Major Ca Research Articles

Abstract 1881: STIM1 and Orai1 control non-Hodgkin lymphoma cells migration

Abstract Non-Hodgkin lymphoma is one of the most common cancer in United States representing 4% of all cancer. Diffuse large B cell lymphoma (DLBCL) is the most common and aggressive non-Hodgkin B lymphoma. One of the characteristic features of this disease is the dissemination of the cancer cells, through the lymphatic system in the secondary lymphoid organs and extranodal sites, leading to the death of patients. Chemokines such as the Stromal Derived Factor 1 (SDF1) control the spread and the homing of the cancer cells. It is known that SDF1 activates various signalling pathways involved in cell proliferation, transcription or migration. Moreover, SDF1 induces an increase in intracellular calcium concentration whose role is still unknown in B cells. Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in this type of cells. By definition, SOCE is activated by Ca2+ release from the endoplasmic reticulum. Two genes are responsible for SOCE activity: Stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that detects store depletion and ORAI1, the pore-forming subunit of Ca2+ release-activated Ca2+ (CRAC) channel. Several studies performed on adherent cells showed that Orai1/STIM1 proteins are involved in cancer cell migration. However, the molecular mechanisms involved in cell migration differ widely between adherent and non-adherent cells. We studied the role of both actors of calcium entry : Orai1 and STIM1 in DLBCL pathology and more precisely in basal and SDF1-induced of DLBCL cell migration. Using Tissue MicroArrays approach we revealed that both Orai1 and STIM1 are under-expressed in DLBCL tumoral tissue compared to normal lymphoid tissue. Next, using calcium imaging experiments we confirmed that SDF-1 triggered Ca2+ responses in two DLBCL cell lines (SUDHL4 and HLY1) involving intracellular Ca2+ store mobilization and extracellular Ca2+ influx. Based on these observations, we investigated the role of Orai1 and STIM1 on SDF1-induced Ca2+ influx using pharmacological and RNA interference approaches. The inhibition of Orai1 by BTP2 as well as the under-expression of Orai1 or STIM1 by shRNA, prevented Ca2+ influx induced by SDF1 suggesting the involvement of Orai1 and STIM1 in this process. Regarding this results , we studied the role of Orai1 and STIM1 on DLBCL cell migration in vitro and in vivo. Our results show that basal or SDF1-induced cell migration was significantly inhibited by underexpression of STIM1 or Orai1 in SUDHL4 and HLY1 cell lines. These results suggest that STIM1 and Orai1 play a key role in the DLBCL cell migration. The identification of STIM1 and Orai1 proteins as key players in the migration of DLBCL cells might provide new therapeutic targets for the treatment of this pathology. Citation Format: Simon Latour, Isabelle Mahouche, Floriane Cherrier, Jean-Philippe Merlio, Sandrine Poglio, Laurence Bresson Bepoldin. STIM1 and Orai1 control non-Hodgkin lymphoma cells migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2017-1881

Dose-Dependent Responses of I3C and DIM on T-Cell Activation in the Human T Lymphocyte Jurkat Cell Line.

Indole-3-carbinol (I3C) and its dimer diindolylmethane (DIM) are bioactive metabolites of a glucosinolate, glucobrassicin, found in cruciferous vegetables. Both I3C and DIM have been reported to possess pro-apoptotic, anti-proliferative and anti-carcinogenic properties via modulation of immune pathways. However, results from these studies remain inconclusive since they lack thorough evaluation of these bioactives’ physiological versus pharmacological effects. In the present study, we investigated I3C and DIM’s dose-dependent effects on cytokines production in human T lymphocytes Jurkat cell line (Clone E6-1). The results showed that I3C and DIM pretreatment, at higher concentrations of 50 and 10 μM, respectively, significantly increased PMA/ionomycin-induced interleukin-2 (IL-2), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) production, measured by real time polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). As a plausible mechanism underlying such pronounced cytokine release, we found robust increase in downstream nuclear factor κB (NF-κB) and nuclear factor of activated T-cells 1 (NFAT1) signaling with I3C pretreatment, whereas DIM pretreatment only significantly induced NF-κB activation, but not NFAT1. We hypothesize that I3C/DIM pretreatment primes the T cells to become hyperresponsive upon PMA/ionomycin stimulation which in turn differentially induces two major downstream Ca2+-dependent inflammatory pathways, NF-κB and NFAT1. Our data show novel insights into the mechanisms underlying induction of pro-inflammatory cytokine release by pharmacological concentrations of I3C and DIM, an effect negligible under physiological conditions.

EVALUATE THE GROUNDWATER SUITABILITY FOR IRRIGATION IN DUJAILA AREA – WASIT GOVERNORATE – MIDDLE OF IRAQ

The selected area is Dujaila project, Wasit governorate, Iraq. Twelve water samples groundwater and drainage were collected during March 2016. Total dissolved solids (TDS), pH, electrical conductivity (EC), major ions Ca2+, Mg2+, Na+, K+, Cl-, SO4 2- and HCO3-, and chemical analysis were determined. To evaluate the suitability of analyzed water for irrigation, Sodium adsorption ratio (SAR), residual sodium carbonate (RSC), sodium percent (Na%) and electrical conductivity were applied. The based on electrical conductivity classification of the analyzed water indicated that both the groundwater and drainage water are unacceptable for irrigation, except for very salt-tolerant plants (EC> 2250 μs/cm). These samples mostly have no harmful effects and no hazard in terms of RSC (RSC < 0), while for SAR, the groundwater samples have no harmful effects of sodium (SAR < 10) but it is unsatisfactory for irrigation for drainage water samples ( SAR > 26) and groundwater samples are good (Na% range from 20 to 40) to permissible (Na% > 40) in terms of Na%, while, the drainage water samples indicate doubtful class for the three samples (Na% > 60). Applying the water-sediment relationship experiments with respect to dry and wet periods show that about 2.2% – 4.8% of the TDS were librated after the drying condition which is believed to be of the water-soluble salts that exist within the sediment column.

Calcium Dynamics Mediated by the Endoplasmic/Sarcoplasmic Reticulum and Related Diseases.

The flow of intracellular calcium (Ca2+) is critical for the activation and regulation of important biological events that are required in living organisms. As the major Ca2+ repositories inside the cell, the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of muscle cells are central in maintaining and amplifying the intracellular Ca2+ signal. The morphology of these organelles, along with the distribution of key calcium-binding proteins (CaBPs), regulatory proteins, pumps, and receptors fundamentally impact the local and global differences in Ca2+ release kinetics. In this review, we will discuss the structural and morphological differences between the ER and SR and how they influence localized Ca2+ release, related diseases, and the need for targeted genetically encoded calcium indicators (GECIs) to study these events.

Calreticulin Is Required for TGF-β-Induced Epithelial-to-Mesenchymal Transition during Cardiogenesis in Mouse Embryonic Stem Cells.

SummaryCalreticulin, a multifunctional endoplasmic reticulum resident protein, is required for TGF-β-induced epithelial-to-mesenchymal transition (EMT) and subsequent cardiomyogenesis. Using embryoid bodies (EBs) derived from calreticulin-null and wild-type (WT) embryonic stem cells (ESCs), we show that expression of EMT and cardiac differentiation markers is induced during differentiation of WT EBs. This induction is inhibited in the absence of calreticulin and can be mimicked by inhibiting TGF-β signaling in WT cells. The presence of calreticulin in WT cells permits TGF-β-mediated signaling via AKT/GSK3β and promotes repression of E-cadherin by SNAIL2/SLUG. This is paralleled by induction of N-cadherin in a process known as the cadherin switch. We show that regulated Ca2+ signaling between calreticulin and calcineurin is critical for the unabated TGF-β signaling that is necessary for the exit from pluripotency and the cadherin switch during EMT. Calreticulin is thus a key mediator of TGF-β-induced commencement of cardiomyogenesis in mouse ESCs.

Plasma membrane calcium ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity.

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca2+ efflux pump, PMCA4. This is the major Ca2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca2+ ] in capacitated and Ca2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca2+ -dependent manner. In Pmca4-/- sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4-/- males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.

Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes.

Astrocytes are non-excitable cells in the brain and their activity largely depends on the intracellular calcium (Ca2+) level. Therefore, maintaining the intracellular Ca2+ homeostasis is critical for proper functioning of astrocytes. One of the key regulatory mechanisms of Ca2+ homeostasis in astrocytes is the store-operated Ca2+ entry (SOCE). This process is mediated by a combination of the Ca2+-store-depletion-sensor, Stim, and the store-operated Ca2+-channels, Orai and TrpC families. Despite the existence of all those families in astrocytes, previous studies have provided conflicting results on the molecular identification of astrocytic SOCE. Here, using the shRNA-based gene-silencing approach and Ca2+-imaging from cultured mouse astrocytes, we report that Stim1 in combination with Orai1 and Orai3 contribute to the major portion of astrocytic SOCE. Gene-silencing of Stim1 showed a 79.2% reduction of SOCE, indicating that Stim1 is the major Ca2+-store-depletion-sensor. Further gene-silencing showed that Orai1, Orai2, Orai3, and TrpC1 contribute to SOCE by 35.7%, 20.3%, 26.8% and 12.2%, respectively. Simultaneous gene-silencing of all three Orai subtypes exhibited a 67.6% reduction of SOCE. Based on the detailed population analysis, we predict that Orai1 and Orai3 are expressed in astrocytes with a large SOCE, whereas TrpC1 is exclusively expressed in astrocytes with a small SOCE. This analytical approach allows us to identify the store operated channel (SOC) subtype in each cell by the degree of SOCE. Our results propose that Stim1 in combination with Orai1 and Orai3 are the major molecular components of astrocytic SOCE under various physiological and pathological conditions.

Physiology and pathophysiology of excitation-contraction coupling: the functional role of ryanodine receptor.

Calcium (Ca2+) release from intracellular stores plays a key role in the regulation of skeletal muscle contraction. The type 1 ryanodine receptors (RyR1) is the major Ca2+ release channel on the sarcoplasmic reticulum (SR) of myocytes in skeletal muscle and is required for excitation-contraction (E-C) coupling. This article explores the role of RyR1 in skeletal muscle physiology and pathophysiology.

Ryanodine receptor modification and regulation by intracellular Ca2 + and Mg2 + in healthy and failing human hearts

RationaleHeart failure is a multimodal disorder, of which disrupted Ca2+ homeostasis is a hallmark. Central to Ca2+ homeostasis is the major cardiac Ca2+ release channel – the ryanodine receptor (RyR2) – whose activity is influenced by associated proteins, covalent modification and by Ca2+ and Mg2+. That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood. ObjectiveTo assess Ca2+ and Mg2+ regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling. Methods and resultsThe function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca2+ (100nM), a decreased cytoplasmic [Ca2+] required for half maximal activation (Ka) and a decrease in inhibition by cytoplasmic Mg2+. This was accompanied by significant hyperphosphorylation of RyR2 S2808 and S2814, reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca2+ or Mg2+. ConclusionDespite different aetiologies, disrupted RyR2 Ca2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca2+ signalling.

QUALITY CHARACTERISTICS OF SURFACE WATERS AT ASPROLAKKAS RIVER BASIN, N.E. CHALKIDIKI, GREECE

The present study reports on a four sampling period (April 2008-July 2008-November 2008-February 2009) study of water quality in streams of NE Chalkidiki. A total of 80 surface water samples were collected and analysed for the major ions Ca2+, Mg2+, K+, Na+, Cl- , HCO3 - , SO4 2-, NO3 - and the trace elements Pb, Fe, Cd, Cu, Cr, Ni, Zn, Mn and As. Also pH, Electrical Conductivity (EC), Total Dissolved Solids (TDS), Temperature, as well as the stream water discharge at each sampling point, were determined in the field. The treatment and the subsequent evaluation of the data showed that the water samples are divided into three separate groups, reflecting the chemistry of water in the three sub-basins of the area i.e., Kokkinolakkas, Kerasia and Piavitsa. The water of the first one is possibly affected by the past mining activities of the area, whereas samples from the other two reflect the influence of sulphide mineralization on the hydrogeochemistry of the corresponding streams. No effect of the seasonal differentiation of stream water discharge was observed.

Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.

Is junctional adhesion molecule A (JAM-A), a sperm protein essential for normal motility, expressed in the murine post-testicular pathway and involved in sperm maturation? JAM-A is present in the prostate and seminal vesicles and in all three regions of the epididymis where it is secreted in epididymosomes in the luminal fluid and can be delivered to sperm in vitro. JAM-A shares with the plasma membrane Ca2+ATPase 4 (PMCA4, the major Ca2+ efflux pump in murine sperm) a common interacting partner, CASK (Ca2+/CaM-dependent serine kinase). JAM-A, like PMCA4, plays a role in Ca2+ regulation, since deletion of Jam-A results in significantly elevated intracellular Ca2+ levels and reduced sperm motility. Recently, PMCA4 was reported to be expressed in the epididymis and along with CASK was shown to be in a complex on epididymosomes where it was transferred to sperm. Because of the association of JAM-A with CASK in sperm and because of the presence of PMCA4 and CASK in the epididymis, the present study was performed to determine whether JAM-A is expressed in the epididymis and delivered to sperm during their maturation. The epididymides, prostate and seminal vesicles were collected from sexually mature C57BL/6J and Institute for Cancer Research mice and antibodies specific for JAM-A and Ser285 -phosphorylated JAM-A (pJAM-A) were used for the analysis. Tissues, sperm and epididymal luminal fluid (ELF) were studied. Epididymosomes were also isolated for study. Caput and caudal sperm were co-incubated with ELF individually to determine their abilities to acquire JAM-A in vitro. Sections of all three regions of the epididymis were subjected to indirect immunofluorescence analysis. Epididymal tissues, fluid, sperm, prostate and seminal vesicle tissues were analyzed for JAM-A and/or pJAM-A via western blotting analysis. The relative amounts of JAM-A and pJAM-A among epididymal tissues, ELF and sperm were detected by western blot via quantification of band intensities. Epididymosomes were isolated by ultracentrifugation of the ELF after it was clarified to remove cells and tissue fragments, and the proteins western blotted for JAM-A and pJAM-A, and exosomal biochemical markers. FACS analysis was used to quantify the amount of JAM-A present on caput and caudal sperm, as well as the amount of JAM-A acquired in vitro after their co-incubation with ELF. Western blots revealed that JAM-A is expressed in all three regions of the epididymis, the prostate and seminal vesicles. As confirmed by indirect immunofluorescence, a western blot showed that JAM-A has a higher expression in the corpus and caudal regions, where it is significantly (P < 0.01) more abundant than in the caput. Both JAM-A and Ser285-phosphorylated JAM-A (pJAM-A) are secreted into the ELF where it is highest in the distal regions. In the ELF, both JAM-A and pJAM-A were detected in epididymosomes. Western blotting of sperm proteins showed a significant (P < 0.01) increase of JAM-A and pJAM-A in caudal, compared with caput, sperm. Flow-cytometric analysis confirmed the increase in JAM-A in caudal sperm where it was 1.4-fold higher than in caput ones. Co-incubation of caput and caudal sperm with ELF demonstrated ~2.3- and ~1.3-fold increases, respectively, in JAM-A levels indicating that epididymosomes transfer more JAM-A to caput sperm that are less saturated with the protein than caudal ones. Not applicable. First, although the ELF was clarified prior to ultracentrifugation for epididymosome isolation, we cannot rule out contamination of the epididymosomal proteins by those from epididymal epithelial cells. Second, the JAM-A detected in the prostate and seminal vesicles might not necessarily be secreted from those organs and may only be present within the tissues, where it would be unable to impact sperm in the ejaculate. Although performed in the mouse the study has implications for humans, as the highly conserved JAM-A is a signaling protein in human sperm. There is physiological significance to the finding that JAM-A, which regulates sperm motility and intracellular Ca2+, exists in elevated levels in the cauda where sperm gain motility and fertilizing ability. The study suggests that the acquisition of JAM-A in the epididymal tract is involved in the mechanism by which sperm gain their motility during epididymal maturation. This increased understanding of sperm physiology is important for aspects of ART. The work was supported by NIH-RO3HD073523 and NIH-5P20RR015588 grants to P.A.M.-D. The authors declare there are no conflicts of interests.

Protein Fragmentation As a Regulatory Mechanism: Insights from Two Different Ca2+ Channels, RyR1 and IP3R.

The ryanodine receptor 1 (RyR1) is the major skeletal muscle Ca2+ release channel and as such a key player in excitation-contraction coupling. Intriguingly, a recent paper reported RyR1 fragmentation 24 h after high-intensity interval exercise (six 30-s all-out cycling bouts with 4 min recoveries in between) in vastus lateralis muscle biopsies taken from recreationally active men (Place et al., 2015). In contrast to what could have been hypothesized based on the role of RyR1 in excitation-contraction coupling, this RyR1 fragmentation did not appear to result in excitation-contraction coupling failure, as both maximal voluntary contraction force and forces evoked by supramaximal electrical stimulations had fully recovered 24 h after exercise. Overall, these results suggest that fragmented RyR1 retain their capacity to release Ca2+ in response to an action potential.

Synaptotagmin 2 Is the Fast Ca2+ Sensor at a Central Inhibitory Synapse

SummaryGABAergic synapses in brain circuits generate inhibitory output signals with submillisecond latency and temporal precision. Whether the molecular identity of the release sensor contributes to these signaling properties remains unclear. Here, we examined the Ca2+ sensor of exocytosis at GABAergic basket cell (BC) to Purkinje cell (PC) synapses in cerebellum. Immunolabeling suggested that BC terminals selectively expressed synaptotagmin 2 (Syt2), whereas synaptotagmin 1 (Syt1) was enriched in excitatory terminals. Genetic elimination of Syt2 reduced action potential-evoked release to ∼10%, identifying Syt2 as the major Ca2+ sensor at BC-PC synapses. Differential adenovirus-mediated rescue revealed that Syt2 triggered release with shorter latency and higher temporal precision and mediated faster vesicle pool replenishment than Syt1. Furthermore, deletion of Syt2 severely reduced and delayed disynaptic inhibition following parallel fiber stimulation. Thus, the selective use of Syt2 as release sensor at BC-PC synapses ensures fast and efficient feedforward inhibition in cerebellar microcircuits.

To Address Suffering That the Majority Can't See: Lessons from Black Women's Leadership in the Workplace

This chapter explores how both historically and in contemporary times of escalating violence against our bodies, minds, and spirits worldwide, Black women lead, love, and live within contexts of suffering.

Suppression of ryanodine receptor function prolongs Ca2+ release refractoriness and promotes cardiac alternans in intact hearts.

Beat-to-beat alternations in the amplitude of the cytosolic Ca2+ transient (Ca2+ alternans) are thought to be the primary cause of cardiac alternans that can lead to cardiac arrhythmias and sudden death. Despite its important role in arrhythmogenesis, the mechanism underlying Ca2+ alternans remains poorly understood. Here, we investigated the role of cardiac ryanodine receptor (RyR2), the major Ca2+ release channel responsible for cytosolic Ca2+ transients, in cardiac alternans. Using a unique mouse model harboring a suppression-of-function (SOF) RyR2 mutation (E4872Q), we assessed the effect of genetically suppressing RyR2 function on Ca2+ and action potential duration (APD) alternans in intact hearts, and electrocardiogram (ECG) alternans in vivo We found that RyR2-SOF hearts displayed prolonged sarcoplasmic reticulum Ca2+ release refractoriness and enhanced propensity for Ca2+ alternans. RyR2-SOF hearts/mice also exhibited increased propensity for APD and ECG alternans. Caffeine, which enhances RyR2 activity and the propensity for catecholaminergic polymorphic ventricular tachycardia (CPVT), suppressed Ca2+ alternans in RyR2-SOF hearts, whereas carvedilol, a β-blocker that suppresses RyR2 activity and CPVT, promoted Ca2+ alternans in these hearts. Thus, RyR2 function is an important determinant of Ca2+, APD, and ECG alternans. Our data also indicate that the activity of RyR2 influences the propensity for cardiac alternans and CPVT in an opposite manner. Therefore, overly suppressing or enhancing RyR2 function is pro-arrhythmic.

Calcium Channels in Vascular Smooth Muscle.

Calcium (Ca2+) plays a central role in excitation, contraction, transcription, and proliferation of vascular smooth muscle cells (VSMs). Precise regulation of intracellular Ca2+ concentration ([Ca2+]i) is crucial for proper physiological VSM function. Studies over the last several decades have revealed that VSMs express a variety of Ca2+-permeable channels that orchestrate a dynamic, yet finely tuned regulation of [Ca2+]i. In this review, we discuss the major Ca2+-permeable channels expressed in VSM and their contribution to vascular physiology and pathology.

A central role for calcineurin in protein misfolding neurodegenerative diseases.

Accumulation of misfolded/unfolded aggregated proteins in the brain is a hallmark of many neurodegenerative diseases affecting humans and animals. Dysregulation of calcium (Ca2+) and disruption of fast axonal transport (FAT) are early pathological events that lead to loss of synaptic integrity and axonal degeneration in early stages of neurodegenerative diseases. Dysregulated Ca2+ in the brain is triggered by accumulation of misfolded/unfolded aggregated proteins in the endoplasmic reticulum (ER), a major Ca2+ storing organelle, ultimately leading to neuronal dysfunction and apoptosis. Calcineurin (CaN), a Ca2+/calmodulin-dependent serine/threonine phosphatase, has been implicated in T cells activation through the induction of nuclear factor of activated T cells (NFAT). In addition to the involvement of several other signaling cascades, CaN has been shown to play a role in early synaptic dysfunction and neuronal death. Therefore, inhibiting hyperactivated CaN in early stages of disease might be a promising therapeutic strategy for treating patients with protein misfolding diseases. In this review, we briefly summarize the structure of CaN, inhibition mechanisms by which immunosuppressants inhibit CaN, role of CaN in maintaining neuronal and synaptic integrity and homeostasis and the role played by CaN in protein unfolding/misfolding neurodegenerative diseases.

Characterization of the ER-Targeted Low Affinity Ca(2+) Probe D4ER.

Calcium ion (Ca2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.

Store-operated calcium entry is required for sustained contraction and Ca2+ oscillations of airway smooth muscle.

Airway hyper-responsiveness in asthma is driven by excessive contraction of airway smooth muscle cells (ASMCs). Agonist-induced Ca2+ oscillations underlie this contraction of ASMCs and the magnitude of this contraction is proportional to the Ca2+ oscillation frequency. Sustained contraction and Ca2+ oscillations require an influx of extracellular Ca2+ , although the mechanisms and pathways mediating this Ca2+ influx during agonist-induced ASMC contraction are not well defined. By inhibiting store-operated calcium entry (SOCE) or voltage-gated Ca2+ channels (VGCCs), we show that SOCE, rather than Ca2+ influx via VGCCs, provides the major Ca2+ entry pathway into ASMCs to sustain ASMCs contraction and Ca2+ oscillations. SOCE may therefore serve as a potential target for new bronchodilators to reduce airway hyper-responsiveness in asthma. Asthma is characterized by airway hyper-responsiveness: the excessive contraction of airway smooth muscle. The extent of this airway contraction is proportional to the frequency of Ca2+ oscillations within airway smooth muscle cells (ASMCs). Sustained Ca2+ oscillations require a Ca2+ influx to replenish Ca2+ losses across the plasma membrane. Our previous studies implied store-operated calcium entry (SOCE) as the major pathway for this Ca2+ influx. In the present study, we explore this hypothesis, by examining the effects of SOCE inhibitors (GSK7975A and GSK5498A) as well as L-type voltage-gated Ca2+ channel inhibitors (nifedipine and nimodipine) on airway contraction and Ca2+ oscillations and SOCE-mediated Ca2+ influx in ASMCs within mouse precision-cut lung slices. We found that both GSK7975A and GSK5498A were able to fully relax methacholine-induced airway contraction by abolishing the Ca2+ oscillations, in a manner similar to that observed in zero extracellular Ca2+ ([Ca2+ ]e ). In addition, GSK7975A and GSK5498A inhibited increases in intracellular Ca2+ ([Ca2+ ]i ) in ASMCs with depleted Ca2+ -stores in response to increased [Ca2+ ]e , demonstrating a response consistent with the inhibition of SOCE. However, GSK7975A and GSK5498A did not reduce Ca2+ release via IP3 receptors stimulated with IP3 released from caged-IP3 . By contrast, nifedipine and nimodipine only partially reduced airway contraction, Ca2+ oscillation frequency and SOCE-mediated Ca2+ influx. These data suggest that SOCE is the major Ca2+ influx pathway for ASMCs with respect to sustaining agonist-induced airway contraction and the underlying Ca2+ oscillations. The mechanisms of SOCE may therefore form novel targets for new bronchodilators.

Moss and Peat Leachate Degradability by Heterotrophic Bacteria: The Fate of Organic Carbon and Trace Metals

ABSTRACTThe respiration of dissolved organic matter (DOM) by aerobic heterotrophic bacterioplankton in boreal surface waters is one of the major factors that regulate CO2 exchange of lakes and rivers with the atmosphere in arctic and subarctic zones. The DOM that originates from topsoil leaching and vegetation degradation is brought to the lakes by surface flow and is subjected to coagulation and degradation by heterotrophic bacteria, which are well-established processes in the majority of boreal aquatic settings. The behavior of colloids and organic complexes of trace metals during this process is virtually unknown. In this work, we studied the interaction of two model heterotrophic bacteria, soil Pseudomonas aureofaciens and aquatic Pseudomonas reactans, with peat and Sphagnum moss leachates from the permafrost region under controlled laboratory conditions in nutrient-free media. The moss leachate was the better substrate for bacterial survival, with P. reactans exhibiting an order of magnitude higher live cell number compared with P. aureofaciens. In eight-day experiments, we analyzed organic carbon and ∼40 major and trace elements (TEs) during heterotrophic bacteria growth. The total net decrease in the concentration of dissolved organic carbon (DOC) was similar for both bacteria and ranged from 30 mg gwet−1 to ≤10 mg gwet−1 during 8 days for the moss and peat leachate, respectively. Despite significant evolutions of pH, DOC, dissolved inorganic carbon (DIC), and cell number, most major (Mg, K, and Ca) and TEs remained nearly constant (within ±30% of the control). Only Fe, Al, P, Zn, Mn, Co, and Ba and to a much lesser extent Cd, Pb, Rare Earth Elements (REEs), U, Ti, and Zr were affected (p < 0.05) by the presence of bacteria relative to the control and exhibited slight to moderate decreases during the experiment. Adsorption onto bacterial surfaces produced fast initial removal of Al, Mn, and Ba and to a lesser degree Cd, Pb, REEs, and U. Intracellular metabolic assimilation mostly affected P, Zn, and Co and progressively decreased their concentrations. Finally, coagulation as individual Fe/Al hydroxides due to DOM removal or pH change could also affect elements that were precipitated with organomineral colloids (Ti and Zr). The degrees of major and TE susceptibility to bacterial activity based on concentration changes during the experiment in both substrates ranged over three orders of magnitude from mg L−1 to µg L−1 and followed the order DOC >> P >> Ba > Zn ≥ Fe ≥ Al > Mn > Cu ≥ Sr > Zr ≥ Ti > Ni ≥ Co > REEs ≥ U > Hf∼Th, which reflected the abundance of the elements in the two substrates. Generally, the soil exopolysaccharide producing bacterium P. aureofaciens in the peat leachate had the greatest impact of the four combinations investigated in this study (two bacteria with two substrates). Under ongoing environmental changes in the boreal zone, the autochthonous processes of bacterioplankton activity are able to decrease the concentrations of a very limited number of TEs, including mainly Fe and several macro- (P) and micro- (Zn, Mn, and Ba) nutrients.