantial Ca2+ release consistent with a large pool of Ca2+ contained within the organelle [2+ release (ca 15% of control), despite the reduction in Ca2+ content of the endoplasmic reticulum. Although the endoplasmic reticular Ca2+ store clearly makes a major contribution to the pool of mobilizable Ca2+, the small thapsigargin-insensitive component to the ATP response led to the suggestion of an additional store, although its identity was unclear. However, in that study, ATP was applied when Ca2+ had barely returned to basal levels, raising the possibility that stores might not have been fully depleted by thapsigargin prior to ATP challenge. In addition to InsP3 receptors, many cell types co-express ryanodine-sensitive Ca2+ release channels. However, neither caffeine nor ryanodine, agonists of these channels, had any effect on cytoplasmic Ca2+ in rabbit tracheal epithelial cells [Airway epithelia have a prominent endoplasmic reticulum from which Carganelle . Howeveral cells .2+ release from the endoplasmic reticulum is important, the limited capacity of the store means that the Ca2+ mobilization phase is transient. To sustain Ca2+ signalling, Ca2+ influx into the cell is necessary. With an external free Ca2+ concentration in the plasma of ca 1.5 mM and a typical resting membrane potential of −70 mV, the electrochemical gradient for Ca2+ entry is large. Therefore, raising membrane permeability by increasing the open probability of Ca2+ channels, is a very effective way to increase Ca2+ flux into the cytoplasm. Airway epithelia, like most other non-excitable cells, do not express functional voltage-gated Ca2+ channels. Ca2+ influx to agonists are unaffected by CaV1.2 (L type) inhibitors such as verapamil and diltiazem [2+ entry provide the main routes for Ca2+ influx.Although Cailtiazem . Instead4.2+ influx in non-excitable cells is through store-operated Ca2+ channels in the plasma membrane [2+ store [3 following hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate. Both G protein-coupled and tyrosine kinase-coupled receptors have been shown to activate store-operated channels [2+-selective, low conductance channel called the Ca2+ release-activated Ca2+ (CRAC) channel that was activated, regardless of how the Ca2+ store was emptied [2+ sensors [2+ content, STIM proteins multimerize and translocate to junctional endoplasmic reticulum that are located less than 20 nm from the plasma membrane. At these sites, they bind to and open the Orai channels, resulting in Ca2+ entry. The crystal structure of Drosophila Orai has been reported at 3.35 Å resolution [A major conduit for Camembrane . These c2+ store . Store dchannels . Electro emptied . CRAC ch emptied ,36. The  sensors . Upon a solution . The chasolution ,40. Gainsolution .2+ signals (Ca2+ microdomains) near open CRAC channels and not a bulk rise in cytoplasmic Ca2+ are essential for activating these processes [2+ microdomains near CRAC channels [2+ channels [2+ microdomains now have a direct line to calcineurin and NFAT, resulting in transcription factor activation in the absence of a bulk Ca2+ rise. Once within the nucleus, NFAT helps regulate gene transcription, often through interaction with the AP-1 complex [2+ microdomains near CRAC channels [2+ microdomains that have built up near CRAC channels signal through different pathways to regulate the activities of different transcription factors, which in turn can operate independently or synergistically.The function of CRAC channels has been studied in detail in immune cells such as mast cells and T lymphocytes ,43. In mrocesses –48. Ca2+channels ,50 or Cachannels  have a pchannels , whereas complex , a heterchannels . This pachannels  but requchannels . Hence, 5.2+ using fluorescent dyes have revealed that airway epithelia possess functional store-operated Ca2+ channels. Challenge of human bronchial epithelia 16-HBE cells with thapsigargin in Ca2+-free solution resulted in Ca2+ store depletion as the Ca2+ that flowed out of the store through the Ca2+ leak pathway was no longer taken back up by SERCA pumps. Readmission of external Ca2+ a few minutes later resulted in a robust cytoplasmic Ca2+ rise as Ca2+ entered the cell through the open CRAC channels [2+ entry, following InsP3-dependent Ca2+ release from the stores [2+ influx but to a lesser extent. Therefore, as in other cell types, activation of Gq-coupled receptors results in store-operated Ca2+ influx in response to physiologically relevant stimuli.Measurements of cytoplasmic Cachannels . In humae stores . Stimula(b)In 16-HBE cells, STIM1 and STIM2 proteins are both expressed as are mRNA transcripts for Orai1 and Orai3 and to a lesser extent Orai2 . Orai1 p2+ influx in response to thapsigargin [2+ entry in response to PAR2 receptor activation [2+ entry [Knockdown of STIM1 using an siRNA-based approach resulted in a reduction in protein expression of 60% and this led to a substantial decrease in store-operated Casigargin . Knockdotivation . Similar2+ entry . STIM1 a(c)2+ that occurred following readmission of external Ca2+ to cells challenged with thapsigargin in Ca2+-free solution was blocked by structurally distinct CRAC channel blockers including Synta66, BTP2, 2-APB as well as the rare earth element La3+ [3+ is reduced by mutating negatively charged aspartates (D110 and D112) in the extracellular loop between transmembrane domains I and II [2+ selectivity [3+. This was indeed the case [50 and Hill coefficient for La3+ block of CRAC channels in airway epithelia  were very similar to that seen in the RBL-1 mast cell line (1.1 and 1.0 μM).The rise in cytoplasmic Caent La3+ . InhibitI and II , which aectivity . If the the case ; the IC5(d)2+), steep inward rectification and high selectivity for Ca2+ [2+ to Na+ is greater than 1000 [2+ and now support large Na+ fluxes [2+-dependent inactivation, which develops bi-expontially with time constants of approximately 10 and approximately 100 ms [2+ ions reduce further Ca2+ entry. Studies investigating the impact of Ca2+ chelators with different on-rates on the kinetics and extent of fast inactivation suggest the incoming Ca2+ ions act within a few nanometres of the channel mouth [CRAC channels have several biophysical characteristics that combine to form a useful molecular fingerprint. These include its tiny unitary conductance , indicative of high selectivity for Ca2+ [2+-dependent inactivation are identical for CRAC channels in airway epithelia and RBL-1 mast cells, over a wide voltage range [Whole cell patch clamp experiments show that the airway epithelial store-operated current has numerous features characteristic of the CRAC channel. Store depletion following dialysis with InsPfor Ca2+ . The stofor Ca2+ . Moreovege range .2+ and Mg2+, the inward current became larger and the current–voltage relationship now reversed at +50 mV [2+ influx.In the absence of external Cat +50 mV , consistt +50 mV , whereast +50 mV , althougt +50 mV , and Ora2+ closely match the channel robustly expressed in immune cells.Nevertheless, several important features of the airway epithelial CRAC channel pore, channel pharmacology and regulation by Ca(e)A major role for CRAC channels in airway epithelia is to regulate gene transcription. Expression of the immediate early gene c-Fos was increased approximately sixfold following stimulation with thapsigargin and this was suppressed by the CRAC channel blockers Synta66 and BTP2 . c-Fos, 2, effects that were all reduced by cyclosporin A. Whereas cyclosporin A abolished IL-6 and TNF-α production, it only partially reduced IL-8 and PGE2 production. By contrast, IL-6 generation to PAR2 receptor activation was only moderately reduced by cyclosporine A, and PGE2 production was not compromised at all [2 production is NFAT-independent following PAR2 receptor activation. PGE2 production following PAR2 receptor activation was partially sensitive to BTP2 suggesting that PGE2 production, although insensitive to NFAT, nonetheless required store-operated Ca2+. The underlying mechanism could involve Ca2+ entry through CRAC channels activating Ca2+-dependent phospholipase A2. This would liberate arachidonic acid, the substrate for the cyclooxygenase enzymes that lead to the production of prostaglandins. Such a mechanism has previously been described in mast cells, where local Ca2+ entry through CRAC channels activated the MEK/ERK pathway, which led phosphorylation and activation of Ca2+-dependent phospholipase A2 as well as stimulation of the 5-lipoxygenase enzyme [2+-dependent phospholipase A2 to leukotriene C4.Activation of PAR2 receptors in airway epithelial cells induced expression of the cytokines interleukin (IL)-6 and IL-8 . This ind at all . These de enzyme ,66. Thro(f)2+ entry pathway or that sufficient levels of STIM1/Orai1 remain after knockdown to maintain basal EGF production.Genetic linkage studies in asthmatic patients revealed a region on chromosome 7 containing the EGF receptor that was associated with airway hyper-reactivity , a step 6.Airway epithelial cells lining the respiratory tract not only provide a first line of defence to air-borne pathogens and irritants, but also play a central role in initiating and orchestrating subsequent innate and adaptive immune responses. In addition to these physiological functions, airway epithelial cells contribute to the remodelling process characteristic of chronic asthma.2+ entry through store-operated CRAC channels. These channels share several traits with their well-defined counterparts in mast cells and T lymphocytes, which help maintain the immune response in asthma. Therefore, targeting CRAC channels is a new and exciting approach to managing chronic airway diseases. By modulating activities of airway epithelial and mast/T cells at the same time, CRAC channel inhibitors would be particularly effective in damping down both immune and remodelling components. Although there are currently no CRAC channel blockers in use in the clinic, the area is extremely active with both small molecule inhibitors [Epithelial cells release cytokines as well as a plethora of other bioactive compounds including EGF and related signalling molecules that are closely associated with the remodelling mechanism. Synthesis and release of many of these signals is triggered by Cahibitors –74 and bhibitors  being pu


However, little is known of the ANO1 activation mechanism by Ca2+. Here, we demonstrate that two helices, “reference” and “Ca2+ sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca2+-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca2+-dependent activation because neutralization of these charges change the Ca2+ sensitivity. We now predict that the Ca2+ sensor helix attaches to the reference helix in the resting state, and as intracellular Ca2+ rises, Ca2+ acts on the sensor helix, which repels it from the reference helix. This Ca2+-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.Anoctamin 1 (ANO1)/TMEM16A is a ClThe online version of this article (doi:10.1007/s00424-014-1603-2) contains supplementary material, which is available to authorized users. To investigate this relationship, physical interaction between the two helices was measured using surface plasmon resonance. A peptide segment (692-NLEPFAGLTPEYMEM-706) spanning the Ca2+ sensor helix of ANO1 was synthesized, biotinylated at the N-terminus, and immobilized on a streptavidin-coated gold sensor chip. Subsequently, a synthetic peptide spanning the reference helix (651-NLFEIGIPKMKKFIRYLKLRR-671) was passed over the immobilized Ca2+ sensor helix. In the absence of Ca2+, the reference helix peptide interacted strongly with the Ca2+ sensor helix  produced a dose-dependent reduction in the interaction between the two synthetic helix peptides. In contrast, a mutant of the reference helix peptide (651-NLFEIGIPAMAAFIAYLALAA-671) in which all Lys and Arg residues were replaced with Ala failed to show any interaction with the Ca2+ sensor helix peptide regardless of Ca2+ concentration  markedly blocked Ca2+-induced ANO1 currents . Replacement of single Lys residues to Gln in the upper part of the reference helix such as 659-KMKK-662 mutant caused a rightward shift of EC50 values, ranging from 3.6 to 6.1 μM , the EC50 increased to 22.8 μM (n = 6). Replacement of Arg or Lys residues with Gly in the lower part of the reference helix such as 665-GYLG-668 mutant shifted the EC50 value to 6.2 μM (n = 6). Furthermore, deletion (ΔKMKKFIRYLK) of the reference helix changed the EC50 values to 51.0 μM (n = 9). A mutation of the two Glu residues in the Ca2+ sensor helix (702-EYME/QYMQ-705) elicited a four-order magnitude of rightward shift in EC50   caused a dramatic rightward shift of EC50   elicited a three-order magnitude of rightward shift in EC50   Fig. . Likewis 8) Fig. . Mutatio 6) Fig. .Fig. 5Muact, a synthetic agonist of ANO1, was synthesized for the purpose of treating cystic fibrosis in an attempt to bypass dysfunctioning CFTR channel i  upon deletion; the ICL3 region is not a site for voltage-induced activation.We reasoned that the positively charged residues in the reference helix may play a role in mediating voltage-dependent activation. Progressive replacement of Lys residues in the upper part of the reference helix shifted the half-maximal activation voltages ) in whole-cell recording. Surprisingly, a mutant (ΔICL3) which hardly responded to sub-millimolar Ca2+ concentration elicited currents when the temperature of bath solution was raised over 44 °C  assay, circular dichroism analysis, as well as mutational study along with electrophysiological experiments support the structure model.With homology modeling, we predict that the ICL3 region in the revised topology of ANO1 has a unique structure composed of two parallel helices that interact with each other in a Ca2+. The two helices are oppositely charged. The role of positive charges in the reference helix appears to hold the Ca2+ sensor helix in the resting state. We imagine that the reference helix pushes the Ca2+-sensor helix away when Ca2+ ions attach to it  and D738  residues beyond the E702 and E705 residues and found that the E734 and D738 are involved in the Ca2+-dependent gating, because mutations in these residues made a large shift in EC50. Thus, Tien and colleagues proposed that the Ca2+ caging with the four Glu and Asp residues is essential for the ANO1 activation. It is not clear how Ca2+ binding to this site gates ANO1. It is conceivable that these additional Glu734 and Asp738 residues may affect the interaction between Ca2+ and the reference Ca2+ sensor helices, because the two Glu and Asp residues are close to the Ca2+ sensor helix. However, to identify the interactions between the two helices and the E734 and D738 residues requires further study.Many studies tried to define the Casitivity . Howevery others . Further variant . Similar448 Fig. . In cont. Amino acid substitution or deletion mutants were prepared using a site-directed mutagenesis kit  or by using the overlap PCR method. Mutations in all mutants were confirmed by sequencing whole nucleotide sequences. HEK 293T cells were transfected with 1 μg of pEGFP-N1-mANO1 or pEGFP-N1-mutants, 0.05 μg pEGFP-N1, and FuGENE  in 35-mm dishes. Transfected cells were incubated in DMEM supplemented with 10 % fetal bovine serum (GIBCO) and penicillin-streptomycin at 37 °C in a 5 % COBorosilicate glass pipettes  with tip resistances of ~2 Mohms were used to form gigaseals on HEK cells. Inside-out membrane patches were excised by pulling the pipette away from the cell. Currents were recorded with a patch-clamp amplifier  with a 1-KHz filter. Data from the amplifier were digitized with Digidata 1440A (Molecular Probes) and stored on a computer.d-glucamine, 2 mM MgCl2, and 10 mM HEPES adjusted to pH 7.2 with HCl. For Ca2+-free solution, 10 mM ethylene glycol tetraacetic acid (EGTA) was added to the control solution. The bath solution contained (in mM) 140 N-methyl d-glucamine, 2 MgCl2, 10 HEPES, 10 chelator  ethylenediamine triacetic acid (HEDTA), or nitrilotriacetic acid (NTA)), and a calculated amount of CaCl2 (adjusted with HCl to pH 7.2). EGTA was used for 0.1 ~ 1.0 μM free Ca2+, whereas HEDTA and NTA were used for 3.0 ~ 30 and 100 ~ 1,000 μM free Ca2+, respectively [2+ greater than 1 mM. To calculate free Ca2+ in pipette solutions, the WEBMAXC program was used (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). Free Ca2+ less than 30 μM in each solution was monitored with Fura-2 fluorometry [The control pipette solution contained 140 mM N-methyl ectively . No chelorometry .A Biacore 3000 was used for kinetic surface plasmon resonance measurement as previously described . Brieflyhttp://salilab.org/modweb), a web server for automated comparative protein structure modeling.The amino acid residues of third intracellular loop (residues 603–663) in ANO2 were selected as the target sequence for homology modeling. Automated homology modeling was implemented by MODWEB  is overlaid (PPTX 170 kb)Below is the link to the electronic supplementary material.