Elevation Of Cytoplasmic Calcium Research Articles

Histone deacetylase OsHDA716 represses rice chilling tolerance by deacetylating OsbZIP46 to reduce its transactivation function and protein stability.

Low temperature is a major environmental factor limiting plant growth and crop production. Epigenetic regulation of gene expression is important for plant adaptation to environmental changes, whereas the epigenetic mechanism of cold signaling in rice (Oryza sativa) remains largely elusive. Here, we report that the histone deacetylase (HDAC) OsHDA716 represses rice cold tolerance by interacting with and deacetylating the transcription factor OsbZIP46. The loss-of-function mutants of OsHDA716 exhibit enhanced chilling tolerance, compared with the wild-type plants, while OsHDA716 overexpression plants show chilling hypersensitivity. On the contrary, OsbZIP46 confers chilling tolerance in rice through transcriptionally activating OsDREB1A and COLD1 to regulate cold-induced calcium influx and cytoplasmic calcium elevation. Mechanistic investigation showed that OsHDA716-mediated OsbZIP46 deacetylation in the DNA-binding domain reduces the DNA-binding ability and transcriptional activity as well as decreasing OsbZIP46 protein stability. Genetic evidence indicated that OsbZIP46 deacetylation mediated by OsHDA716 reduces rice chilling tolerance. Collectively, these findings reveal that the functional interplay between the chromatin regulator and transcription factor fine-tunes the cold response in plant and uncover a mechanism by which HDACs repress gene transcription through deacetylating nonhistone proteins and regulating their biochemical functions.

Cholesterol-load evokes robust calcium response in macrophages: An early event toward cholesterol-induced macrophage death

Macrophages in atherosclerotic lesions accumulate large amounts of unesterified cholesterol. Excess cholesterol load leads to cell death of macrophages, which is associated with the progression of atherosclerotic lesions. Calcium depletion in the endoplasmic reticulum (ER) and subsequent pro-apoptotic aberrant calcium signaling are key events in cholesterol-induced macrophage death. Although these concepts imply cytoplasmic calcium events in cholesterol-loaded macrophages, the mechanisms linking cholesterol accumulation to cytoplasmic calcium response have been poorly investigated. Based on our previous finding that extracellularly applied cholesterol evoked robust calcium oscillations in astrocytes, a type of glial cells in the brain, we hypothesized that cholesterol accumulation in macrophages triggers cytoplasmic calcium elevation. Here, we showed that cholesterol application induces calcium transients in THP-1-derived and peritoneal macrophages. Inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) and l-type calcium channels (LTCCs) prevented cholesterol-induced calcium transients and ameliorated cholesterol-induced macrophage death. These results suggest that cholesterol-induced calcium transients through IP3Rs and LTCCs are crucial mechanisms underlying cholesterol-induced cell death of macrophages.

Gut touch: Molecules, cells, circuits, and purpose.

Most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of luminal contents at this interface to perfect function. Specialized sensory enteroendocrine cells (EECs) in the GI epithelium interact intimately with luminal contents. Recently, we discovered an EEC subpopulation that expresses mechanically gated ion channel Piezo2 and is developmentally and functionally like the population of skin's touch sensors - the Merkel cells. Given these similarities, we hypothesized that the gut has intrinsic tactile sensitivity, which we call gut touch. The Piezo2+ EEC mechanotransduction mechanism involves voltage-gated calcium and sodium ion channels and intracellular calcium stores to amplify and prolong the cytoplasmic calcium elevation, which drives the release of signaling molecules such as serotonin. The released EEC signaling molecules activate multiple downstream intrinsic sensory endings that drive intrinsic reflex motility and secretion circuits. I will end by exploring the physiologic purpose of gut touch and how gut touch disruptions lead to human disease.

Elevation of Cytoplasmic Calcium Suppresses Microtentacle Formation and Function in Breast Tumor Cells.

Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca2+) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca2+ signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca2+ elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca2+, suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca2+ elevation was correlated to Ca2+-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca2+-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca2+-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.

Restraint stress of female mice during oocyte development facilitates oocyte postovulatory aging.

Studies suggest that psychological stress on women can impair their reproduction and that postovulatory oocyte aging (POA) might increase the risk of early pregnancy loss and affect offspring's reproductive fitness and longevity. However, whether psychological stress during oocyte development would facilitate POA is unknown but worth exploring to understand the mechanisms by which psychological stress and POA damage oocytes. This study observed effects of female restraint stress during oocyte development (FRSOD) on oocyte resistance to POA. Female mice were restrained for 48 h before superovulation, and they were sacrificed at different intervals after ovulation to recover aging oocytes for analyzing their early and late aged characteristics. The effects of FRSOD on aging oocytes included: (1) increasing their susceptibility to activation stimulus with elevated cytoplasmic calcium; (2) impairing their developmental potential with downregulated expression of development-beneficial genes; (3) facilitating degeneration, cytoplasmic fragmentation and apoptosis; (4) worsening the disorganization of cortical granules and spindle/chromosomes; and (5) impairing redox potential with increased oxidative stress. In conclusion, FRSOD impairs oocyte resistance to POA, so that stressed oocytes become aged significantly quicker than unstressed controls. Thus, couples wishing to achieve pregnancy should take steps to avoid not only fertilization of aged oocytes but also pregestational stressful life events.

Calcium-sensing receptor acts as an antiviral factor for rotavirus infections and participates in cellular antiviral response.

Objective(s):Rotavirus (RV) is one of the most significant pathogens associated with childhood diarrhoeal deaths worldwide. Elevated cytoplasmic calcium is required for RV replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. The Calcium-sensing receptor (CaSR) is an important Ca2+ sensor that regulates the transport of Ca2+ into or out of the extracellular space by affecting the status of Ca2+ ion channels on the membrane of cells. Currently, the function of CaSR in RV replication is unclear. Materials and Methods: We evaluated the mRNA and protein levels of CaSR in RV-infected cells using qRT-PCR and Western blotting, respectively. Furthermore, we silenced or overexpressed CaSR in Caco-2 cells using siRNA or a CaSR gene contained adenovirus (Adv-CaSR). qRT-PCR, plaque assay, and Western blotting were used to determine the synthesis of virus genomic RNA, production of progeny virion, and the levels of viral proteins. The content of Ca2+ in cells was observed under confocal microscopy. Results:Compared with control cells, the RV-infected cells presented significantly decreased CaSR expression. Moreover, adenoviral-mediated over-expression or induction of CaSR by R568 greatly inhibited the RV RNA synthesis, protein expression, and formation of viroplasm plaques, thereby suppressing RV replication. In contrast, CaSR-silenced cells exhibited significantly enhanced RV replication. Compared with the Adv-Control group, the concentration of cytosolic Ca2+ significantly decreased in the Adv-CaSR group. Conclusion:These findings demonstrated that CaSR is a potential target for inhibition of RV replication. Therefore, enhancing the expression of CaSR might protect hosts from RV infections.

Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy.

Nonalcoholic fatty liver disease (NAFLD) is related to elevated cytoplasmic calcium signaling in hepatocytes, which may be mediated by store-operated calcium channel (SOCC) and inositol triphosphate receptor (IP3R). However, the regulatory effect of calcium signaling on lipid accumulation and degeneration in hepatocytes and the underlying molecular mechanism remain unknown. Autophagy inhibition promotes lipid accumulation and steatosis in hepatocytes. However, the association between elevated calcium signaling and autophagy inhibition in hepatocytes and its effect on hepatocyte fatty lesions remain unclear. Here, we established a mouse hepatocyte fatty gradient model using oleic acid. SOCC and IP3R channel opening and cytoplasmic calcium levels gradually increased with the hepatocyte pimelosis degree, whereas autophagy gradually decreased. We also established an optimal oleic acid (OOA) hepatocyte model, observing significantly increased SOCC and IP3R channel opening and calcium influx alongside significantly decreased autophagy and aggravated cellular fatty lesion. Calcium channel blockers (CCBs) and calcium channel gene silencing reagents (CCGSRs), respectively, reversed these effects, indicating that elevated cytoplasmic calcium signaling promotes NAFLD occurrence and the development by inhibiting hepatocyte autophagy. In the OOA model, upregulated extracellular regulated protein kinases 1/2 (ERK1/2), which can be regulated by SOCC and IP3R proteins transient receptor potential canonical 1 (TRPC1)/IP3R with elevated cytoplasmic calcium signaling, over-inhibited forkhead/winged helix O (FOXO) signaling and over-activated mammalian target of rapamycin complex 1 (mTORC1) signaling. Over-inhibited FOXO signaling significantly downregulated autophagy-related gene 12, which inhibits autophagosome maturation, while over-activated mTORC1 signaling over-inactivated Unc-51 like autophagy activating kinase 1, which inhibits preautophagosome formation. CCBs and CCGSRs recovered autophagy by significantly downregulating ERK1/2 to block abnormal changes in FOXO and mTORC1 signaling. Our findings indicate that upregulated SOCC and IP3R channels and subsequent elevated cytoplasmic calcium signaling in hepatocyte fatty lesions inhibits hepatocyte autophagy through (TRPC1/IP3R)/ERK/(FOXO/mTORC1) signaling pathways, causes lipid accumulation and degeneration in hepatocytes, and promotes NAFLD occurrence and development.

Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice

Low temperature is a major environmental factor that limits plant growth and productivity. Although transient elevation of cytoplasmic calcium has long been recognized as a critical signal for plant cold tolerance, the calcium channels responsible for this process have remained largely elusive. Here we report that OsCNGC9, a cyclic nucleotide-gated channel, positively regulates chilling tolerance by mediating cytoplasmic calcium elevation in rice (Oryza sativa). We showed that the loss-of-function mutant of OsCNGC9 is defective in cold-induced calcium influx and more sensitive to prolonged cold treatment, whereas OsCNGC9 overexpression confers enhanced cold tolerance. Mechanistically, we demonstrated that in response to chilling stress, OsSAPK8, a homolog of Arabidopsis thaliana OST1, phosphorylates and activates OsCNGC9 to trigger Ca2+ influx. Moreover, we found that the transcription of OsCNGC9 is activated by a rice dehydration-responsive element-binding transcription factor, OsDREB1A. Taken together, our results suggest that OsCNGC9 enhances chilling tolerance in rice through regulating cold-induced calcium influx and cytoplasmic calcium elevation.

A cyclic nucleotide-gated channel mediates cytoplasmic calcium elevation and disease resistance in rice.

The transient elevation of cytoplasmic calcium is essential for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). However, the calcium channels responsible for this process have remained unknown. Here, we show that rice CDS1 (CELL DEATH and SUSCEPTIBLE to BLAST 1) encoding OsCNGC9, a cyclic nucleotide-gated channel protein, positively regulates the resistance to rice blast disease. We show that OsCNGC9 mediates PAMP-induced Ca2+ influx and that this event is critical for PAMPs-triggered ROS burst and induction of PTI-related defense gene expression. We further show that a PTI-related receptor-like cytoplasmic kinase OsRLCK185 physically interacts with and phosphorylates OsCNGC9 to activate its channel activity. Our results suggest a signaling cascade linking pattern recognition to calcium channel activation, which is required for initiation of PTI and disease resistance in rice.

Resveratrol Directly Controls the Activity of Neuronal Ryanodine Receptors at the Single-Channel Level

Calcium ion dyshomeostasis contributes to the progression of many neurodegenerative diseases and represents a target for the development of neuroprotective therapies, as reported by Duncan et al. (Molecules 15(3):1168-95, 2010), LaFerla (Nat Rev Neurosci 3(11):862-72, 2002), and Niittykoshi et al. (Invest Ophthalmol Vis Sci 51(12):6387-93, 2010). Dysfunctional ryanodine receptors contribute to calcium ion dyshomeostasis and potentially to the pathogenesis of neurodegenerative diseases by generating abnormal calcium ion release from the endoplasmic reticulum, according to Bruno et al. (Neurobiol Aging 33(5):1001 e1-6, 2012) and Stutzmann et al. (J Neurosci 24(2):508-13, 2004). Since ryanodine receptors share functional and structural similarities with potassium channels, as reported by Lanner et al. (Cold Spring Harb Perspect Biol 2(11):a003996, 2010), and small molecules with anti-oxidant properties, such as resveratrol (3,5,4'-trihydroxy-trans-stilbene), directly control the activity of potassium channels, according to Wang et al. (J Biomed Sci 23(1):47, 2016), McCalley et al. (Molecules 19(6):7327-40, 2014), Novakovic et al. (Mol Hum Reprod 21(6):545-51, 2015), Li et al. (Cardiovasc Res 45(4):1035-45, 2000), Gopalakrishnan et al. (Br J Pharmacol 129(7):1323-32, 2000), and Hambrock et al. (J Biol Chem 282(5):3347-56, 2007), we hypothesized that trans-resveratrol can modulate intracellular calcium signaling through direct binding and functional regulation of ryanodine receptors. The goal of our study was to identify and measure the control of ryanodine receptor activity by trans-resveratrol. Mechanisms of calcium signaling mediated by the direct interaction between trans-resveratrol and ryanodine receptors were identified and measured with single-channel electrophysiology. Addition of trans-resveratrol to the cytoplasmic face of the ryanodine receptor increased single-channel activity at physiological and elevated pathophysiological cytoplasmic calcium ion concentrations. The open probability of the channel increases after interacting with the small molecule in a dose-dependent manner, but remains also dependent on the concentration of its physiological ligand, cytoplasmic-free calcium ions. This study provides the first evidence of a direct functional interaction between trans-resveratrol and ryanodine receptors. Such functional control of ryanodine receptors by trans-resveratrol as a novel mechanism of action could provide additional rationales for the development of novel therapeutic strategies to treat and prevent neurodegenerative diseases.

Cold atmospheric plasma causes a calcium influx in melanoma cells triggering CAP-induced senescence

Cold atmospheric plasma (CAP) is a promising approach in anti-cancer therapy, eliminating cancer cells with high selectivity. However, the molecular mechanisms of CAP action are poorly understood. In this study, we investigated CAP effects on calcium homeostasis in melanoma cells. We observed increased cytoplasmic calcium after CAP treatment, which also occurred in the absence of extracellular calcium, indicating the majority of the calcium increase originates from intracellular stores. Application of previously CAP-exposed extracellular solutions also induced cytoplasmic calcium elevations. A substantial fraction of this effect remained when the application was delayed for one hour, indicating the chemical stability of the activating agent(s). Addition of ryanodine and cyclosporin A indicate the involvement of the endoplasmatic reticulum and the mitochondria. Inhibition of the cytoplasmic calcium elevation by the intracellular chelator BAPTA blocked CAP-induced senescence. This finding helps to understand the molecular influence and the mode of action of CAP on tumor cells.

A Poly(A) Ribonuclease Controls the Cellotriose-Based Interaction between Piriformospora indica and Its Host Arabidopsis.

Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. P. indica fungal cell wall extract induces cytoplasmic calcium elevation in host plant roots. Here, we show that cellotriose (CT) is an elicitor-active cell wall moiety released by P. indica into the medium. CT induces a mild defense-like response, including the production of reactive oxygen species, changes in membrane potential, and the expression of genes involved in growth regulation and root development. CT-based cytoplasmic calcium elevation in Arabidopsis (Arabidopsis thaliana) roots does not require the BAK1 coreceptor or the putative Ca2+ channels TPC1, GLR3.3, GLR2.4, and GLR2.5 and operates synergistically with the elicitor chitin. We identified an ethyl methanesulfonate-induced mutant (cytoplasmiccalcium elevation mutant) impaired in the response to CT and various other cellooligomers (n = 2-7), but not to chitooligomers (n = 4-8), in roots. The mutant contains a single nucleotide exchange in the gene encoding a poly(A) ribonuclease (AtPARN; At1g55870) that degrades the poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our identification of cellotriose as a novel chemical mediator casts light on the complex P. indica-plant mutualistic relationship.

Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat oocytes.

Mechanisms for postovulatory aging (POA) of oocytes and for spontaneous activation (SA) of rat oocytes are largely unknown. Expression of calcium-sensing receptor (CaSR) in rat oocytes and its role in POA remain unexplored. In this study, expression of CaSR in rat oocytes aging for different times was detected by immunofluorescence microscopy, and western blotting and the role of CaSR in POA was determined by observing the effects of regulating its activity on SA susceptibility and cytoplasmic calcium levels. The results showed that CaSR was expressed in rat oocytes. Oocytes recovered 19 h post human chorionic gonadotropin (hCG) injection were more susceptible to SA and expressed more functional CaSR than oocytes recovered 13 h after hCG injection, although both expressed the same level of total CaSR protein. Treatment with CaSR antagonist significantly suppressed cytoplasmic calcium elevation and SA of oocytes. Activation of Na-Ca2+ exchanger with NaCl inhibited SA to a greater extent than suppression of CaSR with NPS-2143, suggesting that calcium sources other than CaSR-controlled channels contributed to the elevation of cytoplasmic calcium. Treatment with T- or L-type calcium channel blockers significantly reduced SA. Suppression of all calcium channels tested reduced SA to minimum. It is concluded that the level of CaSR functional dimer protein, but not that of the total CaSR protein, was positively correlated with the SA susceptibility during POA of rat oocytes confirming that CaSR is involved in POA regulation. Blocking multiple calcium channels might be a better choice for efficient control of SA in rat oocytes.

Abstract 3896: Novel in vivo pan-Hsp90 family protein inhibitor, Panvotinib-401, showed potent anticancer activities without HSF1 activation

Abstract Hsp90 family proteins are implicated in tumorigenesis and many inhibitors have been developed as cancer therapeutics. The inhibitors showed potent inhibitory activities against purified Hsp90 family proteins, Grp94 and TRAP1, as well as Hsp90 in vitro. The mitochondrial TRAP1, however, is not inactivated in vivo due to insufficient drug accumulation in the mitochondria. Here, we showed that simultaneous inactivation of Hsp90 family proteins in different compartments can augment anticancer activity of the Hsp90 inhibitors. When simultaneously inhibited, cytoplasmic calcium elevation and activation of calcineurin inhibited HSF1 triggering heat shock responses such as cytoprotective Hsp70 induction. To improve anticancer efficacy of Hsp90 inhibitors, we developed a small molecule (∼380 Da) mitochondrial inner membrane-permeable Hsp90 inhibitor, and named it as Panvotinib-401, briefly Pan-401. Pan-401 showed much improved cancer-selective cytotoxic activity compared with Hsp90 inhibitors or mitochondria-targeted (accumulating) inhibitor, gamitrinib. Collectively, with improved subcellular distribution, cancer-specific cytotoxic activity of Hsp90 inhibitors can be further enhanced. Citation Format: Byoung Heon Kang. Novel in vivo pan-Hsp90 family protein inhibitor, Panvotinib-401, showed potent anticancer activities without HSF1 activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3896.

Bioelectric Signals and Calcium Waves Coordinate Skin Progenitor Cell Movement Patterns during the Polarization of Feather Buds

A crucial challenge for tissue engineering is how to guide stem cells and their progeny to assemble into the unique organ shapes and structures. Currently known organizers for organogenesis include biochemical molecules, physical forces and bioelectric signals. One manifestation of bioelectric signaling in developing embryos is a relatively steady and long-lasting direct current (DC) and associated electric field. Using a vibrating probe, we detected the endogenous DC currents at different developmental stages of chicken feather buds. Interestingly, reversion of the direction of DC currents was observed at embryonic day 9 in anterior regions of feather buds. Perturbation of the endogenous DC currents by applying pulsed electric fields in the developing feather buds led to reorientation of the feather buds. Molecular studies revealed that T-type voltage-gated calcium channels (VGCCs) were expressed in anterior bud epithelial cells and mesenchymal cells, but rarely in posterior regions. Perfusion of high KCl solution induced cytoplasmic calcium elevations in epithelial and mesenchymal cells. The epithelial cells showed mosaic patterns of calcium fluctuations, while the mesenchymal cells had synchronized calcium fluctuations. Long-term calcium imaging demonstrated brief calcium transients in epithelial cells but systematic calcium waves in mesenchymal cells during feather polarization. Moreover, cell nucleus imaging revealed the movement of anterior bud epithelial cells toward posterior regions and the upward movement of mesenchymal cells during feather polarization. Functional perturbation of T-type VGCCs disrupted the patterns of cell movement and feather polarity, confirming the importance of bioelectric signals and calcium waves in feather polarity establishment.

The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses.

BackgroundVerticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years.ResultsHere we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host.ConclusionsWe conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0268-5) contains supplementary material, which is available to authorized users.

Comparative Analysis of MAMP-induced Calcium Influx in Arabidopsis Seedlings and Protoplasts

Rapid transient elevation of cytoplasmic calcium (Ca(2+)) levels in plant cells is an early signaling event triggered by many environmental cues including abiotic and biotic stresses. Cellular Ca(2+) levels and their alterations can be monitored by genetically encoded reporter systems such as the bioluminescent protein, aequorin. Employment of proteinaceous Ca(2+) sensors is usually performed in transgenic lines that constitutively express the reporter construct. Such settings limit the usage of these Ca(2+) biosensors to particular reporter variants and plant genetic backgrounds, which can be a severe constraint in genetic pathway analysis. Here we systematically explored the potential of Arabidopsis thaliana leaf mesophyll protoplasts, either derived from a transgenic apoaequorin-expressing line or transfected with apoaequorin reporter constructs, as a complementary biological resource to monitor cytoplasmic changes of Ca(2+) levels in response to various biotic stress elicitors. We tested a range of endogenous and pathogen-derived elicitors in seedlings and protoplasts of the corresponding apoaequorin-expressing reporter line. We found that the protoplast system largely reflects the Ca(2+) signatures seen in intact transgenic seedlings. Results of inhibitor experiments including the calculation of IC50 values indicated that the protoplast system is also suitable for pharmacological studies. Moreover, analyses of Ca(2+)signatures in mutant backgrounds, genetic complementation of the mutant phenotypes and expression of sensor variants targeted to different subcellular localizations can be readily performed. Thus, in addition to the prevalent use of seedlings, the leaf mesophyll protoplast setup represents a versatile and convenient tool for the analysis of Ca(2+) signaling pathways in plant cells.

Non-contact High-Frequency Ultrasound Microbeam Stimulation for Studying Mechanotransduction in Human Umbilical Vein Endothelial Cells

We describe how contactless high-frequency ultrasound microbeam stimulation (HFUMS) is capable of eliciting cytoplasmic calcium (Ca2+) elevation in human umbilical vein endothelial cells. The cellular mechanotransduction process, which includes cell sensing and adaptation to the mechanical micro-environment, has been studied extensively in recent years. A variety of tools for mechanical stimulation have been developed to produce cellular responses. We developed a novel tool, a highly focused ultrasound microbeam, for non-contact cell stimulation at a microscale. This tool, at 200 MHz, was applied to human umbilical vein endothelial cells to investigate its potential to elicit an elevation in cytoplasmic Ca2+ levels. It was found that the response was dose dependent, and moreover, extracellular Ca2+ and cytoplasmic Ca2+ stores were involved in the Ca2+ elevation. These results suggest that high-frequency ultrasound microbeam stimulation is potentially a novel non-contact tool for studying cellular mechanotransduction if the acoustic pressures at such high frequencies can be quantified.

Cross-sectional and longitudinal associations between parents’ and preschoolers’ physical activity and TV viewing

colonic tissue mRNA libraries and validated an in vitro human enteroendocrine I-cell model (Hutu80) using a variety of known TAS2R bitter agonists. We show that mRNAs for bitter taste receptors are expressed in human gastric, small intestinal and colonic tissue as well as the Hutu-80 cellline. In addition, Hutu-80 cells respond to a subset of known bitter agonists via a G-protein coupled phospholipase C mediated pathway, elevated cytoplasmic calcium concentrations and subsequent release of CCK. In conclusion, the capability to sense luminal bitter compounds may occur throughout the GI tract. However, not all known lingual bitter agonists stimulate hormone secretion in Hutu-80 cells, suggesting key differences between lingual and GI bitter sensing that may have important implications for design of functional foods to modulate appetite through bitter sensing.

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

Rotavirus nonstructural protein 4 (NSP4) induces dramatic changes in cellular calcium homeostasis. These include increased endoplasmic reticulum (ER) permeability, resulting in decreased ER calcium stores and activation of plasma membrane (PM) calcium influx channels, ultimately causing a 2- to 4-fold elevation in cytoplasmic calcium. Elevated cytoplasmic calcium is absolutely required for virus replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. NSP4 is an ER-localized viroporin, whose activity depletes ER calcium, which ultimately leads to calcium influx. We hypothesized that NSP4-mediated depletion of ER calcium activates store-operated calcium entry (SOCE) through activation of the ER calcium sensor stromal interaction molecule 1 (STIM1). We established and used a stable yellow fluorescent protein-expressing STIM1 cell line (YFP-STIM1) as a biosensor to assess STIM1 activation (puncta formation) by rotavirus infection and NSP4 expression. We found that STIM1 is constitutively active in rotavirus-infected cells and that STIM1 puncta colocalize with the PM-localized Orai1 SOCE calcium channel. Expression of wild-type NSP4 activated STIM1, resulting in PM calcium influx, but an NSP4 viroporin mutant failed to induce STIM1 activation and did not activate the PM calcium entry pathway. Finally, knockdown of STIM1 significantly reduced rotavirus yield, indicating STIM1 plays a critical role in virus replication. These data demonstrate that while rotavirus may ultimately activate multiple calcium channels in the PM, calcium influx is predicated on NSP4 viroporin-mediated activation of STIM1 in the ER. This is the first report of viroporin-mediated activation of SOCE, reinforcing NSP4 as a robust model to understand dysregulation of calcium homeostasis during virus infections.