Potential Mechanism and Involvement of P120-Catenin in the Malignant Biology of Glioma.

Objective: This study analyzed the influence of p120-catenin (CTNND1) on the malignant characteristics of glioma and elucidated the potential underlying mechanism. Methods: The p120 expression level was assessed in the brain tissues of 42 glioma patients and 10 patients with epilepsy by using the immunohistochemical method. Meanwhile, quantitative PCR technology was employed to assess the expression of P120 in the brain tissues of 71 glioma patients and 13 epilepsy patients. LN229, U251, and U87 glioma cells were used for in vitro analysis and categorized into four treatment groups: siRNA-BC group (no RNA sequence was transfected), siRNA-NC group (transfected control RNA sequences with no effect), and siRNA-1 and siRNA-2 groups (two p120-specific interfering RNA transfection). p120 expression in these treatment groups was quantified by western blotting assay. The migratory and invasive capabilities of glioma cells were studied by wound healing assay and Transwell invasion assay, respectively, under different treatment conditions. MTT assay and cell cycle and apoptosis assay were used to determine glioma cell proliferation and apoptosis, respectively. Enzyme-labeled assay was performed to measure intracellular calcium ion concentration. Immunofluorescence assay was performed for determining microtubule formation and glioma cell distribution. Results: Brain tissues of the glioma group exhibited a remarkable increase in the p120 expression level as compared to brain tissues of the nontumor group (P < 0.05). Furthermore, a strong positive correlation was noted between the malignancy degree in glioma brain tissues and p120 expression in Western blotting (r = 0.906, P = 0.00) and QT-PCR (F=830.6, P＜0.01). Compared to the BC and NC groups, the siRNA transfection groups showed a significant suppression in p120 expression in glioma cells (P < 0.05), with a marked attenuation in the invasive, migratory, and proliferative capabilities of glioma cells as well as an increase in apoptotic potential (P < 0.05). Enzyme-labeled assay showed a remarkable increase in calcium concentration in glioma cells after siRNA treatment. Immunofluorescence assay revealed that the microtubule formation ability of glioma cells reduced after siRNA treatment. Conclusion: p120 has a pivotal involvement in facilitating glioma cell invasion and proliferation by potentially modulating these processes through its involvement in microtubule formation and regulation of intracellular calcium ion levels.

Measuring Connexin Hemichannel Opening in Response to an InsP3-Mediated Cytosolic Ca2+ Increase.

The opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulation by a canonical InsP3-mediated [Ca2+]c transient. The protocol relies on a combination of Ca2+ imaging and patch-clamp techniques. The insights gained from our method are expected to make a significant contribution to understanding the structure-function relationship of connexin HCs. The protocol is also suitable to screen candidate therapeutic compounds to treat connexin-related diseases linked to HC dysfunction.

A tale of two pumps: Blue light and abscisic acid alter Arabidopsis leaf hydraulics via bundle sheath cell H + -ATPases.

The bundle sheath cell (BSC) layer tightly enveloping the xylem throughout the leaf is recognized as a major signal-perceiving "valve" in series with stomata, regulating leaf hydraulic conductance (Kleaf) and thereby radial water flow via the transpiring leaf. The BSC blue light (BL) signaling pathway increases Kleaf and the underlying BSC water permeability. Here, we explored the hypothesis that BSCs also harbor a Kleaf-downregulating signaling pathway related to the stress phytohormone abscisic acid (ABA). We employed fluorescence imaging of xylem sap in detached leaves and BSC protoplasts from different genotypes of Arabidopsis (Arabidopsis thaliana) plants, using pH and membrane potential probes to monitor physiological responses to ABA and BL in combination with pharmacological agents. We found that BL-enhanced Kleaf required elevated BSC cytosolic Ca2+. ABA inhibited BL-activated xylem-sap-acidifying BSC H + -ATPase AHA2 (Arabidopsis H + -ATPase 2), resulting in depolarized BSCs and alkalinized xylem sap. ABA also stimulated BSC vacuolar H + -ATPase (VHA), which alkalinized the BSC cytosol. Each pump stimulation, AHA2 by BL and VHA by ABA (under BL), also required Ca2+. ABA stimulated VHA in the dark depending on Ca2+, but only in an alkaline external medium. Taken together with earlier findings on the pH sensitivity of BSC osmotic water permeability (i.e., aquaporin activity), our results suggest a Ca2+-dependent and pH-mediated causative link between the BL- and ABA-regulated activities of two BSC H + -ATPases and Kleaf.

Commonality and heterogeneity of pacemaker mechanisms in the male reproductive organs.

During emission, the first phase of ejaculation, smooth muscle in organs of the male reproductive tract (MRT) vigorously contract upon sympathetic nerve excitation to expel semen consisting of sperm and seminal plasma. During inter-ejaculation phases, the epididymis, seminal vesicles and prostate undergo spontaneous phasic contractions (SPCs), this transporting and maintaining the quality of sperm and seminal plasma. Recent studies have revealed platelet-derived growth factor receptor α-expressing (PDGFRα+) subepithelial interstitial cells in seminal vesicles subserve the role of pacemaker cells that electrically drive SPCs in this organ. PDGFRα+ smooth muscle cells in the epididymis also appear to function as pacemaker cells implicating PDGFRα as a potential signature molecule in MRT pacemaking. The dominant mechanism driving pacemaking in these organs is the cytosolic Ca2+ oscillator. This operates through entrainment of the release-refill cycle of Ca2+ stores, the released Ca2+ ions opening Ca2+-activated chloride channels, including in some cases ANO1 (TMEM16A), with the resultant pacemaker potential activating L-type voltage-dependent Ca2+ channels in the smooth muscle causing contraction (viz. SPCs). A second pacemaker mechanism, namely the membrane oscillator also has a role in specific cases. Further investigations into the commonality and heterogeneity of MRT pacemakers will open an avenue for understanding the pathogenesis of male infertility associated with deterioration of seminal plasma.

Role of cytosolic and endoplasmic reticulum Ca2+ in pancreatic beta-cells: pros and cons.

Pancreatic beta cells utilize Ca2+ to secrete insulin in response to glucose. The glucose-dependent increase in cytosolic Ca2+ concentration ([Ca2+]C) activates a series of insulin secretory machinery in pancreatic beta cells. Therefore, the amount of insulin secreted in response to glucose is determined in a [Ca2+]C-dependent manner, at least within a moderate range. However, the demand for insulin secretion may surpass the capability of beta cells. Abnormal elevation of [Ca2+]C levels beyond the beta-cell endurance capacity can damage them by inducing endoplasmic reticulum (ER) stress and cell death programs such as apoptosis. Therefore, while Ca2+ is essential for the insulin secretory functions of beta cells, it could affect their survival at pathologically higher levels. Because an increase in beta-cell [Ca2+]C is inevitable under certain hazardous conditions, understanding the regulatory mechanism for [Ca2+]C is important. Therefore, this review discusses beta-cell function, survival, ER stress, and apoptosis associated with intracellular and ER Ca2+ homeostasis.

Plant Stomata: An Unrealized Possibility in Plant Defense against Invading Pathogens and Stress Tolerance.

Stomata are crucial structures in plants that play a primary role in the infection process during a pathogen's attack, as they act as points of access for invading pathogens to enter host tissues. Recent evidence has revealed that stomata are integral to the plant defense system and can actively impede invading pathogens by triggering plant defense responses. Stomata interact with diverse pathogen virulence factors, granting them the capacity to influence plant susceptibility and resistance. Moreover, recent studies focusing on the environmental and microbial regulation of stomatal closure and opening have shed light on the epidemiology of bacterial diseases in plants. Bacteria and fungi can induce stomatal closure using pathogen-associated molecular patterns (PAMPs), effectively preventing entry through these openings and positioning stomata as a critical component of the plant's innate immune system; however, despite this defense mechanism, some microorganisms have evolved strategies to overcome stomatal protection. Interestingly, recent research supports the hypothesis that stomatal closure caused by PAMPs may function as a more robust barrier against pathogen infection than previously believed. On the other hand, plant stomatal closure is also regulated by factors such as abscisic acid and Ca2+-permeable channels, which will also be discussed in this review. Therefore, this review aims to discuss various roles of stomata during biotic and abiotic stress, such as insects and water stress, and with specific context to pathogens and their strategies for evading stomatal defense, subverting plant resistance, and overcoming challenges faced by infectious propagules. These pathogens must navigate specific plant tissues and counteract various constitutive and inducible resistance mechanisms, making the role of stomata in plant defense an essential area of study.

Ion Changes and Signaling under Salt Stress in Wheat and Other Important Crops.

High concentrations of sodium (Na+), chloride (Cl-), calcium (Ca2+), and sulphate (SO42-) are frequently found in saline soils. Crop plants cannot successfully develop and produce because salt stress impairs the uptake of Ca2+, potassium (K+), and water into plant cells. Different intracellular and extracellular ionic concentrations change with salinity, including those of Ca2+, K+, and protons. These cations serve as stress signaling molecules in addition to being essential for ionic homeostasis and nutrition. Maintaining an appropriate K+:Na+ ratio is one crucial plant mechanism for salt tolerance, which is a complicated trait. Another important mechanism is the ability for fast extrusion of Na+ from the cytosol. Ca2+ is established as a ubiquitous secondary messenger, which transmits various stress signals into metabolic alterations that cause adaptive responses. When plants are under stress, the cytosolic-free Ca2+ concentration can rise to 10 times or more from its resting level of 50-100 nanomolar. Reactive oxygen species (ROS) are linked to the Ca2+ alterations and are produced by stress. Depending on the type, frequency, and intensity of the stress, the cytosolic Ca2+ signals oscillate, are transient, or persist for a longer period and exhibit specific "signatures". Both the influx and efflux of Ca2+ affect the length and amplitude of the signal. According to several reports, under stress Ca2+ alterations can occur not only in the cytoplasm of the cell but also in the cell walls, nucleus, and other cell organelles and the Ca2+ waves propagate through the whole plant. Here, we will focus on how wheat and other important crops absorb Na+, K+, and Cl- when plants are under salt stress, as well as how Ca2+, K+, and pH cause intracellular signaling and homeostasis. Similar mechanisms in the model plant Arabidopsis will also be considered. Knowledge of these processes is important for understanding how plants react to salinity stress and for the development of tolerant crops.

Cytosolic Sodium Influx in Mesophyll Protoplasts of Arabidopsis thaliana, wt, sos1:1 and nhx1 Differs and Induces Different Calcium Changes.

The sodium influx into the cytosol of mesophyll protoplasts from Arabidopsis thaliana cv. Columbia, wild type, was compared with the influx into sos1-1 and nhx1 genotypes, which lack the Na+/H+ antiporter in the plasma membrane and tonoplast, respectively. Changes in cytosolic sodium and calcium concentrations upon a 100 mM NaCl addition were detected by use of epifluorescence microscopy and the sodium-specific fluorescent dye SBFI, AM, and calcium sensitive Fura 2, AM, respectively. There was a smaller and mainly transient influx of Na+ in the cytosol of the wild type compared with the sos1-1 and nhx1 genotypes, in which the influx lasted for a longer time. Sodium chloride addition to the protoplasts' medium induced a significant increase in cytosolic calcium concentration in the wild type at 1.0 mM external calcium, and to a lesser extent in nhx1, however, it was negligible in the sos1-1 genotype. LiCl inhibited the cytosolic calcium elevation in the wild type. The results suggest that the salt-induced calcium elevation in the cytosol of mesophyll cells depends on an influx from both internal and external stores and occurs in the presence of an intact Na+/H+ antiporter at the plasma membrane. The Arabidopsis SOS1 more effectively regulates sodium homeostasis than NHX1.

Quantitative Analysis of Plant Cytosolic Calcium Signals in Response to Water Activated by Low-Power Non-Thermal Plasma.

Non-thermal plasma technology is increasingly being applied in the plant biology field. Despite the variety of beneficial effects of plasma-activated water (PAW) on plants, information about the mechanisms of PAW sensing by plants is still limited. In this study, in order to link PAW perception to the positive downstream responses of plants, transgenic Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin in the cytosol were challenged with water activated by low-power non-thermal plasma generated by a dielectric barrier discharge (DBD) source. PAW sensing by plants resulted in the occurrence of cytosolic Ca2+ signals, whose kinetic parameters were found to strictly depend on the operational conditions of the plasma device and thus on the corresponding mixture of chemical species contained in the PAW. In particular, we highlighted the effect on the intracellular Ca2+ signals of low doses of DBD-PAW chemicals and also presented the effects of consecutive plant treatments. The results were discussed in terms of the possibility of using PAW-triggered Ca2+ signatures as benchmarks to accurately modulate the chemical composition of PAW in order to induce environmental stress resilience in plants, thus paving the way for further applications in agriculture.

d-Allulose Inhibits Ghrelin-Responsive, Glucose-Sensitive and Neuropeptide Y Neurons in the Arcuate Nucleus and Central Injection Suppresses Appetite-Associated Food Intake in Mice.

d-allulose, a rare sugar, has sweetness with few calories. d-allulose regulates feeding and glycemia, and ameliorates hyperphagia, obesity and diabetes. All these functions involve the central nervous system. However, central mechanisms underlying these effects of d-allulose remain unknown. We recently reported that d-allulose activates the anorexigenic neurons in the hypothalamic arcuate nucleus (ARC), the neurons that respond to glucagon-like peptide-1 and that express proopiomelanocortin. However, its action on the orexigenic neurons remains unknown. This study investigated the effects of d-allulose on the ARC neurons implicated in hunger, by measuring cytosolic Ca2+ concentration ([Ca2+]i) in single neurons. d-allulose depressed the increases in [Ca2+]i induced by ghrelin and by low glucose in ARC neurons and inhibited spontaneous oscillatory [Ca2+]i increases in neuropeptide Y (NPY) neurons. d-allulose inhibited 10 of 35 (28%) ghrelin-responsive, 18 of 60 (30%) glucose-sensitive and 3 of 8 (37.5%) NPY neurons in ARC. Intracerebroventricular injection of d-allulose inhibited food intake at 20:00 and 22:00, the early dark phase when hunger is promoted. These results indicate that d-allulose suppresses hunger-associated feeding and inhibits hunger-promoting neurons in ARC. These central actions of d-allulose represent the potential of d-allulose to inhibit the hyperphagia with excessive appetite, thereby counteracting obesity and diabetes.

D-Allulose cooperates with glucagon-like peptide-1 and activates proopiomelanocortin neurons in the arcuate nucleus and central injection inhibits feeding in mice.

A rare sugar D-Allulose has sweetness without calorie. Previous studies have shown that D-Allulose improves glucose and energy metabolism and ameliorates obesity. However, underlying mechanisms remain elusive. This study explored the effect of central injection of D-Allulose on feeding behavior in mice. We also examined direct effects of D-Allulose on the neurons in the hypothalamic arcuate nucleus (ARC) that regulate feeding, including the anorexigenic glucagon-like peptide-1 (GLP-1)-responsive neurons and proopiomelanocortin (POMC) neurons. Single neurons were isolated from ARC and cytosolic Ca2+ concentration ([Ca2+]i) was measured by fura-2 microfluorometry. Administration of D-Allulose at 5.6, 16.7 and 56 mM concentration-dependently increased [Ca2+]i in ARC neurons. The [Ca2+]i increases took place similarly when the osmolarity of superfusion solution was kept constant. The majority (40%) of the D-Allulose-responsive neurons also responded to GLP-1 with [Ca2+]i increases. D-Allulose increased [Ca2+]i in 33% of POMC neurons in ARC. D-Allulose potentiated the GLP-1 action to increase [Ca2+]i in ARC neurons including POMC neurons. Intracerebroventricular injection of D-Allulose significantly decreased food intake at 1 and 2 h after injection. These results demonstrate that D-Allulose cooperates with glucagon-like peptide-1 and activates the ARC neurons including POMC neurons. Furthermore, central injection of D-Allulose inhibits feeding. These central actions of D-Allulose may underlie the ability of D-Allulose to counteract obesity and diabetes.

ASIC1a senses lactate uptake to regulate metabolism in neurons.

Lactate is a major metabolite largely produced by astrocytes that nourishes neurons. ASIC1a, a Na+ and Ca2+-permeable channel with an extracellular proton sensing domain, is thought to be activated by lactate through chelation of divalent cations, including Ca2+, Mg2+ and Zn2+, that block the channel pore. Here, by monitoring lactate-evoked H+ and Ca2+ transport in cultured mouse cortical and hippocampal neurons, we find that stereo-selective neuronal uptake of L-lactate results in rapid intracellular acidification that triggers H+ extrusion to activate plasma membrane ASIC1a channels, leading to propagating Ca2+ waves into the cytosol and mitochondria. We show that lactate activates ASIC1a at its physiological concentrations, far below that needed to chelate divalent cations. The L-isomer of lactate exerts a much greater effect on ASIC1a-mediated activity than the d-isomer and this stereo-selectivity arises from lactate transporters, which prefer the physiologically common L-lactate. The lactate uptake in turn results in intracellular acidification, which is then followed by a robust acid extrusion. The latter response sufficiently lowers the pH in the vicinity of the extracellular domain of ASIC1a to trigger its activation, resulting in cytosolic and mitochondrial Ca2+ signals that accelerate mitochondrial respiration. Furthermore, blocking ASIC1a led to a robust mitochondrial ROS production induced by L-lactate. Together our results indicate that ASIC1a is a metabolic sensor, which by sensing extracellular pH drop triggered by neuronal lactate uptake with subsequent proton extrusion, transmits a Ca2+ response that is propagated to mitochondria to enhance lactate catabolism and suppress ROS production.

Late Na+ Current Is [Ca2+]i-Dependent in Canine Ventricular Myocytes.

Enhancement of the late sodium current (INaL) increases arrhythmia propensity in the heart, whereas suppression of the current is antiarrhythmic. In the present study, we investigated INaL in canine ventricular cardiomyocytes under action potential voltage-clamp conditions using the selective Na+ channel inhibitors GS967 and tetrodotoxin. Both 1 µM GS967 and 10 µM tetrodotoxin dissected largely similar inward currents. The amplitude and integral of the GS967-sensitive current was significantly smaller after the reduction of intracellular Ca2+ concentration ([Ca2+]i) either by superfusion of the cells with 1 µM nisoldipine or by intracellular application of 10 mM BAPTA. Inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) by KN-93 or the autocamtide-2-related inhibitor peptide similarly reduced the amplitude and integral of INaL. Action potential duration was shortened in a reverse rate-dependent manner and the plateau potential was depressed by GS967. This GS967-induced depression of plateau was reduced by pretreatment of the cells with BAPTA-AM. We conclude that (1) INaL depends on the magnitude of [Ca2+]i in canine ventricular cells, (2) this [Ca2+]i-dependence of INaL is mediated by the Ca2+-dependent activation of CaMKII, and (3) INaL is augmented by the baseline CaMKII activity.

Persistence of the Effects of Se-Fertilization in Olive Trees over Time, Monitored with the Cytosolic Ca2+ and with the Germination of Pollen.

Selenium (Se) is an important micronutrient for living organisms, since it is involved in several physiological and metabolic processes. Biofortification with Se increases the nutritional and qualitative values of foods in Se-deficient regions and increases tolerance to oxidative stress in olive trees. Many studies have shown that Se, in addition to improving the qualitative and nutritional properties of EVO oil, also improves the plant's response to abiotic stress. This study addressed this issue by monitoring the effects of Se on cytosolic Ca2+ and on the germination of olive pollen grains in oxidative stress. The olive trees subjected to treatment with Na-selenate in the field produced pollen with a Se content 6-8 times higher than the controls, even after 20 months from the treatment. Moreover, part of the micronutrient was organic in selenium methionine. The higher selenium content did not produce toxic effects in the pollen, rather it antagonized the undesirable effects of oxidative stress in the parameters under study. The persistence of the beneficial effects of selenium observed over time in pollens, in addition to bringing out an undisputed adaptability of olive trees to the micronutrient, suggested the opportunity to reduce the number of treatments in the field.

Plasma-Activated Water Triggers Rapid and Sustained Cytosolic Ca2+ Elevations in Arabidopsis thaliana.

Increasing evidence indicates that water activated by plasma discharge, termed as plasma-activated water (PAW), can promote plant growth and enhance plant defence responses. Nevertheless, the signalling pathways activated in plants in response to PAW are still largely unknown. In this work, we analysed the potential involvement of calcium as an intracellular messenger in the transduction of PAW by plants. To this aim, Arabidopsis thaliana (Arabidopsis) seedlings stably expressing the bioluminescent Ca2+ reporter aequorin in the cytosol were challenged with PAW generated by a plasma torch. Ca2+ measurement assays demonstrated the induction by PAW of rapid and sustained cytosolic Ca2+ elevations in Arabidopsis seedlings. The dynamics of the recorded Ca2+ signals were found to depend upon different parameters, such as the operational conditions of the torch, PAW storage, and dilution. The separate administration of nitrate, nitrite, and hydrogen peroxide at the same doses as those measured in the PAW did not trigger any detectable Ca2+ changes, suggesting that the unique mixture of different reactive chemical species contained in the PAW is responsible for the specific Ca2+ signatures. Unveiling the signalling mechanisms underlying plant perception of PAW may allow to finely tune its generation for applications in agriculture, with potential advantages in the perspective of a more sustainable agriculture.

Pathological Mechanism of a Constitutively Active Form of Stromal Interaction Molecule 1 in Skeletal Muscle.

Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.

Neglected wardens: T lymphocyte ryanodine receptors.

Ryanodine receptors (RyRs) are intracellular Ca2+ release channels ubiquitously expressed in various cell types. RyRs were extensively studied in striated muscle cells due to their crucial role in muscle contraction. In contrast, the role of RyRs in Ca2+ signalling and functions in non-excitable cells, such as T lymphocytes, remains poorly understood. Expression of different isoforms of RyRs was shown in primary T cells and T cell lines. In T cells, RyRs co-localize with the plasmalemmal store-operated Ca2+ channels of the Orai family and endoplasmic reticulum Ca2+ sensing Stim family proteins and are activated by store-operated Ca2+ entry and pyridine nucleotide metabolites, the intracellular second messengers generated upon stimulation of T cell receptors. Experimental data indicate that together with d-myo-inositol 1,4,5-trisphosphate receptors, RyRs regulate intercellular Ca2+ dynamics by controlling Ca2+ concentration within the lumen of the endoplasmic reticulum and, consequently, store-operated Ca2+ entry. Gain-of-function mutations, genetic deletion or pharmacological inhibition of RyRs alters T cell Ca2+ signalling and effector functions. The picture emerging from the collective data shows that RyRs are the essential regulators of T cell Ca2+ signalling and can be potentially used as molecular targets for immunomodulation or T cell-based diagnostics of the disorders associated with RyRs dysregulation.

Onion component, isoalliin, stimulates feeding and activates the arcuate nucleus neuropeptide Y, ghrelin- and Ninjin'yoeito-responsive neurons.

Appetite loss or anorexia substantially decreases the quality of life in patients with cancer, depression and gastrointestinal disorders, and can lead to sarcopenia and frailty. Foods that restore appetite have been sought-for but are not currently available. Historically, onion intake was adopted to treat a variety of diseases with reduced appetite including cancer and gastrointestinal disturbances. While isoalliin is a core component of onion, the effects of isoalliin on feeding behavior and feeding centers remain unknown. Neuropeptide Y (NPY) and ghrelin are the most potent central and peripheral inducers of appetite. A Japanese kampo medicine Ninjin'yoeito activates ghrelin-responsive NPY neurons in the hypothalamic arcuate nucleus (ARC) and counteracts anorexia induced by an anti-cancer drug cisplatin. This study explored the effects of isoalliin on feeding behavior and activities of ARC neurons in mice. Isoalliin, injected intraperitoneally, dose-dependently increased food intake during dark phase (DP) and daily without altering light phase (LP) food intake. We measured cytosolic Ca2+ concentration ([Ca2+]i) in single ARC neurons including NPY neurons identified by GFP fluorescence. Isoalliin increased [Ca2+]i in 10 of 18 (55.6%) NPY neurons, a majority of which also responded to ghrelin with [Ca2+]i increases, indicating that the ARC ghrelin-responsive NPY neuron is the major target of isoalliin. Isoalliin also increased [Ca2+]i in the ARC neurons that responded to Ninjin'yoeito. These results indicate that isoalliin enhances feeding at the active period and activates ARC ghrelin-responsive NPY neurons and Ninjin'yoeito-responsive neurons. These abilities of isoalliin to stimulate DP feeding and activate ARC orexigenic neurons provide scientific evidence for the health beneficial effects of onion experienced historically and globally.

Forsythiasides-Rich Extract From Forsythiae Fructus Inhibits Mast Cell Degranulation by Enhancing Mitochondrial Ca2+ Uptake.

Mast cells (MCs) activated via IgE/FcεRI or MAS-related G protein coupled receptor (Mrgpr)-mediated pathway can release granules that play prominent roles in hypersensitivity reactions. Forsythiae Fructus, a well-known traditional Chinese medicine, has been clinically used for allergic diseases. Although previous studies indicated that Forsythiae Fructus extract inhibited compound 48/80-induced histamine release from MCs, its effect on IgE-dependent MC degranulation and possible underlying mechanisms remain to be explored. Herein, we prepared the forsythiasides-rich extract (FRE) and investigated its action on MC degranulation and explored its underlying mechanism. Our data showed that FRE could dampen IgE/FcεRI- and Mrgpr-mediated MC degranulation in vitro and in vivo. Mechanism study indicated that FRE decreased cytosolic Ca2+ (Ca2+ [c]) level rapidly and reversibly. Moreover, FRE decreased Ca2+ [c] of MCs independent of plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). While, along with Ca2+ [c] decrease, the increase of mitochondrial Ca2+ (Ca2+ [m]) occurred simultaneously in FRE-treated RBL-2H3 cells. In the isolated mitochondria, FRE also promoted the subcellular organelle to uptake more extramitochondrial Ca2+. In conclusion, by increasing Ca2+ [m] uptake, FRE decreases Ca2+ [c] level to suppress MC degranulation. Our findings may provide theoretical support for the clinical application of Forsythiae Fructus on allergy and other MC-involved diseases.

Aloe-emodin, a naturally occurring anthraquinone, is a highly potent mast cell stabilizer through activating mitochondrial calcium uniporter.

Mast cells play a fundamental role in immune system. Upon stimulation, they are activated via IgE dependent or independent pathway and then release granules which contain plenty of preformed constituents. Mast cell stabilizers are commonly used clinically for inhibiting the degranulation of mast cells. In the current study, we firstly identified aloe-emodin, a naturally occurring anthraquinone, was a prominent mast cell stabilizer. It could strikingly dampen IgE/FcεRI- and MAS-related G protein coupled receptor (Mrgpr)-mediated mast cell degranulation in vitro and in vivo. Mechanism study indicated that aloe-emodin rapidly and reversibly decreased cytosolic Ca2+ (Ca2+[c]) concentration through enhancing the mitochondrial Ca2+ (Ca2+[m]) uptake. After genetically silencing or pharmacologic inhibiting mitochondrial calcium uniporter (MCU), the effects of aloe-emodin on the Ca2+[c] level and mast cell degranulation were significantly weakened. In contrast to six clinical drugs with mast cell stabilizing properties (amlexanox, tranilast, ketotifen, cromolyn disodium salt, dexamethasone and pimecrolimus), aloe-emodin showed an impressive and potent inhibitory action on the mast cell degranulation. Collectively, aloe-emodin is a highly potent mast cell stabilizer. By directly activating MCU, it decreases Ca2+[c] level to suppress mast cell degranulation. Our study may provide a promising candidate for the treatment of mast cell activation-related diseases.