The role of protein kinase C (PKC) in regulating the activity of phospholipase C (PLC) in neutrophils activated with the chemoattractant, platelet-activating factor , was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM) and staurosporine (400 nM).2+, Ca2+ influx, inositol triphosphate (IP3), and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively.Alterations in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase.Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils.Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IPAlthough generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils. IP3 interacts with its receptors on the membranes of calcium storage vesicles releasing Ca2+ into the cytosol  radioreceptor assay procedure , which is a competitive ligand binding assay, and the results expressed as pmol IP3/107 cells.Following 20 min incubation on ice, the tubes were centrifuged at 2000 × 4 in the supernatants of neutrophils activated with PAF (20 and 200 nM) in the absence or presence of GF10903X (1 μM). Neutrophils  in HBSS were preincubated for 10 min at 37°C with the test agent after which PAF was added to the cells and the reactions stopped after 3 min incubation at 37°C (predetermined in preliminary time-course experiments) by the addition of an equal volume of ice-cold HBSS to the tubes which were then held in an ice-bath prior to pelleting the cells by centrifugation. The cell-free supernatants were then assayed for LTB4 using the enzyme immunoassay (EIA) procedure. Supernatants from cells activated with PAF were diluted 1:4 prior to assay. These results are expressed as picograms (pg)/107 cells.A competitive binding enzyme immunoassay procedure  was used to measure LTBn values represent the number of separate experiments in each series for which cells from a minimum of 3 different donors were used) are expressed as the mean value ± standard error of the mean (S.E.M.), with the exception of the fura-2/AM experiments for which the traces are also presented. Levels of statistical significance were calculated using paired Student's t test when comparing two groups, or by analysis of variance (ANOVA) with subsequent Tukey-Kramer multiple comparisons test for multiple groups. A P-value < 0.05 was considered significant.The results of each series of experiments  to neutrophils was accompanied by an abrupt increase in fura-2 fluorescence intensity due to elevation in the cytosolic Ca00 nM to 2+-chelating agent, EGTA, addition of PAF (200 nM), was also accompanied by the characteristic abrupt increase in fura-2 fluorescence, which subsequently declined rapidly towards basal levels without the sustained elevation in fluorescence intensity observed in the absence of EGTA . GF10903X (1 μM) had no effect on thapsigargin-mediated Ca2+ release from intracellular storage vesicles (results not shown).In the presence of the Ca2+ mobilization and IP3 generation by neutrophils [The effects of the phospholipase C (PLC) inhibitor, U73122 (2 μM) added to neutrophils 10 - 15 sec following addition of PAF (200 nM), are shown in Figure trophils , which w2+ into storage vesicles mediated by the protein kinase A (PKA)-sensitive Ca2+-endomembrane ATPase. In the presence of rolipram, cAMP accumulates in neutrophils, activating PKA with consequent upregulation of the activity of the endomembrane Ca2+-ATPase [2+ concentrations were markedly accelerated following attainment of peak fluorescence. Similar effects of rolipram were observed in neutrophils pretreated with the PKC inhibitors, suggesting that these agents do not interfere with endomembrane ATPase-mediated resequestration of Ca2+ into storage vesicles.These results are shown in Figure +-ATPase . NeutropThe consolidated data for all of the fura-2 fluorescence experiments described above are shown in Tables 2+, occurred almost immediately after addition of PAF (20 and 200 nM). An initial abrupt linear decrease in fluorescence intensity over 2 - 3 min, of greater magnitude at the higher concentration of PAF, was followed by a slower decline for a further 2 - 3 min. In the presence of the PKC inhibitors, addition of PAF (20 nM) was followed by a rapid decline in fura-2 fluorescence intensity of significantly greater magnitude  than that observed with untreated cells. In the presence of the PKC inhibitors, addition of PAF (200 nM), resulted in a slight, but insignificant increase in the magnitude of decline in fura-2 fluorescence.These results are shown in Figure The rate and magnitude of decline in fura-2 fluorescence for neutrophils activated with FMLP (1 μM), was significantly increased in the presence of GF10903X (1 μM), (results not shown).2+ following activation of neutrophils with 20 and 200 nM PAF are shown in Table 2+ following activation of the cells with PAF at a concentration of 20 nM. No significant differences were observed for neutrophils activated with higher concentrations of PAF (200 nM). These results correspond closely with those obtained by means of the Mn2+ quenching of fura-2 fluorescence assays.The magnitudes of net influx of Ca2+ from PAF (20 nM)-activated neutrophils measured 5 min following addition of the chemoattractant was 4 ± 2% of the total amount of cell-associated radiolabelled Ca2+ and this increased significantly to 12 ± 2% for cells pretreated with GF10903X, (P < 0.05 for comparison with the untreated control system).The net efflux of Ca3 concentrations increased significantly following exposure of neutrophils to PAF (20 and 200 nM) or FMLP (1 μM), peaking at 10 sec after addition of the chemoattractant. Pre-incubation of the cells with GF10903X (1 μM) resulted in significant increases in IP3 concentrations.These results are shown in Table 4 production by PAF (20 nM)-activated neutrophils was markedly increased in the presence of GF10903X from 175 ± 31 to 794 ± 51 pg/107 cells in the absence or presence of the PKC inhibitor respectively (P < 0.01), rising from a basal value of 24 ± 6 pg/107 for resting cells.LTB2+ homeostasis and down-regulation of Ca2+-dependent pro-inflammatory activity to chemoattractant-activated human neutrophils. Notwithstanding those which target IP3 and its receptor, well-characterized mechanisms which promote efficient clearance of Ca2+ from the cytosol of activated neutrophils include: i) the electrical gradient created by the membrane depolarizing action of NADPH oxidase that restricts the influx of Ca2+ via store-operated Ca2+ channels [2+ pumps, namely the Ca2+-resequestering endomembrane Ca2+- ATPase and the plasma membrane Ca2+-ATPase, that actively transports Ca2+ out of the cell [2+ pumps were considered to be putative targets for PKC in our experimental setting. Firstly, PAF, at the concentrations used in this study, does not activate NADPH oxidase [2+ transients observed with the PKC inhibitors. Secondly, the apparent enhanced Ca2+ efflux in the presence of GF10903X is not compatible with inhibition of the plasma membrane-associated Ca2+-ATPase, which is upregulated by sustained elevations in cytosolic Ca2+ concentrations [2+-ATPase to rolipram was preserved in PAF-activated neutrophils pretreated with the PKC-inhibitors, suggesting that these agents do not significantly interfere with the refilling of Ca2+ stores.The results of the current study have identified a role for PKC in promoting restoration of Cachannels -17 and ithe cell ,19. Howe oxidase , effectitrations . Thirdly3, in chemoattractant-activated neutrophils. The apparent doubling of IP3 concentrations in the presence of the PKC inhibitor observed in the current study likely maintains IP3 receptors in an open state for longer periods, facilitating sustained Ca2+ release by promoting shuttling of the cation between the stores and the cytosol [From a mechanistic perspective however, treatment of neutrophils with GF10903X significantly elevated and prolonged the concentrations of the intracellular second messenger, IP cytosol .2+-chelating agent, EGTA, support this contention, as delayed Ca2+ clearance in the presence of the PKC inhibitors persisted in this setting, and could not be attributed to enhanced Ca2+ influx. Previous reports have suggested that PKC may modulate PAF-mediated activation of PLC by promoting desensitization of the PAF receptor [Experiments performed in the presence of the extracellular Careceptor . This isreceptor ,25.3 receptors at higher concentrations of IP3 not only mobilizes stored Ca2+, but also activates store-operated influx mechanisms [3 activates Ca2+ channels independently of the filling state of Ca2+ stores [3-dependent mechanisms are also likely to contribute to the prolonged cytosolic Ca2+ transients in GF10903X/staurosporine-treated cells. In support of this contention, the magnitudes of Ca2+ reuptake determined by means of both the Mn2+ quenching of fura-2 fluorescence assay and radiometric procedure were markedly increased in the presence of the PKC inhibitors when neutrophils were activated with PAF at 20 nM, but less so at higher concentrations (200 nM). Ca2+ influx mechanisms are clearly submaximally activated at lower PAF concentrations and can be increased by potentiation of the IP3 signal.Sustained activation of IPchanisms . In addi+ stores . These I3 response to chemoattractants reflect a balance between PLC activity and IP3 metabolism by intracellular phosphomonoesterases [3 [2+ mobilization and IP3 generation are complete. U73122 markedly attenuated the prolongation of cytosolic Ca2+ transients in the presence of the PKC inhibitors, suggesting that persistent PLC activity is primarily responsible for the exaggerated IP3 production. Nevertheless, impaired activation of 5'-phosphomonoesterases cannot be conclusively excluded.The magnitude and duration of the IPsterases -8. Becau2+ transients [Further evidence, albeit indirect, that PKC down-regulates PLC activity, is suggested by our previous observations that co-activation of neutrophils with PAF and a phorbol ester, a direct activator of PKC, attenuates PAF-mediated prolongation of peak cytosolic Caansients .2+-dependent pro-inflammatory activities of neutrophils, we measured the effect of GF10903X on PAF-activated leukotriene B4 (LTB4) production. Production of this highly pro-inflammatory eicosanoid was markedly enhanced by treatment of the cells with the PKC inhibitor, underscoring the role of PKC in down-regulating the Ca2+-dependent pro-inflammatory activities of neutrophils. LTB4 recruits and activates not only neutrophils and other types of inflammatory cells, but also amplifies IP3 production via a positive feedback autocrine loop, whereby LTB4 released from the cell, interacts with its receptor on the plasma membrane to activate PLC [3 generation is sustained and this in turn may exaggerate the pro-inflammatory activity of neutrophils.To determine the functional consequences of inactivation of PKC on the Cavate PLC ,29. Cons2+-dependent pro-inflammatory responses of chemoattractant-activated neutrophils, presumably by phosphorylative inactivation of PLC, resulting in termination of IP3 production. This in turn, favours rapid restoration of Ca2+ homeostasis and attenuation of pro-inflammatory activity, a potentially important physiological mechanism of endogenous control of neutrophil inflammation.In conclusion, the current study has demonstrated that PKC down-regulates CaThe authors declare that they have no competing interests.4 and IP3 experiments; LP assisted in formatting and editing the manuscript and RA assisted with experiments, interpretation of results and editing the manuscript.GRT designed and conducted cytosolic calcium experiments and wrote the manuscript; AJT prepared neutrophil suspensions and assisted with experiments; HCS and RC conducted leukotriene BAll of the authors have read and approved the manuscript.


Dinoflagellates are not only important marine primary producers and grazers, but also the major causative agents of harmful algal blooms. It has been reported that many dinoflagellate species can produce various natural toxins. These toxins can be extremely toxic and many of them are effective at far lower dosages than conventional chemical agents. Consumption of seafood contaminated by algal toxins results in various seafood poisoning syndromes: paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), ciguatera fish poisoning (CFP) and azaspiracid shellfish poisoning (ASP). Most of these poisonings are caused by neurotoxins which present themselves with highly specific effects on the nervous system of animals, including humans, by interfering with nerve impulse transmission. Neurotoxins are a varied group of compounds, both chemically and pharmacologically. They vary in both chemical structure and mechanism of action, and produce very distinct biological effects, which provides a potential application of these toxins in pharmacology and toxicology. This review summarizes the origin, structure and clinical symptoms of PSP, NSP, CFP, AZP, yessotoxin and palytoxin produced by marine dinoflagellates, as well as their molecular mechanisms of action on voltage-gated ion channels. Over the past few decades, the occurrence of harmful algal blooms (HABs) has increased both in frequency and in geographic distribution in many regions of the world. This has resulted in adverse impacts on public health and the economy, and has become a global concern –3. It isOf those causative organisms, dinoflagellates, a very large and diverse group of eukaryotic algae in the marine ecosystem, are the major group producing toxins that impact humans , 10. DinIt is known that most dinoflagellate toxins are neurotoxins, which interact with the specific receptors associated with neurotransmitter receptors, or voltage-sensitive ion channels , resultiA. catenella, which resulted in 102 people being ill and six deaths , indicating that brevetoxin analogues display a range of efficacies to neurotoxin site 2 ligands and are activators of neurotoxin site 5, with PbTx-1 being a full agonist and other derivatives acting as partial agonists i by activation of Ca2+ -release from internal stores and Ca2+-influx, while AZA3 induces only Ca2+-influx. AZA5 does not modify intracellular Ca2+ homeostasis. Recent investigation of the effect of AZA4 on cytosolic calcium concentration [Ca2+]i in fresh human lymphocytes demonstrated that AZA4 inhibits store-operated Ca2+ channels (SOC channels) and Ca2+ influx and that this process is reversible i may be activation of phospholipase C [+ conductive pathway which results in activation of the Na+/H+ exchanger [2+]i independently of the activity of voltage dependent Ca2+ channels and Na+/Ca’+ exchange [+ specific and Ca2+ specific channels. Overall, PTX might posses more than one site of action in excitable cells and act as an agonist for low conductance channels conducting Na+/K+, Ca2+ and H+ ions.Over the past few decades much effort has been devoted to define the action mechanisms of PTXs, however these have not been identified. Pharmacological and electrophysiological studies have demonstrated that PTXs act as a haemolysin and alter the function of excitable cells. PTX selectively binds to the Naof 20 pM  and tranof 20 pM –113. Prelipase C  and phoslipase C . There axchanger , 117. Otexchange . The lasThis paper briefly outlines the origin, structure, symptoms and molecular action mechanisms of neurotoxins produced by marine dinoflagellates. These toxins vary in chemical structure and mechanism of action, and produce very distinct biological effects, which provides a potential application of these toxins in pharmacology and toxicology. However, some of them have not been well studied due to the limited supply of pure toxins and their molecular action mechanisms are unknown. Moreover, novel species of neurotoxins produced by dinoflagellates have been found and identified, which provide a challenge for the characterization of their toxin mechanisms and their effects on marine organisms and humans. Further work using the cell-based approach is needed to determine the precise mode of action of these novel neurotoxins from marine dinoflagellates.


This level may fall further in joint disease. Ionic homeostasis is essential for chondrocyte function but, at least in the case of H+ ions, it is sensitive to changes in O2 levels. Ca2+ homeostasis is also critical but the effect of changes in O2 tension has not been investigated on this parameter. Here we define the effect of hypoxia on Ca2+ homeostasis in bovine articular chondrocytes.Articular chondrocytes normally experience a lower O2+ levels ([Ca2+]i) were followed fluorimetrically using Fura-2 to determine the effect of changes in O2 tension. The effects of ion substitution (replacing extracellular Na+ with NMDG+ and chelating Ca2+ with EGTA) were tested. Levels of reactive oxygen species (ROS) and the mitochondrial membrane potential were measured and correlated with [Ca2+]i.Chondrocytes from articular cartilage slices were isolated enzymatically using collagenase. Cytoplasmic Ca2 tension from 20% to 1% for 16-18 h caused [Ca2+]i to approximately double, reaching 105 ± 23 nM (p < 0.001). Ion substitutions indicated that Na+/Ca2+ exchange activity was not inhibited at low O2 levels. At 1% O2, ROS levels fell and mitochondria depolarised. Restoring ROS levels  concomitantly reduced [Ca2+]i.A reduction in O2 tension exerts a significant effect on [Ca2+]i. The proposed mechanism involves ROS from mitochondria. Findings emphasise the importance of using realistic O2 tensions when studying the physiology and pathology of articular cartilage and the potential interactions between O2, ROS and Ca2+.O Cells eviously . When reChondrocytes were loaded with DCF-DA (10 μM) at 37°C for 45 min i was 60 ± 10 nM at 20% O2 compared with 62 ± 10 nM at 1% O2 . At both O2 tensions, therefore, steady state cytoplasmic Ca levels ([Ca2+]i) remained steady at about 60 nM. We went on to study the effects of longer term hypoxia. Chondrocytes were both digested from their matrix and then maintained for 16-18 hours at either 20% or 1% O2 levels before measuring steady state Ca2+ levels at the same O2 tension. At hypoxic levels, 1% O2, a significant elevation in steady state [Ca2+]i was observed . Thus, like pH, steady state Ca2+ levels in articular chondrocytes are sensitive to changes in O2 albeit with a slower time course.Previously published reports on the effects of hypoxia on pH homeostasis in equine articular chondrocytes demonstrated effects within 3 hours when O0% to 1% . Evidenc2+. Chondrocytes were again isolated, and then maintained for 16-18 hours, at either 20% or 1% O2 in standard Ca2+- and Na+- containing saline. Ca2+ levels were then measured in this standard saline and also following transfer to Ca2+-free or Na+-free saline  of the value at 20% O2. Three different protocols were carried out to elevate ROS levels: treatment with the oxidant H2O2 (100 μM), the non-specific ROS generator Co2+ (100 μM) or the mitochondrial complex III inhibitor antimycin A (50 μM). In each case, ROS levels recorded in treated cells incubated at 1% O2 were restored to those observed at 20%, . Using Fura-2 340 nm:380 nm emission ratio (R) as a measure of [Ca2+]i, in cells incubated at 1% but treated to raise ROS levels, it was found that R decreased by a similar amount, reaching values similar to those observed at 20%. For example, R at 1% following addition of H2O2 fell from 1.41 ± 0.001 to 1.06 ± 0.001 (n = 15). For all three protocols, therefore, at 1% O2 when ROS levels were restored, so was [Ca2+]i.Levels of reactive oxygen species (ROS) in equine articular chondrocytes decrease when O0% to 1% . This fi2 and treatment with antimycin A on mitochondrial pd was then investigated. Chondrocytes were isolated at 20% O2 and then incubated at either 20% O2 or 1% O2 for 16-18 hours prior to loading with JC-1. They were also treated with antimycin A (50 μM) at both O2 tensions  reduction in O2 tension from 20% to 1%. Longer exposure to 1% O2, however, caused significant elevation in [Ca2+]i with levels approximately doubling, sufficient to perturb cell function. These effects were associated with both mitochondrial depolarization and a fall in levels of reactive oxygen species (ROS).The present findings are the first to demonstrate an effect of changes in O2+]i can occur through increased entry or decreased removal across the plasma membrane or from intracellular stores. It is not easy to distinguish unequivocally between these possibilities. Despite a decrease in [Ca2+]i in Ca2+-free saline, however, hypoxic chondrocytes still showed higher Ca2+ compared to those at 20% O2. Thus even if increased influx across the plasma membrane was involved, other mechanisms were still able to elevate Ca2+ during hypoxia. Substitution of extracellular Na+ increased [Ca2+]i and exacerbated the difference at the two O2 tensions. This finding is consistent with elevated activity of NCE at low O2, perhaps in an attempt to reduce Ca2+ to levels found at 20% O2. Since NCE activity requires a functional ATP-driven Na+/K+ pump, it is unlikely that ATP was limiting   to rise. The O2 tension at which perturbation of Ca2+ requires further definition, it being particularly important to study the likely physiological levels of between 10% and 1%.As noted above, it is unlikely that articular chondrocytes require Ooduction , growth oduction , proinfloduction  and matroduction . Dedifferylation . Standarrylation . In effehenotype -23. In arthritis -26. O2 adrocytes ,8. The p2+ in chondrocytes, as in other cell types, also has numerous physiological and probably pathological roles i through effects calcium channel gene expression and activity [2, ROS and Ca2+, together with the role of mitochondria [The elevation of intracellular Care (PTP) -48. ROS re (PTP) ,50, as w and NCE -53. Ca2+reported ,55. To areported ,56,57) aactivity ,59. Therchondria ,53,55 bu2+, whilst restoration of ROS levels to those seen at 20% by three disparate reagents  all resulted in decreased Ca2+. Hypoxia also induced depolarization of mitochondria, indicative of a reduction in electron flow through the mitochondrial electron transport chain, and hence ROS production. Addition of antimycin A also blocks electron transport to the terminal complexes, acting at the Qi site of complex III to increase ROS output [2+, as proposed for O2-induced changes in NHE activity and intracellular pH [+, however, perturbed homeostasis on change in O2 tension is observed rapidly, within a few minutes [2+ appear to occur over a much longer time course, despite sharing sensitivity to ROS levels. The reason for this is not immediately apparent. It may be that Ca2+ homeostasis, as a more critical modulator of chondrocyte function, is better protected than pH. Alternatively, it may be that the mechanism involves genomic effects, such as though involving HIF. In addition, a link between Ca2+ and pH in chondrocytes has been shown previously, with alkalinisation causing a rise in Ca2+ [2, however, rather than increasing their pH, the hypoxia-induced rise in Ca2+ cannot be secondary to changes in pH.We show here that a fall in ROS during hypoxia correlated with elevation of CaS output , as alsolular pH . In the  minutes . Effects in Ca2+ . Since c2 tension exerts a significant effect on cytoplasmic Ca2+ levels of articular chondrocytes, with the proposed mechanism involving ROS from mitochondria. Results emphasise the importance of O2 to chondrocyte function and that of using realistic O2 tensions when studying the pathophysiology of articular cartilage.OThe authors declare that they have no competing interests.RW helped plan the experiments, carried them, analysed the data and helped write the manuscript; JSG planned the experiments, analysed data and prepared the manuscript.All authors have read and approved the final manuscript.


The mechanism of atherogenesis is not yet fully understood despite intense study in this area. The effects of high fat diet (HFD) on the changes of trace elements  in several tissues of rabbits have not been documented before. Thus, the aim of this study was to elucidate the changes in trace elements in several tissues of rabbits fed on HFD for a period of feeding of 10 weeks.The HFD group was fed a NOR rabbit chow supplemented with 1.0% cholesterol plus 1.0% olive oil. Fe, Cu and Zn concentrations were measured in four types of tissue from control and HFD rabbits using atomic absorption spectroscopy (AAS). Comparing HFD rabbits to control rabbits, we found that the highest percentage change of increase of Fe was 95% in lung tissue, while the lowest percentage change of increase of Fe was 7% in kidney tissue; the highest percentage change of decrease of Cu was 16% in aortic tissue, while the lowest percentage change of decrease of Cu was 6% in kidney tissue; and the highest percentage change of decrease of Zn was 71% in kidney tissue, while the lowest percentage change of decrease of Zn was 8% in lung tissue.These results suggest that Fe plays a major role in atherogenesis; it may accelerate the process of atherosclerosis probably through the production of free radicals, deposition and absorption of intracellular and extracellular lipids in the intima, connective tissue formation, smooth muscle proliferation, lower matrix degradation capacity and increased plaque stability. Furthermore, inducing anemia in HFD rabbits may delay or inhibit the progression of atherosclerosis. Cu plays a minor role in atherogenesis and Cu supplements may inhibit the progression of atherogenesis, perhaps by reducing the migration of smooth muscle cells from the media to the intima. Zn plays a major role in atherogenesis and that it may act as an endogenous protective factor against atherosclerosis perhaps by reducing lesion Fe content, intracellular and extracellular lipids in the intima, connective tissue formation, and smooth muscle proliferation. These results suggest that it may be possible to use the measurement of changes in trace elements in different tissues of rabbits as an important risk factor during the progression of atherosclerosis. Atherosclerosis is a disease of large- and medium-sized arteries, and is characterized by endothelial dysfunction , vascular inflammation, migration of smooth muscle cells to the inner lining of the artery (intima) and the build-up of lipids, cholesterol and cellular debris within the intima of the vessel wall. The biological mechanisms by which low density lipoprotein (LDL) promote formation of atherosclerotic plaques are still poorly understood. Oxidation of LDL has been found to increase its uptake in macrophages and lead to formation of macrophage foam cells. Other studies have indicated that oxidized LDL may induce vascular inflammation and even give rise to autoimmune reactions in the vascular wall. These activated macrophages produce numerous factors that are injurious to the endothelium, leading to plaque formation. In later stages, calcification in the damaged region leads to hardening of the artery wall, coupled with acute and chronic arterial obstruction -3.Fe may participate in diverse pathological processes by catalyzing the formation of reactive oxygen free radicals. The oxidation of LDL and lipid is believed to be one of the crucial events leading to plaque formation in vasculature. It has been hypothesized that iron-mediated oxidation is involved in this process. Several epidemiological studies have shown that the level of body Fe stores is positively correlated with the incidence of coronary heart disease in human populations. Additional experiments in animals have further revealed that the severity of atherosclerosis can be markedly influenced by Fe overload or deficiency ,5.Cu supplements inhibit the progression of atherosclerosis by increasing superoxide dismutase (SOD) expression, thereby reducing the interaction of nitric oxide (NO) with superoxide, and hence potentiating NO-mediated pathways that may protect against atherosclerosis .Zn supplementation decreased the elevated levels of cholesterol oxidation products in the aorta and plasma caused by eating a high-cholesterol diet. Several studies have shown that Zn reduced oxidative damage and the risk of cardiovascular disease. Scientists have suggested that because Zn supplementation both reduced the formation of atheromas and lowered lipid peroxidation it may have antioxidant activity. Since Zn is not redox active, it may not act directly as a scavenging antioxidant but instead may act as an indirect antioxidant by competing with pro-oxidant metals such as Fe and Cu for strategic binding sites -9. The rin vivo situation and to avoid autolysis changes and bacterial growth, the aortas, hearts, lungs and kidneys were carefully removed in a manner which avoided any damage to the tissues. Each segment was rapidly flushed with deionized water to remove any residual blood. The tissue samples were flash-frozen in liquid nitrogen and stored at -85°C until analysis.The atherosclerotic model used in this study was the New Zealand white rabbit , obtained from the Laboratory Animal Center . Twenty rabbits were individually caged, and divided into control group and HFD group. The control group (n = 8) was fed on 100 g/day of NOR diet  for a period of 10 weeks. Chemical composition of the laboratory NOR rabbit diet (Purina Certified Rabbit Chow # 5321) is shown in Table Various rabbit tissue samples were wet digested with nitric acid and converted into acidic digest solutions for analysis by AAS method. The tissue was freeze dried in order to minimize loss of analytes and to facilitate subsequent sample preparation steps, and then homogenized to a fine powder by ball-milling in plastic containers. Approximately 0.20 to 0.25 g of powdered tissue was weighed into a Teflon reaction vessel and 3 ml of HNO3 were added. The closed reaction vessel was heated in a 130°C oven until digestion was completed. Samples were then diluted to a final volume of 20 ml with quartz distilled water and stored in 1 oz. polyethylene bottles for later analysis by instrumental techniques.AAS determines the presence and concentration of trace elements  in different tissues of rabbits. Fe, Cu and Zn absorbed ultraviolet (UV) light when they were excited by heat. The AAS instrument looks for a particular metal by focusing a beam of UV light at a specific wavelength through a flame and into a detector. The sample of interest was aspirated into the flame. If that metal is present in the sample, it will absorb some of the light, thus reducing its intensity. The instrument measures the change in intensity. A computer data system converted the change in intensity into an absorbance. As concentration goes up, absorbance goes up. A calibration curve was constructed by running standards of various concentrations  on the AAS and observing the corresponding absorbance. A calibration curve was made and then samples were tested and measured against this curve. AAS measurements were carried out at the Research Center for Girls, King Saud University. Trace elements  were measured using a Specter AA-220 series double-beam digital atomic absorption spectrophotometer. The concentration of trace elements in each tissue sample was calculated by comparing the absorbance produced by the sample with that produced by a series of standards as follows:The results were expressed as mean ± standard error (SE). To assess the significance of the differences between the control group and HFD group of rabbits, statistical analysis was performed using one-way analysis of variance (ANOVA) for repeated measurements, with significance assessed at 5% confidence level.Figure Figure Figure Figures Figure Figure In this study, HFD rabbits were fed normal rabbit chow supplemented with 1.0% cholesterol plus 1.0% olive oil for a period of 10 weeks. We found that in HFD rabbits, the percentage change of Fe was significantly increased in lung by 95%, kidney by 7% heart by 25% and aorta by 33% compared with control rabbits. These results suggest that Fe plays a major role in atherogenesis, probably through the production of free radicals, and that inducing anemia in HFD rabbits may delay or inhibit the progression of atherosclerosis. This study proposes that percentage increase in Fe in several tissues of rabbit may enhance deposition and absorption of intracellular and extracellular lipids in the intima, promote connective tissue formation, and accelerate smooth muscle proliferation leading to lower matrix degradation capacity and increased plaque stability. To clarify the degree of fatty streaks and fibrous plaques, specimens from the aorta of NOR and CHO were stained with Sudan. The aorta of NOR rabbits were completely free of fatty streaks and fibrous plaques, and were characterized by a barely visible intima. On the contrary, the aortic specimens from CHO rabbits exhibited lesions which comprised of fatty streaks and fibrous plaques. Furthermore, to clarify the degree of atherosclerotic lesions, specimens from the aorta of NOR and CHO were stained with hematoxylin and eosin. the aortic specimens from CHO rabbits illustrates marked intimal thickening with focal loss of medial architecture. The intima contains intracellular and extracellular lipids, connective tissue formation, and smooth muscle proliferation.Several epidemiological studies have investigated the role of Fe as a potential risk factor in coronary heart disease, although many of this type of study have conflicting conclusions . It has In this study, percentage change of Cu was significantly decreased in lung by 11%, kidney by 6% heart by 9% and aorta by 16% in HFD rabbits compared with control rabbits. These results suggest that Cu may catalyze free radicals through the Fenton reaction, and that it plays a minor role in atherogenesis because its percentage change is lower than the percentage change of Fe in most of the tissues of HFD rabbits. Furthermore, these results suggest that Cu supplements may inhibit the progression of atherogenesis, perhaps by reducing the migration of smooth muscle cells from the media to the intima. Watt et al.  have repWe found in this study that percentage change of Zn was significantly decreased in lung by 8%, kidney by 71% heart by 14% and aorta by 18% compared with control rabbits. These results suggest that Zn may act as an endogenous protective factor against atherosclerosis, perhaps by reducing lesion Fe content, intracellular and extracellular lipids in the intima, connective tissue formation, and smooth muscle proliferation. Furthermore, our results suggest that Zn supplements may completely inhibit the progression of atherogenesis, perhaps by reducing the percentage change of Fe in most of the tissues of HFD rabbits. A study has shown that Zn can reduce the effects of carotid artery injury induced in rats by balloon dilatation, by reducing smooth muscle cell proliferation and intimal thickening . Zn is bIt becomes evident from this study that the changes in trace elements would alter the initiation and progression of atherosclerosis in HFD rabbits. The evidence for the same can be found only in aortic tissue , but notWe used AAS to elucidate the role of a HFD on trace elements  in different tissues of HFD rabbits. The findings of this study can be summarized as follows: 1) Percentage change of Fe was significantly increased in lung by 95%, kidney by 7%, heart by 25% and aorta by 33% compared with control rabbits. 2) Percentage change of Cu was significantly decreased in lung by 11%, kidney by 6% heart by 9% and aorta by 16% in HFD rabbits compared with control rabbits. 3) Percentage change of Zn was significantly decreased in lung by 8%, kidney by 71% heart by 14% and aorta by 18% compared with control rabbits.These results suggest that Fe plays a major role in atherogenesis; it may accelerate the process of atherosclerosis probably through the production of free radicals, deposition and absorption of intracellular and extracellular lipids in the intima, connective tissue formation, smooth muscle proliferation, lower matrix degradation capacity and increased plaque stability. Furthermore, inducing anemia in HFD rabbits may delay or inhibit the progression of atherosclerosis. Cu plays a minor role in atherogenesis and Cu supplements may inhibit the progression of atherogenesis, perhaps by reducing the migration of smooth muscle cells from the media to the intima. Zn plays a major role in atherogenesis and that it may act as an endogenous protective factor against atherosclerosis perhaps by reducing lesion Fe content, intracellular and extracellular lipids in the intima, connective tissue formation, and smooth muscle proliferation. These results suggest that it may be possible to use the measurement of changes in trace elements in different tissues of rabbits as an important risk factor during the progression of atherosclerosis.Fe: Iron; Cu: Copper; Zn: Zinc; HFD (CHO): high fat diet; AAS: atomic absorption spectroscopy; LDL: low density lipoprotein; SOD: superoxide dismutase; NO: nitric oxide; UV: ultraviolet; NOR: normal.The authors declare that they have no competing interests.MAKA, HAA and SAM equally participated in all experiments, analysis and data interpretation and helped to draft the manuscript. The atherosclerotic model used in this study was obtained from the Laboratory Animal Center . The control and HFD was prepared by Research Diet Inc., New Jersey, USA. MAKA and HAA conceived the study and its design, obtained research grants for its development, supervised all technical activities, coordinated data interpretation and wrote the final version of the manuscript. All authors read and approved the final manuscript.


Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.Human d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of KTogether the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion. Tyrosine kinase signaling pathways play a major role in the regulation of cell growth, division and motility. It is unsurprising, therefore, that aberrations of these pathways can underlie cancerous phenotypes ,2 and thC. elegans gene required for migration of neuronal cells in embryonic development, suggesting a role for the Grb7 family in cell migration [Grb7 is a member of a family of adapter proteins that includes Grb10 and Grb14, and serves to couple activated tyrosine kinases to downstream signaling pathways . These pigration ,9. Grb7 igration  includinigration ,13 whoseigration .Although the precise downstream activities of Grb7 are not yet known, there is compelling evidence that Grb7 represents an important new cancer target . Grb7 isbetween the PH and SH2 domains termed the BPS domain [Members of the Grb7 family share a conserved multi-domain structure comprising an N-terminal proline rich domain, a Ras-associating-like (RA) domain, a plekstrin homology (PH) domain, a C-terminal src-homology 2 (SH2) domain and a region S domain ,22. The S domain ,24. PhosS domain ,26. SubsS domain .Despite the SH2 domains of Grb7, Grb10 and Grb14 proteins exhibiting between 62 – 72% sequence identity, the domains recognise different phosphotyrosine containing sequences. The Grb7 SH2 domain displays a strong preference for phosphotyrosines contained within a pYXN motif, where X is any amino acid . The preGrb7-peptide18-No Arms Thioether), was developed using phage display screening for peptides able to specifically bind Grb7 SH2 [in vivo, but to significantly attenuate the migration of pancreatic cancer cells [Due to the implication of Grb7 in cancer cell progression, an inhibitor specific for Grb7 SH2 domain has been developed that targets the Grb7 SH2 pY peptide binding site . Such a Grb7 SH2 . G7-18NAer cells . In addier cells . G7-18NAAs a step towards determining the means by which Grb7 SH2 recognizes its specific pY peptide substrates and the G7-18NATE peptide inhibitor we report the crystal structure of the human Grb7 SH2 domain to 2.1 Å resolution. This facilitates the first high resolution comparison of the Grb7 SH2 domain with Grb10 and Grb 14 SH2 domains, allowing us to better understand the basis for their differential binding specificities ,37. We afree = 25.5 %) . The fiet al. [The monomer fold of the Grb7 SH2 domain is typical of the SH2 domain family, forming a βαβββββαβ configuration  Figure . The staet al. is adopteThe Grb7 SH2 domain superposes with Grb14  and GrbThe Grb7 SH2 structure differs markedly from that in complex with the ErbB2 receptor derived peptide, pY1139 determined using NMR spectroscopy  with thapo-SH2 domain structures usually exhibits poorly defined electron density [The ligand binding site within SH2 domains comprises the phosphotyrosine (pY) binding pocket and adjacent peptide binding site that confers specificity for the residues immediately C-terminal to the pY . The Grb density , the GrbThe adjacent peptide binding site of the Grb7 SH2 domain differs from that observed in typical SH2 domains, such as the archetypal Src SH2 domain, which bind phosphopeptides in an extended conformation . In thes2 per monomer, representing approximately 7 % of the monomer surface area. The intermolecular interactions between the two protomers that make up the dimer may help define the positions of residues in the BG and EF loops. This is reflected in the B-factors of these residues (amino acids 496 – 499 and 516 – 524), which are lower than the B-factors determined for residues in other loop regions.The Grb7 SH2 domain crystallised as a dimer, forming a dimerisation interface (between chains A and B and between chains C and D in the asymmetric unit) analogous to that observed for Grb10 and Grb14 domain structures ,37. The 2 per monomer and involves contacts between the AA and BC loops and the αA helix of the A chain, and the βD, βD' and βE strands and the BC, DE and EF loops of the D chain. This buried surface area is smaller than that between the AB and CD dimers and is peculiar to this Grb7 SH2 domain crystal form. Since this interface does not involve Phe 511, shown to be critical to dimerisation in solution, we propose that this is not the dimer that predominates in solution.A second dimerisation interface (between chains A and D) was also observed within the asymmetric unit. This interface is not formed within the crystal packing of other Grb7 family member SH2 domains. It occurs with a buried surface area of approximately 400 Åin vivo via its SH2 domain, as also thought to occur for the Grb10 and Grb14 molecules [The observation of dimer formation in the crystal structure prompted us to investigate the strength of the interaction between Grb7 SH2 domains as well as between full-length Grb7 molecules. The dimer dissociation constant was calculated for both the Grb7 SH2 domain and full-length Grb7 at 20°C using analytical ultracentrifugation. Figure olecules ,37.in vivo [d = ~35.7 μ M. This is only an order of magnitude less than the Kd = 2.3 μM measured by Lyons and co-workers for the binding of the Grb7 SH2 domain to pY1139, a native phosphopeptide ligand derived from the ErbB2 receptor [The G7-18NATE peptide Figure  is the fin vivo . These i1H-15N-HSQC spectra were acquired for 15N-labelled Grb7 SH2 as G7-18NATE was titrated in to slight excess  were considered significant. Upon mapping these residues onto the structure of the Grb7 SH2 domain, they clustered into two regions on the protein surface  occurs along the αB helix and BG loop, which forms part of the dimerisation interface of Grb7 SH2, where direct contacts with the peptide ligand would not be expected to take place. Such perturbations may result from impact of G7-18NATE binding on the Grb7 SH2 domain dimer-monomer equilibrium. A shift from dimeric to monomeric Grb7 SH2 domain has previously been reported upon binding by the pY1139 peptide . This waOther Grb7 SH2 residues perturbed by G7-18NATE binding were located outside these two domains and may result from distal structural rearrangements in these regions resulting from the peptide binding. Interestingly, no major perturbation of the Grb7 SH2 DE loop region was observed in either our study of G7-18NATE, or the study of pY1139 binding. It is thus unlikely that the conformational rearrangement of the DE loop reported in the NMR derived structure of the Grb7 SH2/pY1139 peptide complex occurs upon to peptide binding.Together the structural data has allowed a model of the G7-18NATE peptide interacting with the Grb7 SH2 domain to be constructed. In separate studies we have shown that the G7-18NATE peptide has an intrinsic propensity for a turn conformation about the YDN motif . The curThe Grb7 SH2 domain has been identified as a potential target for the development of agents to reduce the invasive potential of cancer cells in which Grb7 is overexpressed. Its crystal structure to 2.1 Å resolution provides structural detail for understanding the basis of interaction with natural targets and designed inhibitors. We have shown that the Grb7 SH2 domain adopts the classic SH2 domain fold excepting, as also observed for Grb10 and Grb14 SH2 domains, an extension of the βD' and βE strands resulting in a longer DE loop ,37,42. ADespite the similarity of the Grb7/10/14 SH2 domains only Grb7 shows preferential binding for a YXN motif ,28,53. GThe current structural data allows the impact of the βD6 residue on the structure of Grb7 SH2 domain to be examined. The βD6 residue occurs at the peptide binding interface at the "specificity determining region" alongside the phosphotyrosine binding pocket. By analogy to the interaction between the Grb2 SH2 domain and pYXN containing phosphopeptides, the βD6 residue forms critical hydrogen bond contacts with the side-chain functionalities of the +2 Asn via both its backbone NH and carbonyl moieties ,52,56. Td of the same order of magnitude as that obtained for the SH2 domain alone. This suggests that the Grb7 SH2 domain forms the principle dimerisation interface within the Grb7 dimer. A similar conclusion was reached for Grb10 by Hubbard and co-workers who found that mutation of Phe 515 to an Arg in full-length Grb10γ (the mutation which produced monomeric Grb10 SH2 domain) generates a monomeric form of the full-length protein [The Grb7 SH2 domain was found to be dimeric in the crystal structure and in solution, forming an analogous dimerisation interface to that observed for Grb10 and Grb14 SH2 domains -39. Dime protein .The physiological importance of Grb7 SH2 domain dimerisation is still uncertain. It has been suggested for Grb10 and Grb14, that the dimeric state of the protein may contribute to a cooperative association of two Grb molecules to form a dimeric activated tyrosine kinase receptor ,37. Mored = ~35.7 μM. Only an order of magnitude lower affinity than that measured for the pY1139 peptide with Grb7 SH2 domain [4 – 105 times weaker than the affinity measured for the binding of the SH2 domain to a phosphorylated form of the peptide, with the difference in the total free energy of complex formation largely the result of the electrostatic interactions between the SH2 domain and the phosphate moiety [The structure of the Grb7 SH2 domain, elucidated by our crystallographic study, has permitted its interaction with a recently developed inhibitor peptide to be examined more closely. The G7-18NATE peptide is an important lead peptide in the development of novel therapeutics targeting cancer cell migration ,28. We re moiety ,58. The in vivo.A modeled interaction of the G7-18NATE peptide with the Grb7 SH2 structure, consistent with both the crystallographic and NMR derived data, provides a starting point for understanding the structural basis for the binding specificity observed. As discussed above, the positioning of Tyr 5 against the βD6 (Leu 481) sidechain of Grb7 SH2 may be critical for constructive interactions with the YDN motif. The modeled interaction also demonstrates that the lack of a phosphate moiety on the Tyr5 of G7-18NATE may be partly compensated for by electrostatic interactions between the Glu3 and the positively charged cleft. Whilst glutamate is often used as a pY mimetic, here it may contribute to binding alongside a non-phosphorylated tyrosine. Asp 6 at the +1 position is also likely to be favoured due to its accommodation without steric hindrance in a pocket of the Grb7 SH2 surface with potential for the formation of hydrogen bond contacts. Based on these observations, second generation peptides and peptidmimetic molecules are currently being developed to potently and selectively inhibit Grb7 in vivo. The interaction of Grb7 SH2 domain with a non-phosphorylated inhibitor peptide (G7-18NATE) has been examined, revealing that this peptide binds with unexpectedly high affinity at the classical peptide binding site and that this impacts upon the dimerisation status of the molecule. The structural basis for their interaction has been modeled based on the biophysical data and represents a starting point for the development of second generation inhibitors of Grb7 based cancer cell migration and proliferation.The crystallographically determined structure and dimer formation of the Grb7 SH2 domain has been described providing a new level of insight into Grb7 target specificity and the likely basis of Grb7 dimer formation Escherichia coli strain BL21(DE3).pLysS as previously described [15N-labelled Grb7 SH2 was overexpressed in minimal media containing 15NH4Cl as its only nitrogen source, using a method similar to that described by Cai and co-workers [expected = 13,672 g/mol, Mobserved = 13,687 g/mol,).The pGex2T plasmid containing the Grb7 SH2 insert (encoding residues 415-532 of human Grb7) was obtained from Dr. Roger Daly . Unlabelescribed . 15N-lab-workers . Both GrE. coli strain BL21(DE3).pLysS. All cultures were grown in LB containing 100 μg/ml ampicillin and 25 μg/ml chloramphenicol. Full-length Grb7 was expressed as a GST fusion and purified using glutathione affinity chromatography. Full-length Grb7 was removed by on column cleavage with PreScision protease (Amersham) and further purified using cation exchange chromatography. The molecular mass of the purified full-length Grb7 was confirmed using MALDI-TOF mass spectrometry .Full-length Grb7 (residues 1 – 532) was subcloned from the mammalian expression vector pRc/CMV into the pGex6P2 expression vector. The identity of the construct was confirmed by DNA sequencing and the construct was used to transform the 280 using an extinction coefficient of 8250 M-1 (Grb7 SH2) and 49500 M-1 (full-length Grb7) [The purified proteins were dialyzed into 50 mM MES (pH 6.6), 100 mM NaCl and 1 mM DTT, concentrated and stored at 4°C. The final concentrations were determined spectrophotometrically at Ath Grb7) .2CO-WFEGYDNTFPC)-amide) was synthesised manually using Fmoc chemistry and standard solid phase peptide synthesis using RINK resin [et al. [et al. [expected = 1417.6 g/mol, Mmeasured = 1417.8 g/mol).G7-18NATE  equipped with focusing optics and a Mar345dtb image plate detector. X-ray diffraction data were measured to a resolution of 2.1 Å and integrated and scaled with DENZO and SCALEPACK [P212121, with unit cell dimensions a = 62.6 Å, b = 63.9 Å, c = 105.7 Å. The data collection statistics are given in Table The Grb7 SH2 domain was crystallized using the hanging drop vapor diffusion method. 2 μl of protein solution (6.5 mg/ml) was mixed with 1 μl of reservoir solution (100 mM sodium citrate (pH 6.1), 22.5% PEG 4000, 0.2 M ammonium sulphate and 5% glycerol) and incubated over 1 ml of reservoir solution at 22°C. Crystals typically grew within 4 days as square plates with dimensions of 100 × 100 × 30 μmCALEPACK . StructuCALEPACK . The difO| - |Fc| electron density map. Ordered water molecules were initially added to the model using ARP waters from within REFMAC5  and lat280 values of 0.1, 0.3 and 0.6. Sedimentation equilibrium experiments were performed at 20°C in an Optima XL-A analytical ultracentrifuge  at rotor speeds of 14,000, 16,600, 24,300 and 28,800 rpm for Grb7 SH2 and 11,800 and 17,300 rpm for full-length Grb7. Cells fitted with conventional Yphantis 12-mm six-channel equilibrium centrepieces [Grb7 SH2 domain and full-length Grb7 were analysed at loading concentrations of Arepieces  and quarrepieces ,49. The repieces .d) and heat of binding (ΔH) were allowed to float.ITC experiments were performed on a VP-ITC Microcalorimeter . Grb7 SH2 domain was dialysed extensively against 50 mM MES (pH 6.6), 100 mM NaCl and 1 mM DTT at 4°C. Lyophilised G7-18NATE was dissolved in the final dialysate and the pH adjusted to 6.6 using 0.1 M NaOH. The reference power was set to 10 μCal/sec and the cell contents were stirred continuously at 310 rpm throughout the titrations. G7-18NATE (440 μM) was titrated into Grb7 SH2 (25 μM) in 30 8 μL injections at 25 °C, with a 5 min delay between each injection, and the heat changes were monitored. A binding isotherm was generated by plotting the heat change evolved per injection against the molar ratio of G7-18NATE to Grb7 SH2. Following baseline correction for heats of dilution , the binding isotherm was fit by a single binding site model using a non-linear least squares method, using Origin . During fitting the stoichiometry (n) was fixed at 1 while the binding constant .Purified 1H resonance frequency of 600.13 MHz using a 5 mm TXI probe  equipped with XYZ-triple gradient capabilities (Bruker). 1H,15N-HSQC spectra were acquired using adapted versions of the published pulse sequences [15N-decoupling during acquisition was achieved using the GARP decoupling scheme [1H carrier frequency was set to that of the water resonance and the 15N carrier frequency was set at a frequency between the Arg Nε and backbone amide resonances. Spectral widths of 12.25 ppm (1H) and 40.0 ppm (15N) were used. Between 32 – 64 scans per increment were collected over 512 t1increments of 1024 complex data points. The spectra were processed on a Silicon graphics O2 workstation using XWINNMR software (Bruker). The 1H frequency was referenced to the water signal at 4.80 ppm, calibrated externally using TSP. The digital resolution was enhanced by zero filling to 2048 × 2048 (F2 × F1) real data points. The spectral resolution of the 15N,1H-HSQC spectra was enhanced by apodisation with a Lorentz-Gauss function in both dimensions. Baselines corrections were applied by subtraction of an automatically calculated polynomial. Cross-peak volume was calculated using the integration function within the XWINNMR software (Bruker).NMR experiments were performed at 298 K on a Bruker Avance 600 operating at a equences ,76. Wateequences . The 15Ng scheme . The 1H The G7-18NATE peptide was constructed using O  with theThe author(s) declares that there are no competing interests.in vivo activities. MCJW solved the crystal structure and supervised building and refinement. JAW conceived the study, instructed in protein preparation, peptide synthesis, NMR and AU analysis and wrote the manuscript with CJP. All authors read, contributed to and approved the final manuscript.CJP prepared proteins for biophysical studies, crystallised the Grb7 SH2 domain, built the crystal structure, analysed the AU data, ran and interpreted the NMR experiments and built the protein/peptide model. JMM designed, conducted and analysed the ITC experiment. SEP and JPM designed and conducted the AU experiments. JWS advised and assisted with x-ray data collection and model building. PJL provided initial protein constructs, protocols and expert advise. SCP and DNK are the developers of the G7-18NATE peptide inhibitor of Grb7 and provided expert advise on its preparation and


When CBS cells were grown in Ca2+-free medium and then switched to medium supplemented with 1.4 mM Ca2+, proliferation was reduced and morphologic features of differentiation were expressed. E-cadherin, which was minimally expressed in nonsupplemented medium, was rapidly induced in response to Ca2+ stimulation. Sustained activation of the extracellular signal-regulated kinase (ERK) occured in Ca2+-supplemented medium. When an inhibitor of ERK activation (10 μM U0126) was included in the Ca2+-supplemented culture medium, ERK-activation did not occur. Concomitantly, E-cadherin was not induced, cell proliferation remained high and differentiation was not observed. The same level of Ca2+ supplementation that induced MAP kinase activation also stimulated CaSR upregulation in CBS cells. A clonal isolate of the CBS line that did not upregulate CaSR expression in response to extracellular Ca2+ was isolated from the parent cells. This isolate failed to produce E-cadherin or undergo growth inhibition/induction of differentiation when exposed to Ca2+ in the culture medium. However, ERK-activation occurred as efficiently in this isolate as in parent CBS cells or in a cloned isolate that underwent growth reduction and differentiation in response to Ca2+ stimulation. Together, these data indicate that CaSR upregulation and MAP kinase signalling are both intermediates in the control of colon carcinoma cell growth and differentiation. They appear to function, at least in part, independently of one another.In the present study, we demonstrate that Ca The CaS2+. Here, we show that the extracellular signal-regulated kinase (ERK) cascade of the mitogen-activated protein (MAP) kinase family is activated in a sustained fashion by extracellular Ca2+ in a line of colon carcinoma cells (CBS), concomitant with growth suppression and induction of differentiation. Interference with ERK activation using the inhibitor U0126 prevents Ca2+-induced alterations in growth and differentiation. A CBS clone that fails to upregulate CaSR in response to Ca2+ also fails to demonstrate growth reduction or induction of differentiation under the same conditions. However, when the CaSR-defective cells are exposed to extracellular Ca2+, ERK activation occurs as efficiently as in the parent cells. Taken together, these data indicate that both CaSR upregulation and MAP kinase signalling are critical intermediates in the control of colon carcinoma cell growth and differentiation. The two events appear to be (at least in part) independent of each other.The present studies further our efforts to understand mechanisms of growth regulation in colon epithelium by extracellular Caβ-tubulin, obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Signalling inhibitors included U0126 (ERK inhibitor), obtained from Cell Signaling Technologies Inc.   supplemented with sodium bicarbonate, amino acids, and 5% foetal bovine serum (FBS). Growth was at 37°C in an atmosphere of 95% air and 5% CO2. Cells were subcultured by brief exposure to trypsin/EDTA solution.The moderately-differentiated colon carcinoma cell line, CBS, was used in these studies. This cell line was described in our past reports dealing with CaSR expression and colon carcinoma growth control . The celμl of CBS-conditioned medium. When individual colonies were of sufficient size, they were subcultured into 25-cm2 flasks. When the flasks reached confluence, there were sufficient cells for use in experiments. Cloned isolates were maintained exactly as the parent cells.Random cell cloning was used to obtain several isolates of the CBS line. Briefly, a single-cell suspension of the parent cells was fractionated using a flow cytometer  and one cell per well deposited into wells of a 96-well dish containing 100 4 cells per well using Ca2+-free SMEM with 5% FBS as culture medium. After allowing the cells to attach overnight, they were washed twice with Ca2+-free SMEM without FBS and then incubated in Ca2+-free SMEM with 5% dialysed FBS. Different amounts of Ca2+ were then added to individual wells as indicated in the Results. Proliferation was measured by releasing the cells with trypsin/EDTA and enumerating them using a particle counter . For dose–response studies, cells were harvested on day-3. For time-course studies, cell counts were made on days 1–4.Cells were plated in 24-well tissue culture dishes at 4 × 10Immunoperoxidase staining for E-cadherin was performed as described previously using formalin-fixed CBS cells . After incubation for the desired amount of time, cells were lysed in 1 × cell lysis buffer consisting of 20 mM Tris-HCl (pH 7.4), 2 mM sodium vanadate, 1.0 mM sodium fluoride, 100 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 25 μg ml−1 each aprotinin, leupeptin and pepstatin, and 2 mM EDTA and EGTA. Lysis was performed at 4°C by scraping the cells into lysis buffer and sonicating the samples. Cell lysates were incubated on ice for 15 min and then cleared by microcentrifugation at 16 000 g for 15 min. The supernatant fluids were collected and protein concentration was estimated using the BioRad DC protein assay kit .CBS cells were plated at 3 × 10μg of total protein per lane) were examined by Western blotting for E-cadherin, CaSR, and signalling intermediates  using procedures described previously  were expressed as mean±s.d. For experiments in which multiple groups were included, statistical analyses were carried out by ANOVA followed by paired group comparisons. For experiments in which two groups were compared to one another, the Student's 2+ levels on proliferation of CBS cells was studied. As shown in M extracellular Ca2+. Growth was inhibited by approximately 45% in the presence of 1.4 mM Ca2+. As shown in the lower panel of 2+ (1.4 mM)-supplemented medium. Statistically significant inhibition was observed on days 2–4. A change in the morphological appearance of the cells accompanied Ca2+-induced growth inhibition. Whereas CBS cells grew in small, grape-like clusters under low-Ca2+ conditions, they became flattened in the presence of Ca2+-supplemented medium  extracellular Ca2+. As compared to cells maintained in SMEM+dialysed FBS (without Ca2+ supplementation) throughout the 3-day incubation period, a reduction in cell growth was observed in the presence of 1.4 mM Ca2+. Concomitantly, cells maintained in Ca2+-supplemented medium underwent a change in morphological appearance (i.e. increased flattening) consistent with differentiation. Both the suppression of proliferation and induction of differentiated features were substantially reversed when U0126 (10 μM) was included in the culture medium throughout the incubation period. These data indicate that Ca2+ modulation of growth and differentiation occurs in a MAP kinase-dependent manner.Following the observation of growth inhibition with increasing concentrations of exogenous Ca2+ on the expression of E-cadherin both by immunostaining and immunoblotting techniques. As shown in 2+-free SMEM plus dialysed FBS for 3 days showed little or no staining for E-cadherin. However, cells grown in the same medium supplemented with 1.4 mM Ca2+ showed significant E-cadherin expression. It could be seen that much of the reactivity in the presence of Ca2+ was at the cell periphery. When cell lysates were prepared and expression of E-cadherin assessed by Western blotting, a 3.5-fold increase in the expression of E-cadherin was observed in cells grown in Ca2+-supplemented medium as compared to the cells grown in Ca2+-free medium (μM) was included in Ca2+-supplemented medium, increased E-cadherin expression was largely abolished , there was no effect on cell growth under low-Ca2+ conditions and only a modest  increase in proliferation compared to control cells in high-Ca2+ medium  (μM) and the PI3K inhibitor  strongly inhibited proliferation of CBS cells under low-Ca2+ conditions but little effect under high-Ca2+ conditions  as well as signalling through the PI+ medium . Consistndition) . Resultsnditions . Consister agent .2+ on CBS growth and differentiation appeared to involve ERK activation, experiments were conducted to determine if the same treatment influenced CaSR expression. Consistent with our recent studies , but the expression level was low. When the extracellular Ca2+ concentration was increased to 1.4 mM, expression of CaSR was substantially increased  while the other was incubated in the same culture medium supplemented with extracellular Ca2+ to a final concentration of 1.4 mM. After 3 days, lysates were prepared from cells in each well and examined for E-cadherin expression by Western blotting. In none of the 12 isolates was there significant E-cadherin expression in the absence of Ca2+-supplementation. In Ca2+-supplemented medium, six of the isolates expressed high levels of E-cadherin while in the other six isolates there was little or no E-cadherin expression. Western blots from representative Ca2+-responsive and -nonresponsive isolates  are shown in 2+-stimulation underwent morphological differentiation in Ca2+-supplemented medium and demonstrated reduced growth as compared to cells in nonsupplemented medium . In contrast, the cells that did not express E-cadherin in response to Ca2+-stimulation failed to differentiate  in Ca2+-supplemented medium and did not demonstrate a significant reduction in growth .In all, 12 separate isolates of randomly-cloned CBS cells were incubated in duplicate wells. One well of each duplicate was maintained in Ca2+-responsive isolate R1 and Ca2+-nonresponsive isolate NR1 were maintained for 3 days in SMEM plus dialyzed FBS with or without added Ca2+. At the end of the incubation period, lysates were prepared and examined for CaSR expression by Western blotting. Both isolates expressed detectable levels of CaSR. In nonsupplemented medium, the levels expressed by the two isolates were similar to the level expressed by the uncloned parent population  suppressed levels of phosphorylated ERK to below baseline levels in both isolates in Ca2+-supplemented medium (not shown).In a final set of experiments, the phosphorylation pattern of ERK was assessed in R1 and NR1 isolates grown for 1 day in SMEM plus dialysed FBS with or without added Ca2+ is a critical regulator of epithelial cell growth and differentiation, but the mechanistic events that lead from an increase in extracellular Ca2+ to growth inhibition and onset of differentiation are not fully understood. Induction of E-cadherin is known to be important. E-cadherin is minimally expressed in rapidly-growing epithelial cells in vitro but is upregulated during differentiation  and downregulation of TCF4 transcription. Additional events associated with Ca2+ treatment in CBS cells included increased production of the cell cycle inhibitors P21 and P27, increased γ-catenin expression and increased cytokeratin expression (Extracellular Ca2+-induced E-cadherin expression (and the subsequent changes in growth and differentiation) in cells derived from human colon cancer. The two events appear to be, at least in part, independent of one another. Using a line of colon carcinoma cells, we found that cells equilibrated in Ca2+-free medium and then exposed to 1.4 mM Ca2+ underwent a (relatively) slow but sustained rise in phosphorylated ERK. The same conditions that led to a rise in ERK phosphorylation (activation) also resulted in expression of E-cadherin and with reduced growth and increased differentiation. U0126, a potent inhibitor of MEK-dependent ERK-activation, prevented Ca2+-induced E-cadherin expression. Concomitantly, the reduction in cell proliferation and onset of differentiation normally seen in Ca2+-supplemented medium did not occur. U0126 was effective at 10 μM, a concentration at which the inhibitor is known to be highly specific for MEK-dependent ERK activation . Activation tends to be rapid (peaks within 2–5 min) and transient, with the active kinases falling to some intermediate level within 15–30 min. Under such conditions, a proliferative response is seen .2+ was shown to induce ERK activation in an immortalised epidermal keratinocyte line  that downstream signalling events would not be required for induction. Additional studies will be needed to verify this suggestion and to fully explore the mechanism of Ca2+-induced CaSR expression.Induction of CaSR appears to be a second critical event leading from Caitamin D . Respons2+ concentration resulted in induction of E-cadherin and inhibition of growth , raising the extracellular Caf growth .


TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored.The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway.Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [CaThis is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis. Signaling cascades involving intracellular Ca2+]i. Voltage dependent Ca2+ channels (Cavs) are important players during signaling and in fact several Cavs have been detected in sperm from different species 2+ signaling in different cell types. TRPs are classified into seven groups  displaying a large variety of gating and regulatory mechanisms, and therefore participate in diverse cellular processes. In sperm, however, only the TRPC subfamily has been explored and indeed several of its members have been detected Cells utilize different mechanisms to regulate their i changes were measured in cell population or single cell experiments in HSM with 2 mM CaCl2 final unless otherwise specified.Motile sperm were adjusted to a concentration of 5−10×106 cells/ml) were placed in a 600 µl cuvette with continuous stirring. Fluorescence intensity was measured with an Aminco 8000 spectrofluorimeter . Data were collected during 300 s at a frequency of 0.5 Hz.Cells (5−10×10Sperm were immobilized on poly-L-lysine (0.1% w/v) coated round coverslips (poly-L-lysine drops were air dried followed by one rinse with water) which were mounted on a chamber and placed on the stage of an inverted Nikon Diaphot 300 microscope. Fluorescence illumination was supplied by a Luxeon V Star Lambertian Cyan LED part # LXHL-LE5C  attached to a custom-built stroboscopic control box. The LED was mounted into a FlashCube40 assembly with dichroic mirror M40-DC400  . LED output was synchronized to the Exposure Out signal of a Cool Snap CCD camera via the control box to produce a single flash of 1 ms duration per individual exposure. The camera exposure time was set equivalent to flash duration (1 ms). Images were collected every 500 ms using IQ software  and a Plan Apo 60x/1.40 -oil- Nikon objective.Coverslips with cells attached were placed in a micro-incubator chamber and then filled with 1 ml of the appropriate medium (HSM) previously warmed at 37°C. The temperature was then lowered to 25°C and data was collected while lowering the temperature from 25 to 13°C with a Bipolar Temperature Controller .http://rsb.info.nih.gov/ij/). Background subtraction was performed using a region of interest placed as close as possible to the sperm of interest. Any incompletely adhered sperm that moved during the course of any experiment were discarded. Fluorescence measurements in individual sperm were made by manually drawing a region of interest around the head for each sperm or a group of cells. Results are presented as pseudo color [Ca2+]i images as indicated on the individual figures.Images were processed using IQ software  and Image J  and self written Igor Programs. A lasso was drawn around the head of each spermatozoon in the field of view. Cells were removed from analysis if the spermatozoon moved out of the lasso. Raw intensity values were imported into Microsoft Excel and normalized using the next equations:Where F is fluorescence intensity at time t and F0 is the mean of F taken during the control period. The total series of (F/F0) – 1 was then plotted vs. time.Where % R is the relative fluorescence normalized to the fluorescence obtained after the menthol or progesterone addition.2+ signaling regulates various important sperm functions. Although several Cavs and TRPC channels have been detected in sperm from different species, little is known about the presence and role of TRPM members of the TRP family in human sperm. In other cell types, these channels sense and respond to different environmental cues such as temperature, osmolarity, pH, mechanical stress, etc., all of which are present during sperm transit through the female tract.CaConsidering the availability, at the time, of specific antibodies, agonists and antagonists against TRPM8 channel, we explored its presence and functional role in human sperm. First, we detected mRNA for TRPM8 in cDNA prepared from human semen. Using anti-TRPM8 we observed the protein in the head (irregular staining) and in the flagella (punctuate pattern) of human sperm . We usedAs TRPM8 was immunologically found uniformly distributed along the sperm flagella, we evaluated if menthol, the principal TRPM8 agonist, influenced the motility pattern of non-capacitated and capacitated sperm using a CASA system. Even at the highest menthol concentration used (1 mM) we did not observe significant changes in the main motility parameters of non-capacitated or capacitated sperm  and capsazepine (20 µM) inhibited the AR by 80% and 70% respectively  and the number of responsive cells to about 15% . The menthol induced [Ca2+]i increase observed in these experiments was always transient and recovered to basal levels after about 40 seconds. Consistent with the AR inhibition observed with capsazepine, this TRPM8 antagonist blocked 60% of the [Ca2+]i rise; similar results were obtained with BCTC (2+]i independently of TRPM8 due to release from intracellular stores 2+ concentrations were lowered until reaching those found intracellularly (∼100 nM). The results showed that under this condition the signal was completely abolished .The menthol induced i responses (B and D) to a decreasing temperature ramp from 25°C to 13°C. A temperature dependent [Ca2+]i increase was clearly observed between 23°C–21°C, which was blocked by capsazepine (2+]i change was above 20 (not shown), confirming the temperature dependence of this process. Representative traces of the temperature-induced [Ca2+]i responses and their inhibition by capsazepine are shown in It is well known that temperature is the universal agonist of TRPM8, varying within the 18°C–25sazepine . The Q102+]i increases and the AR in sperm, it seemed worth asking if this channel participates in the ZP or progesterone induced AR. For this purpose, recombinant human ZP3 (10 ng/µl) and progesterone (4 µM) were used to trigger the AR and BCTC and capsazepine were tested for their ability to inhibit the AR in these conditions. 2+]i increase was also insensitive to capsazepine  that helps sperm penetrate the extracellular egg's matrix and reach the plasma membrane. The egg's extracellular coat is composed of several glycoproteins; human eggs express ZP1, ZP2, ZP3 and ZP4. Most current models consider that ZP3 is the physiological AR inducer and that ZP3 binding to its sperm receptor(s) (not yet fully characterized) produces a biphasic rise in [Ca2+]i. This Ca2+ entry is essential for AR completion. Artificially raising [Ca2+]i with ionophores can trigger AR. At least three different Ca2+ channels have been implicated in the ZP3-induced AR. The pharmacology and kinetics of the first, fast (∼200 ms) [Ca2+]i transient are consistent with opening of Cav channels  v channels usually require a depolarization to open. Though the identity of the depolarizing channel is still unknown, a Cl− channel such as the Glycine receptor has been a proposed candidate 2+-sensitive PLC and IP3 and diacylglycerol production. IP3 then binds to its receptor  located in the acrosome 2+ from this intracellular store which activates a plasma membrane Store Operated Channel (SOC) (third channel) leading to a sustained [Ca2+]i increase lasting several minutes. These SOCs may be constituted by TRP family members As in other exocytotic processes, [Ca2+]i increase, its signaling cascade is different from that of ZP3 2+]i and also induces the AR; however, the pharmacology of these responses differs from that of the physiological inducers, indicating a distinct signaling cascade. In agreement with this notion, TRPM8 null mice are fertile The signaling model described is based mainly on results from mouse sperm, but a similar sequence of events has been proposed for the human AR Finally, TRP channels integrate multiple signals such as temperature, osmolarity, pH changes, mechanical stress, etc. Their heteromultimeric nature contributes to their diverse regulation Figure S1Menthol does not influence human sperm motility. Human sperm separated by swim up were tracked and analyzed with the Hobson Tracker computer-aided semen analysis system. Thirty frames were acquired at a frame rate of 60 Hz. The following parameters are shown: % of motile sperm (%Mot), % of sperm with active motility (%Act), % of sperm with hyperactivated motility (%Hyper), amplitude of lateral head displacement , curvilinear velocity , and the derived parameters of linearity  and straightness . Sperm were exposed to up to 1 mM menthol (white bars) and none of the parameters were significantly different from the control (black bars) in non capacitating (top panel) or capacitating (bottom panel) conditions(n = 7).(0.81 MB TIF)Click here for additional data file.Figure S2Positive control for the TRPM8 antibody. Total protein homogenates from human sperm and mouse brain were subjected to Western Blot experiments with anti-TRPM8 (Santa Cruz Biotechnology) and a band of the appropriate molecular weight (arrow head) was detected in both samples. The additional bands of lower molecular weight probably represent degradation products of the same protein.(0.09 MB TIF)Click here for additional data file.Figure S32+]i responses when exposed to menthol (500 µM) (A) or decreasing temperature (B) in the absence (black) or presence (gray) of 20 µM Cz. This antagonist not only diminishes the magnitude of the responses Click here for additional data file.


Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination.2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca2+-independent signal drifts due to change in cell shape, focus shift, etc.We applied BRET to develop an autoluminescent Ca2+ imaging not only in single live cells but also in small living subjects.The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca Renilla reniformis luciferase (RLuc8) 2+ so it has been used as a Ca2+ indicator to monitor free Ca2+ concentration ([Ca2+]) in living specimens 2+ binding, which takes more than several tens of minutes 2+ indicator was developed based on split RLuc and Gluc 2+-induced interaction between CaM and M13. Although these indicators can detect Ca2+-dependent signal changes in living cells, they could not calibrate [Ca2+] because imaging of these indicators is based on single-emission measurement and thus the signal intensity change is highly dependent on the stoichiometric composition of the N- and C-terminal halves of the split luciferase in the cells. Also, these indicators cannot cancel out signal drift mainly caused by change in cell shape, focus drift, and uptake and consumption of coelenterazine-h due to auto or catalytic oxidization. To allow quantitative long-term real-time Ca2+ imaging, we designed a Ca2+ indicator based on BRET between RLuc8 and Venus, a brighter version of EYFP 2+-sensing domain, we used a Ca2+ binding protein, calmodulin (CaM), and its target peptide, M13, as in the case of the well-known FRET-based Ca2+ indicator, cameleon 2+ from CaM induces reversible conformational change of the CaM-M13 fusion domain between an extended and compact form, which, in turn, changes the distance between Venus and RLuc8 whereby their emission is reciprocally changed due to BRET. Thus, this indicator enables ratiometric observation that can cancel out artifactual signal drift. To obtain a large BRET change, we tried several circularly permuted Venus variants 2+ binding. Due to the brightness and large dynamic range, we successfully visualized Ca2+ oscillation induced by agonist stimulation of Hela cells with improved time resolution and high signal-to-noise ratio.Bioluminescent proteins such as luciferase are a powerful tool for monitoring biological processes including gene expression in living organisms since bioluminescent signals can be acquired without an external light source; bioluminescence imaging is thus completely free from phototoxicity, photo-induced physiological reaction, and autofluorescence from the specimen, enabling signal detection from deep inside the tissue with high signal-to-noise ratio. These properties make bioluminescent proteins potentially superior to fluorescent proteins as a bioimaging tool. However, bioluminescence signals are too dim to be measured in real time, i.e., bioluminescence imaging generally requires longer exposure  than fluorescence imaging that takes less than 1 second. To overcome this drawback, an auto-illuminating fluorescent protein, eBAF-Y, has been developed 2+ indicator, we chose the CaM-M13 chimeric protein for the Ca2+-sensing domain as in the case of the FRET-based Ca2+ indicator, cameleon YC3.60 (see Ref. 12). ECFP in YC3.60 was first replaced with RLuc8, the brightest version of luciferase 2+. The dynamic range of the BRET signal change in RLuc8-CaM-M13-cp173Venus was 10% (BRet-based Autoluminescent Ca2+ indicator), had the largest dynamic range (60%) among the constructs that we tested  which were thought to be derived from both Ca2+ and coelenterazine-h binding to BRAC. Because both kinetics were quite similar, we could not distinguish which component was derived from Ca2+ binding to BRAC. Thus, we then measured Ca2+-dissociation kinetics of BRAC (ι = 0.21 sec) was independent on Ca2+ concentration and much smaller than that of YC3.60  (ι = 0.19 sec) 2+ affinity of BRAC (Kd = 1.9 µM) is about 10 times lower than that of YC3.60 (Kd = 0.25 µM), and indicated that association/dissociation kinetics of BRAC are fast enough to detect Ca2+ dynamics in living cells and organelles.To evaluate the chemical properties of BRAC, Caf 1.9 µM . We alsof 1.9 µM . We then of BRAC . The dis.34 s−1)  and comp2, respectively. The dynamic range obtained was 44% which was comparable to that measured in vitro.In order to determine the validity of BRAC in living cells, we transfected Hela cells with the cDNA encoding BRAC. Direct excitation of the Venus moiety with blue light revealed that BRAC was localized in the cytoplasmic compartment, and slightly in the nucleus . Figure 2+ indicator, we compared performance of BRAC with other BRET-based Ca2+ indicator, G5A 2+ binding. However, G5A failed to report histamine-induced Ca2+ oscillations that were reproducibly observed by using BRAC  are common early event in plant defense signaling, and Ca2+ plays a essential role in activating the plant's surveillance system against attempted microbial invasion Arabidopsis thaliana has resistance to Pseudomonas syringae pv. Tomato (Pst) DC3000 having the avirulence gene avrRpm1 (Pst DC3000/avrRpm1) but susceptible to Pst DC3000 2+]i increase during the plant defense signaling, we inoculated bacterial pathogens into the leaves of BRAC-expressing Arabidopsis lines. We monitored the changes in [Ca2+]i during 30–45 and 80–95 minutes after the bacterial inoculation , Ni-NTA agarose from Qiagen GmbH , Lipofectamine 2000 from Invitrogen , Dulbecco's Modified Eagle's Medium (DMEM) from Sigma , ionomycin from Calbiochem  and fetal bovine serum from BioWest .2+ indicators was constructed by replacing ECFP and cp173Venus in YC3.60 with Venus or circularly permuted Venus variants, and RLuc8, respectively. G5A gene was constructed by fusion of GFP gene with aequorin coding sequences through 5 times repeating SGGSGSGGQ peptide coding sequence as shown previously pGL4.70 [hRLuc] (Promega) was used as the template for introducing eight mutations, A55T/C124A/S130A/K136R/A143M/M185V/M253L/S287L, by means of a site-directed mutagenesis method to yield RLuc8 Escherichia coli [JM109(DE3)] at 23°C, purified using an Ni-NTA column (Qiagen). Emission spectra of BRAC were measured using a spectrophotometer  and a microplate reader . Final concentration of 1–10 µM coelenterazine-h (Promega) was used as the luminescent substrate for RLuc8. Ca2+ titration was performed by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared using O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (EGTA), N-(2-Hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (EDTA-OH), or Nitrilotriacetic acid (NTA) in 100 mM KCl, 10 mM MOPS (pH 7.2). Free Ca2+ concentrations were calculated by using 0.15, 4.3 and 170 µM as the Kd value of EGTA, EDTA-OH and NTA for Ca2+, respectively 2+ titration curve was used to calculate apparent Kd value by non-linear regression analysis. The averaged data from eight independent measurements were fitted to the Hill equation using Origin7 software (OriginLab).Recombinant BRAC protein with N-terminal polyhistidine tags was expressed in 2+ binding kinetics of BRAC were performed by using stopped-flow photometry system consisting of RX.2000 rapid mixing stopped-flow unit  and FP-750 spectrophotometer . Emission intensity of Venus (530 nm) from BRAC were monitored at 1 kHz just after rapid mixing of 5 nM BRAC protein with 20 µM coelenterazine-h in various concentration of Ca2+ buffer. In this experiment, we did not mix coelenterazine-h with BRAC prior to measurement to avoid undesirable consumption of coelenterazine-h by Rluc8 in BRAC during sample preparation. Thus, time course of emission intensity in the stopped-flow experiments consists of three components of kinetics derived from Ca2+ binding to BRAC, coelenterazine-h binding to BRAC, and catalytic oxidation of coelenterazine-h by BRAC. To estimate the catalytic oxidation of coelenterazine-h by Rluc8 in BRAC, we measured time course of emission intensity change after mixing BRAC with 20 µM coelenterazine-h in Ca2+-free solution, and used the obtained data as a “base line”. Then, we measured time course of both association and dissociation of Ca2+ to and from BRAC by mixing 1 volume of BRAC in Ca2+-free buffer with 25 volume of solution containing 1.69 µM Ca2+, and 1 volume of BRAC in 1.69 µM Ca2+ solution with 25 volume of Ca2+-free buffer, respectively. The averaged data from at least 5 independent measurements were used for following analysis. The averaged time course data for association and dissociation kinetics were subtracted by the base line to remove the fraction derived from autonomous catalytic oxidation of coelenterazine-h by BRAC. Then, the time constants (ι) were calculated by means of curve fitting in single exponential equation using the data from 0.2 sec to 2.0 sec to minimize contribution of signal derived from association of coelenterazine-h with BRAC just after mixing. Measurements of Ca2+ binding kinetics of YC3.60 were performed as shown previously 2+ concentration was controlled by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared using O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (EGTA) in 100 mM KCl, 10 mM MOPS (pH 7.2). Free Ca2+ concentration in every solution was confirmed with Ca2+-sensitive electrode  which is calibrated with a CaCl2 standard solution .Measurements of CapH titration was performed using a series of 20 mM buffers with 100 mM KCl in pH 5.7 and 6.2 (MES), 6.6 and 7.0 (MOPS), 7.4 (Tris), 7.8 and 8.0 (HEPES), and 8.6 (Glycin).Hela cells were cultured in a homemade 35-mm glass-bottom dish in DMEM (Sigma) containing 10% fetal bovine serum (BioWest). Cells were transfected with plasmids by means of Lipofectamine 2000 (Invitrogen). At 1 to 2 days after transfection, cells expressing BRAC or G5A were subjected to imaging. 10 µM coelenterazine-h were added to the culture medium just before observation of BRAC and 1–4 hours before observation of G5A.Arabidopsis thaliana  expressing BRAC under control of the 35S promoter. Plants were grown at 22°C under the 8-h-light in a day for 5–6 weeks. For pathology tests, we used Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and an avirulent strain (Pst DC3000/avrRpm1) that was kindly provided by Dr. Jeffery L. Dangl. We cultured the bacteria in KB medium with kanamycin (30 µg/ml) and rifampicin (100 µg/ml) 2, we suspended them at 5×107 cfu/ml for pathology tests. We inoculated the suspensions into the abaxial surfaces of leaves with needleless syringes.We used homozygous 2+, respectively.An inverted microscope  was used to observe BRAC and G5A signals in living cells maintained at 37°C with a continuous supply of 95% air and 5% carbon dioxide by using a stage-top incubator . Light emission from the samples was focused by a 40× oil-immersion objective lens  on a cooled electron-multiplying charge-coupled device (EM-CCD) camera . A 2×2 binning setting was used for every 1-second imaging. To simultaneously acquire both RLuc8 and Venus images, we used a W-View system (Hamamatsu Photonics) composed of 510-nm short pass and 510-nm long pass dichroic mirrors, and F01-479/40 and F01-536/40 bandpass filters (both from Semrock) to split the RLuc8 and Venus images. Image analysis was performed using Aquacosmos (Hamamatsu Photonics). Maximum and minimum BRET ratio values in Hela cells were measured after treatment with 5 µM ionomycin/1 mM EGTA, and 5 µM ionomycin/5 mM CaWe used macro zoom microscopy  with 1× objective lens  on a cooled electron-multiplying charge-coupled device (EM-CCD) camera . A 1×1 binning setting was used for every 10-second imaging. To simultaneously acquire both RLuc8 and Venus images, we used W-View system and mirrors as aforementioned in section of living cell imaging.GenBank accession number for BRAC is GU180352.Video S1Video showing cytosolic Ca2+ oscillation in HeLa cells expressing BRAC in response to the application of 10 (micro)M hisitamine. Video corresponds to the data in (3.60 MB AVI)Click here for additional data file.