Sample records for genetically-encoded calcium indicator

  1. Chronic imaging of cortical sensory map dynamics using a genetically encoded calcium indicator


    Minderer, M; Liu, W.; Sumanovski, L. T.; Kügler, S; Helmchen, F; Margolis, D. J.


    Abstract  In vivo optical imaging can reveal the dynamics of large-scale cortical activity, but methods for chronic recording are limited. Here we present a technique for long-term investigation of cortical map dynamics using wide-field ratiometric fluorescence imaging of the genetically encoded calcium indicator (GECI) Yellow Cameleon 3.60. We find that wide-field GECI signals report sensory-evoked activity in anaesthetized mouse somatosensory cortex with high sensitivity and spatiotemporal ...

  2. Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS. (United States)

    Borges-Pereira, Lucas; Budu, Alexandre; McKnight, Ciara A; Moore, Christina A; Vella, Stephen A; Hortua Triana, Miryam A; Liu, Jing; Garcia, Celia R S; Pace, Douglas A; Moreno, Silvia N J


    Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca(2+) oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca(2+) enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca(2+) changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca(2+) oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca(2+) influx. This is the first study showing, in real time, Ca(2+) signals preceding egress and their direct link with motility, an essential virulence trait.

  3. Chronic imaging of cortical sensory map dynamics using a genetically encoded calcium indicator. (United States)

    Minderer, Matthias; Liu, Wenrui; Sumanovski, Lazar T; Kügler, Sebastian; Helmchen, Fritjof; Margolis, David J


    In vivo optical imaging can reveal the dynamics of large-scale cortical activity, but methods for chronic recording are limited. Here we present a technique for long-term investigation of cortical map dynamics using wide-field ratiometric fluorescence imaging of the genetically encoded calcium indicator (GECI) Yellow Cameleon 3.60. We find that wide-field GECI signals report sensory-evoked activity in anaesthetized mouse somatosensory cortex with high sensitivity and spatiotemporal precision, and furthermore, can be measured repeatedly in separate imaging sessions over multiple weeks. This method opens new possibilities for the longitudinal study of stability and plasticity of cortical sensory representations.

  4. Imaging the response of the retina to electrical stimulation with genetically encoded calcium indicators. (United States)

    Weitz, Andrew C; Behrend, Matthew R; Lee, Nan Sook; Klein, Ronald L; Chiodo, Vince A; Hauswirth, William W; Humayun, Mark S; Weiland, James D; Chow, Robert H


    Epiretinal implants for the blind are designed to stimulate surviving retinal neurons, thus bypassing the diseased photoreceptor layer. Single-unit or multielectrode recordings from isolated animal retina are commonly used to inform the design of these implants. However, such electrical recordings provide limited information about the spatial patterns of retinal activation. Calcium imaging overcomes this limitation, as imaging enables high spatial resolution mapping of retinal ganglion cell (RGC) activity as well as simultaneous recording from hundreds of RGCs. Prior experiments in amphibian retina have demonstrated proof of principle, yet experiments in mammalian retina have been hindered by the inability to load calcium indicators into mature mammalian RGCs. Here, we report a method for labeling the majority of ganglion cells in adult rat retina with genetically encoded calcium indicators, specifically GCaMP3 and GCaMP5G. Intravitreal injection of an adeno-associated viral vector targets ∼85% of ganglion cells with high specificity. Because of the large fluorescence signals provided by the GCaMP sensors, we can now for the first time visualize the response of the retina to electrical stimulation in real-time. Imaging transduced retinas mounted on multielectrode arrays reveals how stimulus pulse shape can dramatically affect the spatial extent of RGC activation, which has clear implications in prosthetic applications. Our method can be easily adapted to work with other fluorescent indicator proteins in both wild-type and transgenic mammals.

  5. Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells. (United States)

    Perry, Jacob L; Ramachandran, Nina K; Utama, Budi; Hyser, Joseph M


    Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.

  6. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

    Directory of Open Access Journals (Sweden)

    Jasper eAkerboom


    Full Text Available Genetically encoded calcium indicators (GECIs are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, RCaMPs, engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+] and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2 or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

  7. Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation

    Directory of Open Access Journals (Sweden)

    J. Michael eGee


    Full Text Available Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators, have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson’s disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (< 5 kb. In utero electroporation offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal

  8. Imaging intracellular Ca²⁺ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators. (United States)

    Jiang, Ruotian; Haustein, Martin D; Sofroniew, Michael V; Khakh, Baljit S


    Astrocytes display spontaneous intracellular Ca(2+) concentration fluctuations ([Ca(2+)]i) and in several settings respond to neuronal excitation with enhanced [Ca(2+)]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca(2+)]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca(2+)]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca(2+)]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca(2+)]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca(2+)]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca(2+)]i signals in the striatal microcircuitry.

  9. Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator. (United States)

    Miyamoto, Akitoshi; Bannai, Hiroko; Michikawa, Takayuki; Mikoshiba, Katsuhiko


    Monitoring the pattern of intracellular Ca(2+) signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca(2+) indicators (GECIs) are used for monitoring intracellular Ca(2+) changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca(2+) signals using GECIs. We here compared the Ca(2+) signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca(2+) responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca(2+) signals.

  10. Monitoring activity in neural circuits with genetically encoded indicators

    Directory of Open Access Journals (Sweden)

    Gerard Joseph Broussard


    Full Text Available Recent developments in genetically encoded indicators of neural activity (GINAs have greatly advanced the field of systems neuroscience. As they are encoded by DNA, GINAs can be targeted to genetically defined cellular populations. Combined with fluorescence microscopy, most notably multi-photon imaging, GINAs allow chronic simultaneous optical recordings from large populations of neurons or glial cells in awake, behaving mammals, particularly rodents. This large-scale recording of neural activity at multiple temporal and spatial scales has greatly advanced our understanding of the dynamics of neural circuitry underlying behavior—a critical first step toward understanding the complexities of brain function, such as sensorimotor integration and learning.Here, we summarize the recent development and applications of the major classes of GINAs. In particular, we take an in-depth look at the design of available GINA families with a particular focus on genetically encoded calcium indicators, sensors probing synaptic activity, and genetically encoded voltage indicators. Using the family of the genetically encoded calcium indicator GCaMP as an example, we review established sensor optimization pipelines. We also discuss practical considerations for end users of GINAs about experimental methods including approaches for gene delivery, imaging system requirements, and data analysis techniques. With the growing toolbox of GINAs and with new microscopy techniques pushing beyond their current limits, the age of light can finally achieve the goal of broad and dense sampling of neuronal activity across time and brain structures to obtain a dynamic picture of brain function.

  11. Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice

    Directory of Open Access Journals (Sweden)

    Henry Lütcke


    Full Text Available Fluorescent calcium (Ca2+ indicator proteins (FCIPs are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60 in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 - in combination with various optical techniques - thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.

  12. Dual optical recordings for action potentials and calcium handling in induced pluripotent stem cell models of cardiac arrhythmias using genetically encoded fluorescent indicators. (United States)

    Song, LouJin; Awari, Daniel W; Han, Elizabeth Y; Uche-Anya, Eugenia; Park, Seon-Hye E; Yabe, Yoko A; Chung, Wendy K; Yazawa, Masayuki


    Reprogramming of human somatic cells to pluripotency has been used to investigate disease mechanisms and to identify potential therapeutics. However, the methods used for reprogramming, in vitro differentiation, and phenotyping are still complicated, expensive, and time-consuming. To address the limitations, we first optimized a protocol for reprogramming of human fibroblasts and keratinocytes into pluripotency using single lipofection and the episomal vectors in a 24-well plate format. This method allowed us to generate multiple lines of integration-free and feeder-free induced pluripotent stem cells (iPSCs) from seven patients with cardiac diseases and three controls. Second, we differentiated human iPSCs derived from patients with Timothy syndrome into cardiomyocytes using a monolayer differentiation method. We found that Timothy syndrome cardiomyocytes showed slower, irregular contractions and abnormal calcium handling compared with the controls. The results are consistent with previous reports using a retroviral method for reprogramming and an embryoid body-based method for cardiac differentiation. Third, we developed an efficient approach for recording the action potentials and calcium transients simultaneously in control and patient cardiomyocytes using genetically encoded fluorescent indicators, ArcLight and R-GECO1. The dual optical recordings enabled us to observe prolonged action potentials and abnormal calcium handling in Timothy syndrome cardiomyocytes. We confirmed that roscovitine rescued the phenotypes in Timothy syndrome cardiomyocytes and that these findings were consistent with previous studies using conventional electrophysiological recordings and calcium imaging with dyes. The approaches using our optimized methods and dual optical recordings will improve iPSC applicability for disease modeling to investigate mechanisms underlying cardiac arrhythmias and to test potential therapeutics.

  13. Genetically Encoded Voltage Indicators in Circulation Research. (United States)

    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter


    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

  14. Genetically Encoded Voltage Indicators in Circulation Research

    Directory of Open Access Journals (Sweden)

    Lars Kaestner


    Full Text Available Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

  15. Visualizing presynaptic calcium dynamics and vesicle fusion with a single genetically encoded reporter at individual synapses

    Directory of Open Access Journals (Sweden)

    Rachel E Jackson


    Full Text Available Synaptic transmission depends on the influx of calcium into the presynaptic compartment, which drives neurotransmitter release. Genetically encoded reporters are widely used tools to understand these processes, particularly pHluorin-based reporters that report vesicle exocytosis and endocytosis through pH dependent changes in fluorescence, and genetically encoded calcium indicators (GECIs that exhibit changes in fluorescence upon binding to calcium. The recent expansion of the color palette of available indicators has made it possible to image multiple probes simultaneously within a cell. We have constructed a single molecule reporter capable of concurrent imaging of both presynaptic calcium influx and exocytosis, by fusion of sypHy, the vesicle associated protein synaptophysin containing a GFP-based pHluorin sensor, with the red-shifted GECI R-GECO1. Due to the fixed stoichiometry of the two probes, the ratio of the two responses can also be measured, providing an all optical correlate of the calcium dependence of release. Here, we have characterized stimulus-evoked sypHy-RGECO responses of hippocampal synapses in vitro, exploring the effects of different stimulus strengths and frequencies as well as variations in external calcium concentrations. By combining live sypHy-RGECO imaging with post-hoc fixation and immunofluorescence, we have also investigated correlations between structural and functional properties of synapses.

  16. Optogenetic Monitoring of Synaptic Activity with Genetically Encoded Voltage Indicators (United States)

    Nakajima, Ryuichi; Jung, Arong; Yoon, Bong-June; Baker, Bradley J.


    The age of genetically encoded voltage indicators (GEVIs) has matured to the point that changes in membrane potential can now be observed optically in vivo. Improving the signal size and speed of these voltage sensors has been the primary driving forces during this maturation process. As a result, there is a wide range of probes using different voltage detecting mechanisms and fluorescent reporters. As the use of these probes transitions from optically reporting membrane potential in single, cultured cells to imaging populations of cells in slice and/or in vivo, a new challenge emerges—optically resolving the different types of neuronal activity. While improvements in speed and signal size are still needed, optimizing the voltage range and the subcellular expression (i.e., soma only) of the probe are becoming more important. In this review, we will examine the ability of recently developed probes to report synaptic activity in slice and in vivo. The voltage-sensing fluorescent protein (VSFP) family of voltage sensors, ArcLight, ASAP-1, and the rhodopsin family of probes are all good at reporting changes in membrane potential, but all have difficulty distinguishing subthreshold depolarizations from action potentials and detecting neuronal inhibition when imaging populations of cells. Finally, we will offer a few possible ways to improve the optical resolution of the various types of neuronal activities. PMID:27547183

  17. Imaging the awake visual cortex with a genetically encoded voltage indicator. (United States)

    Carandini, Matteo; Shimaoka, Daisuke; Rossi, L Federico; Sato, Tatsuo K; Benucci, Andrea; Knöpfel, Thomas


    Genetically encoded voltage indicators (GEVIs) promise to reveal the membrane potential of genetically targeted neuronal populations through noninvasive, chronic imaging of large portions of cortical space. Here we test a promising GEVI in mouse cortex during wakefulness, a challenging condition due to large hemodynamic activity, and we introduce a straightforward projection method to separate a signal dominated by membrane voltage from a signal dominated by hemodynamic activity. We expressed VSFP-Butterfly 1.2 plasmid in layer 2/3 pyramidal cells of visual cortex through electroporation in utero. We then used wide-field imaging with two cameras to measure both fluorophores of the indicator in response to visual stimuli. By taking weighted sums and differences of the two measurements, we obtained clear separation of hemodynamic and voltage signals. The hemodynamic signal showed strong heartbeat oscillations, superimposed on slow dynamics similar to blood oxygen level-dependent (BOLD) or "intrinsic" signals. The voltage signal had fast dynamics similar to neural responses measured electrically, and showed an orderly retinotopic mapping. We compared this voltage signal with calcium signals imaged in transgenic mice that express a calcium indicator (GCaMP3) throughout cortex. The voltage signal from VSFP had similar signal-to-noise ratios as the calcium signal, it was more immune to vascular artifacts, and it integrated over larger regions of visual space, which was consistent with its reporting mostly subthreshold activity rather than the spiking activity revealed by calcium signals. These results demonstrate that GEVIs provide a powerful tool to study the dynamics of neural populations at mesoscopic spatial scales in the awake cortex. Copyright © 2015 Carandini et al.

  18. Genetically encoded Ca2+ indicators; expanded affinity range, color hue and compatibility with optogenetics

    Directory of Open Access Journals (Sweden)

    Takeharu eNagai


    Full Text Available Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs such as derivatives of yellow cameleons (YCs and GCaMPs/pericams (Miyawaki et al., 1997; Nakai et al., 2001; Nagai et al., 2001 are a highly advanced class of indicators. Continued efforts for improvement of the performance of GECIs have resulted in brighter indicators with better photo-stability and expanded dynamic range, thus improving the sensitivity of detection. Fine-tuning of other properties, including Ca2+ affinity and Hill constant, have also contributed to increase the detectability of Ca2+ dynamics. Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca2+ indicators and bioluminescence-based Ca2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment.

  19. Use of genetically encoded calcium indicators (GECIs combined with advanced motion tracking techniques to examine the behavior of neurons and glia in the enteric nervous system of the intact murine colon

    Directory of Open Access Journals (Sweden)

    Grant Willem Hennig


    Full Text Available Genetically encoded Ca2+ indicators (GECIs have been used extensively in many body systems to detect Ca2+ transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca2+ indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We developed several GCaMP3 mice: 1 Wnt1-GCaMP3, all enteric neurons and glia; 2 GFAP-GCaMP3, enteric glia; 3 nNOS-GaMP3, enteric nitrergic neurons, and 4 ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC, using low power Ca2+ imaging. In this preliminary study, we observed neuronal and glial cell Ca2+ transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100-1000 active cells through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca2+ transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca2+ transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca2+ transients some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural

  20. Genetically encoded pH‐indicators reveal activity‐dependent cytosolic acidification of Drosophila motor nerve termini in vivo

    National Research Council Canada - National Science Library

    Rossano, Adam J; Chouhan, Amit K; Macleod, Gregory T


    .... •  At the fruit fly larval neuromuscular junction, fluorescent genetically encoded pH‐indicators (GEpHIs) revealed significant cytosolic acidification of presynaptic termini during nerve activity...

  1. Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain

    Directory of Open Access Journals (Sweden)

    Yukiko eMishina


    Full Text Available Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviours. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP prototypical design or on the voltage dependent state transitions of microbial opsins.We recently introduced a new VSFP design in which the voltage-sensing domain (VSD is sandwiched between a FRET pair of fluorescent proteins (termed VSFP-Butterflies and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.

  2. Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals. (United States)

    Ohkura, Masamichi; Sasaki, Takuya; Sadakari, Junko; Gengyo-Ando, Keiko; Kagawa-Nagamura, Yuko; Kobayashi, Chiaki; Ikegaya, Yuji; Nakai, Junichi


    Imaging the activities of individual neurons with genetically encoded Ca(2+) indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca(2+) signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F(max)/F(min) = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca(2+) imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca(2+) responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.

  3. Genetically encoded pH-indicators reveal activity-dependent cytosolic acidification of Drosophila motor nerve termini in vivo. (United States)

    Rossano, Adam J; Chouhan, Amit K; Macleod, Gregory T


    All biochemical processes, including those underlying synaptic function and plasticity, are pH sensitive. Cytosolic pH (pH(cyto)) shifts are known to accompany nerve activity in situ, but technological limitations have prevented characterization of such shifts in vivo. Genetically encoded pH-indicators (GEpHIs) allow for tissue-specific in vivo measurement of pH. We expressed three different GEpHIs in the cytosol of Drosophila larval motor neurons and observed substantial presynaptic acidification in nerve termini during nerve stimulation in situ. SuperEcliptic pHluorin was the most useful GEpHI for studying pH(cyto) shifts in this model system. We determined the resting pH of the nerve terminal cytosol to be 7.30 ± 0.02, and observed a decrease of 0.16 ± 0.01 pH units when the axon was stimulated at 40 Hz for 4 s. Realkalinization occurred upon cessation of stimulation with a time course of 20.54 ± 1.05 s (τ). The chemical pH-indicator 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein corroborated these changes in pH(cyto). Bicarbonate-derived buffering did not contribute to buffering of acid loads from short (≤ 4 s) trains of action potentials but did buffer slow (~60 s) acid loads. The magnitude of cytosolic acid transients correlated with cytosolic Ca(2+) increase upon stimulation, and partial inhibition of the plasma membrane Ca(2+)-ATPase, a Ca(2+)/H(+) exchanger, attenuated pH(cyto) shifts. Repeated stimulus trains mimicking motor patterns generated greater cytosolic acidification (~0.30 pH units). Imaging through the cuticle of intact larvae revealed spontaneous pH(cyto) shifts in presynaptic termini in vivo, similar to those seen in situ during fictive locomotion, indicating that presynaptic pH(cyto) shifts cannot be dismissed as artifacts of ex vivo preparations.

  4. Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.

    Directory of Open Access Journals (Sweden)

    Haruki Odaka

    Full Text Available Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.

  5. Genetically-Encoded Yellow Fluorescent cAMP Indicator with an Expanded Dynamic Range for Dual-Color Imaging (United States)

    Odaka, Haruki; Arai, Satoshi; Inoue, Takafumi; Kitaguchi, Tetsuya


    Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics. PMID:24959857

  6. Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.

    Directory of Open Access Journals (Sweden)

    Kenta Saito

    Full Text Available BACKGROUND: Efficient bioluminescence resonance energy transfer (BRET from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. METHODOLOGY/PRINCIPAL FINDINGS: We applied BRET to develop an autoluminescent Ca(2+ indicator, BRAC, which is composed of Ca(2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca(2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca(2+-independent signal drifts due to change in cell shape, focus shift, etc. CONCLUSIONS/SIGNIFICANCE: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca(2+ imaging not only in single live cells but also in small living subjects.

  7. Fast kinetics of calcium signaling and sensor design. (United States)

    Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J


    Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change.

  8. Genetically Encoded Sensors for Metabolites (United States)

    Deuschle, Karen; Fehr, Marcus; Hilpert, Melanie; Lager, Ida; Lalonde, Sylvie; Looger, Loren L.; Okumoto, Sakiko; Persson, Jörgen; Schmidt, Anja; Frommer, Wolf B.


    Background Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels. Methods We therefore developed genetically encoded nanosensors for several metabolites to provide a basic set of tools for the determination of cytosolic and subcellular metabolite levels in real time by using fluorescence microscopy. Results Prototypes of these sensors were successfully used in vitro and also in vivo, i.e., to measure sugar levels in fungal and animal cells. Conclusions One of the future goals will be to expand the set of sensors to a wider spectrum of substrates by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets in conjunction with mutagenesis. This toolbox can then be applied for four-dimensional imaging of cells and tissues to elucidate the spatial and temporal distribution of metabolites as a discovery tool in functional genomics, as a tool for high-throughput, high-content screening for drugs, to test metabolic models, and to analyze the interplay of cells in a tissue or organ. PMID:15688353

  9. Toward Better Genetically Encoded Sensors of Membrane Potential. (United States)

    Storace, Douglas; Sepehri Rad, Masoud; Kang, BokEum; Cohen, Lawrence B; Hughes, Thom; Baker, Bradley J


    Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because individual brain regions can include a multitude of different cell types. Optical imaging allows for simultaneous recording from numerous neurons or brain regions. Optical signals of membrane potential are useful because membrane potential changes are a direct sign of both synaptic and action potentials. Here we describe recent improvements in the in vitro and in vivo signal size and kinetics of genetically encoded voltage indicators (GEVIs) and discuss their relationship to alternative sensors of neural activity.

  10. Evaluating a genetically encoded optical sensor of neural activity using electrophysiology in intact adult fruit flies

    Directory of Open Access Journals (Sweden)

    Gilles Laurent


    Full Text Available Genetically encoded optical indicators hold the promise of enabling non-invasive monitoring of activity in identified neurons in behaving organisms. However, the interpretation of images of brain activity produced using such sensors is not straightforward. Several recent studies of sensory coding used G-CaMP 1.3-a calcium sensor-as an indicator of neural activity; some of these studies characterized the imaged neurons as having narrow tuning curves, a conclusion not always supported by parallel electrophysiological studies. To better understand the possible cause of these conflicting results, we performed simultaneous in vivo 2-photon imaging and electrophysiological recording of G-CaMP 1.3 expressing neurons in the antennal lobe (AL of intact fruitflies. We find that G-CaMP has a relatively high threshold, that its signal often fails to capture spiking response kinetics, and that it can miss even high instantaneous rates of activity if those are not sustained. While G-CaMP can be misleading, it is clearly useful for the identification of promising neural targets: when electrical activity is well above the sensor's detection threshold, its signal is fairly well correlated with mean firing rate and G-CaMP does not appear to alter significantly the responses of neurons that express it. The methods we present should enable any genetically encoded sensor, activator, or silencer to be evaluated in an intact neural circuit in vivo in Drosophila.

  11. Mouse redox histology using genetically encoded probes. (United States)

    Fujikawa, Yuuta; Roma, Leticia P; Sobotta, Mirko C; Rose, Adam J; Diaz, Mauricio Berriel; Locatelli, Giuseppe; Breckwoldt, Michael O; Misgeld, Thomas; Kerschensteiner, Martin; Herzig, Stephan; Müller-Decker, Karin; Dick, Tobias P


    Mapping the in vivo distribution of endogenous oxidants in animal tissues is of substantial biomedical interest. Numerous health-related factors, including diet, physical activity, infection, aging, toxins, or pharmacological intervention, may cause redox changes. Tools are needed to pinpoint redox state changes to particular organs, tissues, cell types, and subcellular organelles. We describe a procedure that preserves the in vivo redox state of genetically encoded redox biosensors within histological tissue sections, thus providing "redox maps" for any tissue and comparison of interest. We demonstrate the utility of the technique by visualizing endogenous redox differences and changes in the context of tumor growth, inflammation, embryonic development, and nutrient starvation. Copyright © 2016, American Association for the Advancement of Science.

  12. Imaging calcium in neurons. (United States)

    Grienberger, Christine; Konnerth, Arthur


    Calcium ions generate versatile intracellular signals that control key functions in all types of neurons. Imaging calcium in neurons is particularly important because calcium signals exert their highly specific functions in well-defined cellular subcompartments. In this Primer, we briefly review the general mechanisms of neuronal calcium signaling. We then introduce the calcium imaging devices, including confocal and two-photon microscopy as well as miniaturized devices that are used in freely moving animals. We provide an overview of the classical chemical fluorescent calcium indicators and of the protein-based genetically encoded calcium indicators. Using application examples, we introduce new developments in the field, such as calcium imaging in awake, behaving animals and the use of calcium imaging for mapping single spine sensory inputs in cortical neurons in vivo. We conclude by providing an outlook on the prospects of calcium imaging for the analysis of neuronal signaling and plasticity in various animal models.

  13. Chronic calcium imaging of neurons in the mouse visual cortex using a troponin C-based indicator. (United States)

    Santos, Alexandre Ferrão; Hübener, Mark


    This protocol describes the use of the genetically encoded troponin C-based calcium indicator TN-XXL to chronically monitor the functional properties of single neocortical neurons in the mouse visual cortex. A cranial window is implanted over the brain of a mouse expressing TN-XXL in pyramidal neurons of the cerebral cortex. Several days later, the visual cortex is mapped and photographed to facilitate repeated imaging of the same region using two-photon microscopy. Initial two-photon imaging may be done ∼2 wk after the window is implanted. We show the application of this technique for long-term in vivo imaging of stimulus response properties. Beyond providing functional information, long-term imaging of TN-XXL-labeled neurons also enables the simultaneous monitoring of structural properties down to the level of single dendritic spines.

  14. New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging. (United States)

    Oheim, Martin; van 't Hoff, Marcel; Feltz, Anne; Zamaleeva, Alsu; Mallet, Jean-Maurice; Collot, Mayeul


    Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca(2+) probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca(2+) probes. In response to this demand more red-emitting chemical and FP-based Ca(2+)-sensitive indicators have been developed since 2009 than in the thirty years before. In this topical review, we survey the physicochemical properties of these red-emitting Ca(2+) probes and discuss their utility for biological Ca(2+) imaging. Using the spectral separability index Xijk (Oheim M., 2010. Methods in Molecular Biology 591: 3-16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca(2+)-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca(2+) indicators that ideally should be made available for each probe. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck

  15. Relating a calcium indicator signal to the unperturbed calcium concentration time-course

    Directory of Open Access Journals (Sweden)

    Abarbanel Henry DI


    Full Text Available Abstract Background Optical indicators of cytosolic calcium levels have become important experimental tools in systems and cellular neuroscience. Indicators are known to interfere with intracellular calcium levels by acting as additional buffers, and this may strongly alter the time-course of various dynamical variables to be measured. Results By investigating the underlying reaction kinetics, we show that in some ranges of kinetic parameters one can explicitly link the time dependent indicator signal to the time-course of the calcium influx, and thus, to the unperturbed calcium level had there been no indicator in the cell.

  16. Optimization of a GCaMP calcium indicator for neural activity imaging. (United States)

    Akerboom, Jasper; Chen, Tsai-Wen; Wardill, Trevor J; Tian, Lin; Marvin, Jonathan S; Mutlu, Sevinç; Calderón, Nicole Carreras; Esposti, Federico; Borghuis, Bart G; Sun, Xiaonan Richard; Gordus, Andrew; Orger, Michael B; Portugues, Ruben; Engert, Florian; Macklin, John J; Filosa, Alessandro; Aggarwal, Aman; Kerr, Rex A; Takagi, Ryousuke; Kracun, Sebastian; Shigetomi, Eiji; Khakh, Baljit S; Baier, Herwig; Lagnado, Leon; Wang, Samuel S-H; Bargmann, Cornelia I; Kimmel, Bruce E; Jayaraman, Vivek; Svoboda, Karel; Kim, Douglas S; Schreiter, Eric R; Looger, Loren L


    Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

  17. A genetically encoded, high-signal-to-noise maltose sensor

    Energy Technology Data Exchange (ETDEWEB)

    Marvin, Jonathan S.; Schreiter, Eric R.; Echevarría, Ileabett M.; Looger, Loren L. (Puerto Rico); (HHMI)


    We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes.

  18. Relating a calcium indicator signal to the unperturbed calcium concentration time-course

    National Research Council Canada - National Science Library

    Borst, Alexander; Abarbanel, Henry D I


    .... By investigating the underlying reaction kinetics, we show that in some ranges of kinetic parameters one can explicitly link the time dependent indicator signal to the time-course of the calcium...

  19. Genetically encoded optical sensors for monitoring of intracellular chloride and chloride-selective channel activity

    Directory of Open Access Journals (Sweden)

    Piotr Bregestovski


    Full Text Available This review briefly discusses the main approaches for monitoring chloride (Cl−, the most abundant physiological anion. Noninvasive monitoring of intracellular Cl− ([Cl−]i is a challenging task owing to two main difficulties: (i the low transmembrane ratio for Cl−, approximately 10:1; and (ii the small driving force for Cl−, as the Cl− reversal potential (ECl is usually close to the resting potential of the cells. Thus, for reliable monitoring of intracellular Cl−, one has to use highly sensitive probes. From several methods for intracellular Cl− analysis, genetically encoded chloride indicators represent the most promising tools. Recent achievements in the development of genetically encoded chloride probes are based on the fact that yellow fluorescent protein (YFP exhibits Cl−-sensitivity. YFP-based probes have been successfully used for quantitative analysis of Cl− transport in different cells and for high-throughput screening of modulators of Cl−-selective channels. Development of a ratiometric genetically encoded probe, Clomeleon, has provided a tool for noninvasive estimation of intracellular Cl− concentrations. While the sensitivity of this protein to Cl− is low (EC50 about 160 mM, it has been successfully used for monitoring intracellular Cl− in different cell types. Recently a CFP–YFP-based probe with a relatively high sensitivity to Cl− (EC50 about 30 mM has been developed. This construct, termed Cl-Sensor, allows ratiometric monitoring using the fluorescence excitation ratio. Of particular interest are genetically encoded probes for monitoring of ion channel distribution and activity. A new molecular probe has been constructed by introducing into the cytoplasmic domain of the Cl−-selective glycine receptor (GlyR channel the CFP–YFP-based Cl-Sensor. This construct, termed BioSensor-GlyR, has been successfully expressed in cell lines. The new genetically encoded chloride probes offer means of screening

  20. Relating a calcium indicator signal to the unperturbed calcium concentration time-course


    Abarbanel Henry DI; Borst Alexander


    Abstract Background Optical indicators of cytosolic calcium levels have become important experimental tools in systems and cellular neuroscience. Indicators are known to interfere with intracellular calcium levels by acting as additional buffers, and this may strongly alter the time-course of various dynamical variables to be measured. Results By investigating the underlying reaction kinetics, we show that in some ranges of kinetic parameters one can explicitly link the time dependent indicat...

  1. Genetically-encoded biosensors for monitoring cellular stress in bioprocessing. (United States)

    Polizzi, Karen M; Kontoravdi, Cleo


    With the current wealth of transcriptomic data, it is possible to design genetically-encoded biosensors for the detection of stress responses and apply these to high-throughput bioprocess development and monitoring of cellular health. Such biosensors can sense extrinsic factors such as nutrient or oxygen deprivation and shear stress, as well as intrinsic stress factors like oxidative damage and unfolded protein accumulation. Alongside, there have been developments in biosensing hardware and software applicable to the field of genetically-encoded biosensors in the near future. This review discusses the current state-of-the-art in biosensors for monitoring cultures during biological manufacturing and the future challenges for the field. Connecting the individual achievements into a coherent whole will enable the application of genetically-encoded biosensors in industry.

  2. In vivo characterization of genetic expression of virus-transduced calcium indicators in retinal ganglion cells using a low-cost funduscope. (United States)

    Chang, Yao-Chuan; Walston, Steven T; Chow, Robert H; Weiland, James D


    Virus-transduced calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. To track the level of in vivo expression of genetically encoded calcium indicators (GECIs) in rodent retina, we developed a noninvasive imaging approach based on a custom-modified low-cost and simple fundus system that enabled us to monitor and characterize in vivo bright-field and fluorescence retinal image. The system clearly resolves individual retinal ganglion cells (RGCs) and axons. RGC fluorescence intensity and number of observable fluorescent cells show a consistent rising trend from week 1 to week 3 after viral injection, indicating a uniform increase of GCaMP6f expression. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array (MEA) and used calcium imaging to identify the optimal time for studying the responsiveness of RGCs to external electrical stimulation. The results show that the fluorescence-endoscopy fundus system is a powerful and widely accessible tool for evaluating in vivo fluorescence reporter expression.

  3. Photoacoustic imaging using genetically encoded reporters: a review (United States)

    Brunker, Joanna; Yao, Junjie; Laufer, Jan; Bohndiek, Sarah E.


    Genetically encoded contrast in photoacoustic imaging (PAI) is complementary to the intrinsic contrast provided by endogenous absorbing chromophores such as hemoglobin. The use of reporter genes expressing absorbing proteins opens the possibility of visualizing dynamic cellular and molecular processes. This is an enticing prospect but brings with it challenges and limitations associated with generating and detecting different types of reporters. The purpose of this review is to compare existing PAI reporters and signal detection strategies, thereby offering a practical guide, particularly for the nonbiologist, to choosing the most appropriate reporter for maximum sensitivity in the biological and technological system of interest.

  4. Recent Advances in Development of Genetically Encoded Fluorescent Sensors. (United States)

    Sanford, Lynn; Palmer, Amy


    Genetically encoded fluorescent sensors are essential tools in modern biological research, and recent advances in fluorescent proteins (FPs) have expanded the scope of sensor design and implementation. In this review we compare different sensor platforms, including Förster resonance energy transfer (FRET) sensors, fluorescence-modulated single FP-based sensors, translocation sensors, complementation sensors, and dimerization-based sensors. We discuss elements of sensor design and engineering for each platform, including the incorporation of new types of FPs and sensor screening techniques. Finally, we summarize the wide range of sensors in the literature, exploring creative new sensor architectures suitable for different applications.

  5. Calcium Green FlAsH as a genetically targeted small-molecule calcium indicator. (United States)

    Tour, Oded; Adams, Stephen R; Kerr, Rex A; Meijer, Rene M; Sejnowski, Terrence J; Tsien, Richard W; Tsien, Roger Y


    Intracellular Ca(2+) regulates numerous proteins and cellular functions and can vary substantially over submicron and submillisecond scales, so precisely localized fast detection is desirable. We have created a approximately 1-kDa biarsenical Ca(2+) indicator, called Calcium Green FlAsH (CaGF, 1), to probe [Ca(2+)] surrounding genetically targeted proteins. CaGF attached to a tetracysteine motif becomes ten-fold more fluorescent upon binding Ca(2+), with a K(d) of approximately 100 microM, <1-ms kinetics and good Mg(2+) rejection. In HeLa cells expressing tetracysteine-tagged connexin 43, CaGF labels gap junctions and reports Ca(2+) waves after injury. Total internal reflection microscopy of tetracysteine-tagged, CaGF-labeled alpha(1C) L-type calcium channels shows fast-rising depolarization-evoked Ca(2+) transients, whose lateral nonuniformity suggests that the probability of channel opening varies greatly over micron dimensions. With moderate Ca(2+) buffering, these transients decay surprisingly slowly, probably because most of the CaGF signal comes from closed channels feeling Ca(2+) from a tiny minority of clustered open channels. With high Ca(2+) buffering, CaGF signals decay as rapidly as the calcium currents, as expected for submicron Ca(2+) domains immediately surrounding active channels. Thus CaGF can report highly localized, rapid [Ca(2+)] dynamics.

  6. Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution. (United States)

    Liu, Xueliang; Lopez, Paola A; Giessen, Tobias W; Giles, Michael; Way, Jeffrey C; Silver, Pamela A


    Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.

  7. Deconvolution of calcium fluorescent indicator signal from AFM cantilever reflection. (United States)

    Lopez-Ayon, G Monserratt; Oliver, David J; Grutter, Peter H; Komarova, Svetlana V


    Atomic force microscopy (AFM) can be combined with fluorescence microscopy to measure the changes in intracellular calcium levels (indicated by fluorescence of Ca²⁺ sensitive dye fluo-4) in response to mechanical stimulation performed by AFM. Mechanical stimulation using AFM is associated with cantilever movement, which may interfere with the fluorescence signal. The motion of the AFM cantilever with respect to the sample resulted in changes of the reflection of light back to the sample and a subsequent variation in the fluorescence intensity, which was not related to changes in intracellular Ca²⁺ levels. When global Ca²⁺ responses to a single stimulation were assessed, the interference of reflected light with the fluorescent signal was minimal. However, in experiments where local repetitive stimulations were performed, reflection artifacts, correlated with cantilever motion, represented a significant component of the fluorescent signal. We developed a protocol to correct the fluorescence traces for reflection artifacts, as well as photobleaching. An added benefit of our method is that the cantilever reflection in the fluorescence recordings can be used for precise temporal correlation of the AFM and fluorescence measurements.

  8. Protein ligand-tethered synthetic calcium indicator for localization control and spatiotemporal calcium imaging in plant cells. (United States)

    Takaoka, Yousuke; Shigenaga, Miyuki; Imai, Masaki; Nukadzuka, Yuuki; Ishimaru, Yasuhiro; Saito, Kei; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Ueda, Minoru


    In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.

  9. Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

    Institute of Scientific and Technical Information of China (English)

    Min Chen; Yanru Wang; Tingting Hou; Huiliang Zhang; Aijuan Qu; Xianhua Wang


    Mitochondrial calcium plays a crucial role in mitochondriai metabolism,cell calcium handling,and cell death.However,some mechanisms concerning mitochondrial calcium regulation are still unknown,especially how mitochondrial calcium couples with cytosolic calcium.In this work,we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation.Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester,a mitochondrial membrane potential indicator.The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2.The apparent Kd of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes.Furthermore,we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria.In HeLa cells,we found that mitochondrial calcium ([Ca2+]mito)responded to the changes of cytosolic calcium ([Ca2+]cyto)induced by histamine or thapasigargin.Moreover,external Ca2+ (100 μmol/L) directly induced an increase of [Ca2+]mito in permeabilized HeLa cells.However,in rat cardiomyocytes [Ca2+]mito did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine.In permeabilized cardiomyocytes,600 nmol/L free Ca2+ repeatedly increased the fluorescent signals of mito-GCaMP2,which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria.These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

  10. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging. (United States)

    Jahn, Karolina; Hille, Carsten


    For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+)-free and Ca(2+)-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+)-dependent way, unraveling in vitro dissociation constants K(D) of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca(2+)-free and Ca(2+)-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D) of 180 nM was determined. Thus, quantitative [Ca(2+)]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+)]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+) indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

  11. Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

    Directory of Open Access Journals (Sweden)

    Karolina Jahn

    Full Text Available For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR and Asante Calcium Green (ACG for two-photon (2P-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720-900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca(2+-free and Ca(2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+-dependent way, unraveling in vitro dissociation constants K(D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns and long (2.44 ns decay time components attributable to the Ca(2+-free and Ca(2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D of 180 nM was determined. Thus, quantitative [Ca(2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

  12. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences (United States)

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.


    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  13. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Directory of Open Access Journals (Sweden)

    Bernhard Hochreiter


    Full Text Available Fluorescence- or Förster resonance energy transfer (FRET is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a cleavage; (b conformational-change; (c mechanical force and (d changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  14. Fluorescent proteins as genetically encoded FRET biosensors in life sciences. (United States)

    Hochreiter, Bernhard; Garcia, Alan Pardo; Schmid, Johannes A


    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  15. Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.

    Directory of Open Access Journals (Sweden)

    John G Yamauchi

    Full Text Available We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC by developing sensor cells stably expressing a Ca(2+ permeable LGIC and a genetically encoded Förster (or fluorescence resonance energy transfer (FRET-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A serotonin receptors and a chimera of human α7/mouse 5-HT(3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

  16. Synthesis, Characterization and Biological Activities of a New Fluorescent Indicator for the Intracellular Calcium Ions

    Institute of Scientific and Technical Information of China (English)

    HE Huaizhen; LEI Lei; LI Jianli; SHI Zhen


    A novel calcium-selective fluorescent indicator Fluo-3M AM was synthesized by introduction of a methyl group into the Ca2+-chelating moiety and adequately characterized by spectral methods (1H NMR, GC-MS, IR and MALDI-TOF MS). Meanwhile, its fluorescence spectra and some biological activities have been also studied. The results indicate that the new fluorescent indicator has relatively high affinity to calcium and a strong fluorescence signal, which should be useful for biomedical researchers to investigate the effects of calcium ions in biosystems.

  17. Calcium signalling indicates bilateral power balancing in the Drosophila flight muscle during manoeuvring flight. (United States)

    Lehmann, Fritz-Olaf; Skandalis, Dimitri A; Berthé, Ruben


    Manoeuvring flight in animals requires precise adjustments of mechanical power output produced by the flight musculature. In many insects such as fruit flies, power generation is most likely varied by altering stretch-activated tension, that is set by sarcoplasmic calcium levels. The muscles reside in a thoracic shell that simultaneously drives both wings during wing flapping. Using a genetically expressed muscle calcium indicator, we here demonstrate in vivo the ability of this animal to bilaterally adjust its calcium activation to the mechanical power output required to sustain aerodynamic costs during flight. Motoneuron-specific comparisons of calcium activation during lift modulation and yaw turning behaviour suggest slightly higher calcium activation for dorso-longitudinal than for dorsoventral muscle fibres, which corroborates the elevated need for muscle mechanical power during the wings' downstroke. During turning flight, calcium activation explains only up to 54 per cent of the required changes in mechanical power, suggesting substantial power transmission between both sides of the thoracic shell. The bilateral control of muscle calcium runs counter to the hypothesis that the thorax of flies acts as a single, equally proportional source for mechanical power production for both flapping wings. Collectively, power balancing highlights the precision with which insects adjust their flight motor to changing energetic requirements during aerial steering. This potentially enhances flight efficiency and is thus of interest for the development of technical vehicles that employ bioinspired strategies of power delivery to flapping wings.

  18. Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor. (United States)

    Nakanishi, Yoichi; Iida, Syuntaro; Ueoka-Nakanishi, Hanayo; Niimi, Tomoaki; Tomioka, Rie; Maeshima, Masayoshi


    Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

  19. Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors. (United States)

    Lee, Sungmoo; Piao, Hong Hua; Sepheri-Rad, Masoud; Jung, Arong; Sung, Uhna; Song, Yoon-Kyu; Baker, Bradley J


    Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

  20. Inter-population differences in otolith morphology are genetically encoded in the killifish Aphanius fasciatus (Cyprinodontiformes

    Directory of Open Access Journals (Sweden)

    Ali Annabi


    Full Text Available Inter-population differences in otolith shape, morphology and chemistry have been used effectively as indicators for stock assessment or for recognizing environmental adaptation in fishes. However, the precise parameters that affect otolith morphology remain incompletely understood. Here we provide the first direct support for the hypothesis that inter-population differences in otolith morphology are genetically encoded. The study is based on otolith morphology and two mitochondrial markers (D-loop, 16S rRNA of three natural populations of Aphanius fasciatus (Teleostei: Cyprinodontidae from Southeast Tunisia. Otolith and genetic data yielded congruent tree topologies. Divergence of populations likely results from isolation events in the course of the Pleistocene sea level drops. We propose that otolith morphology is a valuable tool for resolving genetic diversity also within other teleost species, which may be important for ecosystem management and conservation of genetic diversity. As reconstructions of ancient teleost fish faunas are often solely based on fossil otoliths, our discoveries may also lead to a new approach to research in palaeontology.

  1. Genetically encoded tools: bridging the gap between neuronal identity and function. (United States)

    Cho, Yong Ku


    Genetically encoded tools are positioned to serve a unique and critical role in bridging the gap between the genetic identity of neurons and their functional properties. However, the use of these tools is limited by our current understanding of cell-type identity. As we make technological advances that focus on capturing functional aspects of neurons such as connectivity, activity, and metabolic states, our understanding of neuronal identity will deepen and may enable the use of genetically encoded tools for modulating disease-specific circuits for therapeutic purposes.

  2. Genetic encoding of the post-translational modification 2-hydroxyisobutyryl-lysine


    Knight, William A.; Cropp, T.Ashton


    We report the synthesis and genetic encoding of a recently discovered posttranslational modification, 2-hydroxyisobutryl-lysine, to the genetic code of E. coli. The production of homogeneous proteins containing this amino acid will facilitate the study of modification in full-length proteins.

  3. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    Directory of Open Access Journals (Sweden)

    Jordan S. Leyton-Mange


    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  4. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

    Directory of Open Access Journals (Sweden)

    Mazahir T Hasan


    Full Text Available Genetically encoded fluorescent calcium indicator proteins (FCIPs are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs and synaptic and sensory stimulation can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

  5. Calcium (United States)

    ... in luck if you like sardines and canned salmon with bones. Almond milk. previous continue Working Calcium ... drinks, and cereals. Other Considerations for Building Bones Vitamin D is essential for calcium absorption, so it's ...

  6. Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

    Directory of Open Access Journals (Sweden)

    Mojca Benčina


    Full Text Available Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments.

  7. Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3. (United States)

    Perez-Terzic, C; Stehno-Bittel, L; Clapham, D E


    The fluorescent indicator Fluo-3 is widely used to monitor the calcium concentration ([Ca2+]) in the cytoplasm and nucleus of various cells. Estimates of nuclear [Ca2+] are based on the assumption of identical behavior of Fluo-3 in different cellular compartments. The assumption is not valid if the fluorescence properties of the dye are altered by the nuclear environment, independent of the [Ca2+]. To determine the effects of the nucleoplasm on the behavior of Fluo-3, we applied laser scanning confocal microscopy and spectrophotometry to measure fluorescence intensity as well as emission and absorbance spectra of the Ca2+ indicator, Fluo-3. Spectra were measured in intact Xenopus oocytes, neuroblastoma cells, and cytoplasmic and nucleoplasmic homogenates. The fluorescence signal in intact cells loaded with Fluo-3 was approximately 2-times higher in the nucleus when compared to the cytoplasm. The fluorescence intensity of Fluo-3 in nucleoplasmic homogenates was higher than in cytoplasmic homogenates or internal buffers even when [Ca2+] was clamped. Despite identical [Ca2+], pH, and temperature, the emission and absorbance spectra of Fluo-3 from nuclear homogenates displayed a higher fluorescence at each wavelength measured when compared to spectra from cytoplasmic homogenates or internal buffer solutions, and saturated above 100 nM. These findings demonstrate that the composition of the nucleoplasm changes the fluorescence properties of the calcium indicator Fluo-3. Consequently, analysis of nuclear calcium dynamics must take into account the distinct behavior of Fluo-3 in different cellular compartments.

  8. Controlling Macromolecular Topology with Genetically Encoded SpyTag-SpyCatcher Chemistry


    Zhang, Wen-Bin; Sun, Fei; Tirrell, David A.; Arnold, Frances H.


    Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag–SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyT...

  9. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction


    Lang, Kathrin; Davis, Lloyd; Torres-Kolbus, Jessica; Chou, Chungjung; Deiters, Alexander; Chin, Jason W.


    The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetr...

  10. Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps. (United States)

    Reeves, Alexander M B; Shigetomi, Eiji; Khakh, Baljit S


    Calcium signaling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes, and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signaling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis (Sholl, 1953) of astrocytes loaded with common calcium indicator dyes and compared these data with Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving ∼90% of the area of an astrocyte and its extensive processes unsampled. By using morphological reconstructions as "maps" after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.

  11. Calibration and functional analysis of three genetically encoded Cl−/pH sensors

    Directory of Open Access Journals (Sweden)

    Marat eMukhtarov


    Full Text Available Monitoring of the intracellular concentrations of Cl− and H+ requires sensitive probes that allow reliable quantitative measurements without perturbation of cell functioning. For these purposes the most promising are genetically encoded fluorescent biosensors, which have become powerful tools for non-invasive intracellular monitoring of ions, molecules and enzymatic activity. A ratiometric CFP/YFP-based construct with a relatively good sensitivity to Cl− has been developed (Markova et al., 2008; Waseem et al., 2010. Recently, a combined Cl−/pH sensor (ClopHensor opened the way for simultaneous ratiometric measurement of these two ions (Arosio et al., 2010. ClopHensor was obtained by fusion of a red-fluorescent protein (DsRed-monomer to the E2GFP variant that contains a specific Cl−-binding site. This construct possesses pKa = 6.8 for H+ and Kd in the 40-50 mM range for Cl− at physiological pH (~7.3 As in the majority of cell types the intracellular Cl− concentration ([Cl−]i is about 10 mM, the development of sensors with higher sensitivity is highly desirable. Here we report the intracellular calibration and functional characterization of ClopHensor and its two derivatives: the membrane targeting PalmPalm-ClopHensor and the H148G/V224L mutant with improved Cl− affinity, reduced pH dependence and pKa shifted to more alkaline values. For functional analysis, constructs were expressed in CHO cells and [Cl−]i was changed by using pipettes with different Cl− concentrations during whole-cell recordings. Kd values for Cl− measured at 33°C and pH ~ 7.3 were, respectively, 39 mM, 47 mM and 21 mM for ClopHensor, PalmPalm-ClopHensor and the H148G/V224L mutant. PalmPalm-ClopHensor resolved responses to activation of Cl−-selective glycine receptor channels better than did ClopHensor. Our observations indicate that these different ClopHensor constructs are promising tools for non-invasive measurement of [Cl−]i in various living

  12. Real-time visualization of calcium ion activity in shallow benthic foraminiferal cells using the fluorescent indicator Fluo-3 AM (United States)

    Toyofuku, Takashi; Jan de Nooijer, Lennart; Yamamoto, Hiroyuki; Kitazato, Hiroshi


    Calcium ion storage and movements within foraminiferal cells were observed using the fluorescent cell-permeant calcium indicator Fluo-3 AM. Living specimens of the shallow-water benthic foraminifer Ammonia beccarii were incubated with 8 μM Fluo-3 AM in both natural seawater and calcium-free artificial seawater. No fluorescence was observed in specimens that were incubated in Fluo-3 AM/calcium-free seawater, while fluorescent emission was identified in all foraminifera that were incubated in Fluo-3 AM/natural seawater. In another series of experiments, juvenile specimens were incubated with Fluo-3 AM to trace calcium ion activity during the process of chamber formation. The method described here is a potential tool to observe the flow of calcium ions within living foraminiferal cells and may yield valuable information on the process of calcite precipitation.

  13. Imaging of calcium dynamics in pollen tube cytoplasm. (United States)

    Barberini, María Laura; Muschietti, Jorge


    Cytoplasmic calcium [(Ca(2+))cyt] is a central component of cellular signal transduction pathways. In plants, many external and internal stimuli transiently elevate (Ca(2+))cyt, initiating downstream responses that control different features of plant development. In pollen tubes the establishment of an oscillatory gradient of calcium at the tip is essential for polarized growth. Disruption of the cytosolic Ca(2+) gradient by chelators or channel blockers inhibits pollen tube growth. To quantify the physiological role of (Ca(2+))cyt in cellular systems, genetically encoded Ca(2+) indicators such as Yellow Cameleons (YCs) have been developed. The Cameleons are based on a fluorescence resonance energy transfer (FRET) process. Here, we describe a method for imaging cytoplasmic Ca(2+) dynamics in growing pollen tubes that express the fluorescent calcium indicator Yellow Cameleon 3.6 (YC 3.6), using laser-scanning confocal microscopy.

  14. StrigoQuant: A genetically encoded biosensor for quantifying strigolactone activity and specificity

    KAUST Repository

    Samodelov, S. L.


    Strigolactones are key regulators of plant development and interaction with symbiotic fungi; however, quantitative tools for strigolactone signaling analysis are lacking. We introduce a genetically encoded hormone biosensor used to analyze strigolactone-mediated processes, including the study of the components involved in the hormone perception/signaling complex and the structural specificity and sensitivity of natural and synthetic strigolactones in Arabidopsis, providing quantitative insights into the stereoselectivity of strigolactone perception. Given the high specificity, sensitivity, dynamic range of activity, modular construction, ease of implementation, and wide applicability, the biosensor StrigoQuant will be useful in unraveling multiple levels of strigolactone metabolic and signaling networks.

  15. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo. (United States)

    Tada, Mayumi; Takeuchi, Atsuya; Hashizume, Miki; Kitamura, Kazuo; Kano, Masanobu


    Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

  16. FRET-based genetically-encoded sensors for quantitative monitoring of metabolites. (United States)

    Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad


    Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

  17. Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo. (United States)

    Yang, Helen H; St-Pierre, François; Sun, Xulu; Ding, Xiaozhe; Lin, Michael Z; Clandinin, Thomas R


    A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

  18. Shining light on signaling and metabolic networks by genetically encoded biosensors (United States)

    Lalonde, Sylvie; Ehrhardt, David W; Frommer, Wolf B


    Fluorescent labels have revolutionized cell biology. Signaling intermediates and metabolites can be measured in real time with subcellular spatial resolution. Most of these sensors are based on fluorescent proteins, and many report fluorescence resonance energy transfer. Because the biosensors are genetically encoded, a toolbox for addressing cell biological questions at the systems level is now available. Fluorescent biosensors are able to determine the localization of proteins and their dynamics, to reveal the cellular and subcellular localization of the respective interactions and activities, and to provide complementary data on the steady state levels of ions, metabolites, and signaling intermediates with high temporal and spatial resolution. They represent the basis for cell-based high-throughput assays that are necessary for a systems perspective on plant cell function. PMID:16188489

  19. Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics. (United States)

    Sharma, Vangmayee; Wang, Yane-Shih; Liu, Wenshe R


    Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme's catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.

  20. pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH (United States)

    Robertson, J. Brian; Johnson, Carl Hirschie


    We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H+ specific; neither Ca++, Mg++, Na+, nor K+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate. PMID:22905204

  1. A fully genetically encoded protein architecture for optical control of peptide ligand concentration (United States)

    Schmidt, Daniel; Tillberg, Paul W.; Chen, Fei; Boyden, Edward S.


    Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.

  2. Organoids and the genetically encoded self-assembly of embryonic stem cells. (United States)

    Turner, David A; Baillie-Johnson, Peter; Martinez Arias, Alfonso


    Understanding the mechanisms of early embryonic patterning and the timely allocation of specific cells to embryonic regions and fates as well as their development into tissues and organs, is a fundamental problem in Developmental Biology. The classical explanation for this process had been built around the notion of positional information. Accordingly the programmed appearance of sources of Morphogens at localized positions within a field of cells directs their differentiation. Recently, the development of organs and tissues from unpatterned and initially identical stem cells (adult and embryonic) has challenged the need for positional information and even the integrity of the embryo, for pattern formation. Here we review the emerging area of organoid biology from the perspective of Developmental Biology. We argue that the events underlying the development of these systems are not purely linked to self-organization, as often suggested, but rather to a process of genetically encoded self-assembly where genetic programs encode and control the emergence of biological structures.

  3. Development and properties of genetically encoded pH sensors in plants.

    Directory of Open Access Journals (Sweden)

    Alexandre eMartinière


    Full Text Available Fluorescent proteins (FPs have given access to a large choice of live imaging techniques and has thereby profoundly modified our view of plant cells. Together with technological improvement of imaging, they have opened the possibility to monitor physico-chemical changes within cells. For this purpose, a new generation of fluorescent proteins has been engineered. For instance, pHluorin, a point mutated version of GFP, allows to get local pH estimates. In this paper, we will describe how genetically encoded sensors can be used to measure pH in the microenvironment of living tissues and subsequently discuss the role of pH in (i exocytosis, (ii ion uptake by plant roots, (iii cell growth and (iv protein trafficking.

  4. pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.

    Directory of Open Access Journals (Sweden)

    Yunfei Zhang

    Full Text Available We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+ specific; neither Ca(++, Mg(++, Na(+, nor K(+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

  5. Reversibly switchable photoacoustic tomography using a genetically encoded near-infrared phytochrome (United States)

    Yao, Junjie; Kaberniuk, Andrii A.; Li, Lei; Shcherbakova, Daria M.; Zhang, Ruiying; Wang, Lidai; Li, Guo; Verkhusha, Vladislav V.; Wang, Lihong V.


    Optical imaging of genetically encoded probes has revolutionized biomedical studies by providing valuable information about targeted biological processes. Here, we report a novel imaging technique, termed reversibly switchable photoacoustic tomography (RS-PAT), which exhibits large penetration depth, high detection sensitivity, and super-resolution. RS-PAT combines advanced photoacoustic imaging techniques with, for the first time, a nonfluorescent photoswitchable bacterial phytochrome. This bacterial phytochrome is the most near-infrared shifted genetically encoded probe reported so far. Moreover, this bacterial phytochrome is reversibly photoconvertible between its far-red and near-infrared light absorption states. Taking maximum advantage of the powerful imaging capability of PAT and the unique photochemical properties of the phytochrome, RS-PAT has broken through both the optical diffusion limit for deep-tissue imaging and the optical diffraction limit for super-resolution photoacoustic microscopy. Specifically, with RS-PAT we have achieved an unprecedented detection sensitivity of ~2 μM, or as few as ~20 tumor cells, at a centimeter depth. Such high sensitivity is fully demonstrated in our study by monitoring tumor growth and metastasis at whole-body level with ~100 μm resolution. Moreover, our microscopic implementation of RS-PAT is capable of imaging mammalian cells with a sub-diffraction lateral resolution of ~140 nm and axial resolution of ~400 nm, which are respectively ~2-fold and ~75-fold finer than those of our conventional photoacoustic microscopy. Overall, RS-PAT is a new and promising imaging technology for studying biological processes at different length scales.

  6. Controlling enzyme inhibition using an expanded set of genetically encoded amino acids. (United States)

    Zheng, Shun; Kwon, Inchan


    Enzyme inhibition plays an important role in drug development, metabolic pathway regulation, and biocatalysis with product inhibition. When an inhibitor has high structural similarities to the substrate of an enzyme, controlling inhibitor binding without affecting enzyme substrate binding is often challenging and requires fine-tuning of the active site. We hypothesize that an extended set of genetically encoded amino acids can be used to design an enzyme active site that reduces enzyme inhibitor binding without compromising substrate binding. As a model case, we chose murine dihydrofolate reductase (mDHFR), substrate dihydrofolate, and inhibitor methotrexate. Structural models of mDHFR variants containing non-natural amino acids complexed with each ligand were constructed to identify a key residue for inhibitor binding and non-natural amino acids to replace the key residue. Then, we discovered that replacing the key phenylalanine residue with two phenylalanine analogs (p-bromophenylalanine (pBrF) and L-2-naphthylalanine (2Nal)) enhances binding affinity toward the substrate dihydrofolate over the inhibitor by 4.0 and 5.8-fold, respectively. Such an enhanced selectivity is mainly due to a reduced inhibitor binding affinity by 2.1 and 4.3-fold, respectively. The catalytic efficiency of the mDHFR variant containing pBrF is comparable to that of wild-type mDHFR, whereas the mDHFR variant containing 2Nal exhibits a moderate decrease in the catalytic efficiency. The work described here clearly demonstrates the feasibility of selectively controlling enzyme inhibition using an expanded set of genetically encoded amino acids.

  7. Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents

    Directory of Open Access Journals (Sweden)

    Jennifer H Hou


    Full Text Available The cardiac action potential (AP and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf. We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 – 102 hours post fertilization (hpf, the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

  8. Study on the Ratio of Solid Calcium Indicator%固体钙指示剂的配比研究

    Institute of Scientific and Technical Information of China (English)

    宋秀丽; 易伟


    To study the best ratio of solid calcium indicator.Experimental results under certain conditions have been compared from the determination of calcium iron with different ratio solid calcium indicator.Experimental results showed that the best mass ratio was 1:100 of solid calcium indicator and sodium chloride.At this ratio the sharp color changed in end point,the accuracy was significantly improved.This method has been applied to the determination of calcium sample with satisfactory result.The determination results was the same as national standard method consistent with the recovery of 99.9%~107.7% RSD was less than 1.53%.So this method can be used to determine the calcium sample and monitor the quality products.%研究固体钙指示剂的最佳配制比例,对不同配比的固体钙指示剂在一定条件下测定钙离子的实验结果进行比较.实验结果表明:固体钙指示剂的最佳配比是钙指示剂与氯化钠的质量比为1∶100,此比例下终点变色敏锐,准确度显著提高,稳定性好.在该配比下测定了样品的含量,其回收率在99.9%~107.7%的范围内,相对标准偏差在1.53%以内,测定结果与用国家标准方法所测结果一致.该方法可用于测定含钙试样,对产品质量进行监控.

  9. Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways. (United States)

    Michener, Joshua K; Thodey, Kate; Liang, Joe C; Smolke, Christina D


    Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.

  10. Engineering genetically encoded nanosensors for real-time in vivo measurements of citrate concentrations.

    Directory of Open Access Journals (Sweden)

    Jennifer C Ewald

    Full Text Available Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET. We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.

  11. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening. (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam


    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

  12. EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels. (United States)

    Schmidt, Moritz J; Fedoseev, Artem; Bücker, Dennis; Borbas, Julia; Peter, Christine; Drescher, Malte; Summerer, Daniel


    The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.

  13. Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor. (United States)

    Ahmad, Mohammad; Ameen, Seema; Siddiqi, Tariq Omar; Khan, Parvez; Ahmad, Altaf


    Glycine betaine (GB) is one of the key compatible solutes that accumulate in the cell at exceedingly high level under the conditions of high salinity. It plays a crucial role in the maintenance of osmolarity of the cell without affecting the physiological processes. Analysis of stress-induced physiological conditions in living cells, therefore, requires real-time monitoring of cellular GB level. Glycine Betaine Optical Sensor (GBOS), a genetically-encoded FRET-based nanosensor developed in this study, allows the real-time monitoring of GB levels inside living cells. This nanosensor has been developed by sandwiching GB binding protein (ProX) between the Förster resonance energy transfer (FRET) pair, the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Conformational change in ProX, which was used as sensory domain, reported the change in the level of this compatible solute in in vitro and in vivo conditions. Binding of the GB to the sensory domain fetches close to both the fluorescent moieties that result in the form of increased FRET ratio. So, any change in the concentration of GB is correlated with change in FRET ratio. This sensor also reported the GB cellular dynamics in real-time in Escherichia coli cells after the addition of its precursor, choline. The GBOS was also expressed in yeast and mammalian cells to monitor the intracellular GB. Therefore, the GBOS represents a unique FRET-based nanosensor which allows the non-invasive ratiometric analysis of the GB in living cells.

  14. Probing Protein-Protein Interactions with Genetically Encoded Photoactivatable Cross-Linkers. (United States)

    Cooley, Richard B; Sondermann, Holger


    Fundamental to all living organisms is the ability of proteins to interact with other biological molecules at the right time and location, with the proper affinity, and to do so reversibly. One well-established technique to study protein interactions is chemical cross-linking, a process in which proteins in close spatial proximity are covalently tethered together. An emerging technology that overcomes many limitations of traditional cross-linking methods is one in which photoactivatable cross-linking noncanonical amino acids are genetically encoded into a protein of interest using the cell's native translational machinery. These proteins can then be used to trap interacting biomolecules upon UV illumination. Here, we describe a method for the site-specific incorporation of photoactivatable cross-linking amino acids into fluorescently tagged proteins of interest in E. coli. Photo-cross-linking and analysis by SDS-PAGE using in-gel fluorescence detection, which provides rapid, highly sensitive, and specific detection of cross-linked adducts even in impure systems, are also described. An example expression and cross-linking experiment involving transmembrane signaling of a bacterial second messenger receptor system that controls biofilm formation is shown. All reagents needed to carry out these experiments are commercially available, and do not require special or unique technology to perform, making this method tractable to a broad community studying protein structure and function.

  15. Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells. (United States)

    Uprety, Rajendra; Luo, Ji; Liu, Jihe; Naro, Yuta; Samanta, Subhas; Deiters, Alexander


    We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

  16. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.

    Directory of Open Access Journals (Sweden)

    Xiaokun Shu


    Full Text Available Electron microscopy (EM achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator, a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

  17. Correlation of mandibular radiomorphometric indices with serum calcium and serum estradiol in pre- and post-menopausal women

    Directory of Open Access Journals (Sweden)

    Lina Govind Chandak


    Full Text Available Background: Osteoporosis is a disease that is seen commonly with increasing age. The purpose of this study was to compare the bone quality of pre- and post-menopausal women using the quantitative indices determined by measurements on panoramic radiographs (mental index, inferior and superior panoramic mandibular indices, antegonion index [AGI], and gonion index and to determine the effects of serum calcium and serum estradiol levels on alveolar bone loss. Materials and Methods: Sixty female patients in the age group of 25–55 years were included in the study. The patients were divided into three equal groups, i.e., control Group A (twenty - premenopausal women, study Group B (twenty - postmenopausal women with healthy periodontium, study Group C (twenty - postmenopausal women with periodontitis. Quantitative indices were measured on digital panoramic radiographs of the patients and serum calcium and estradiol levels were determined. Results: Correlation of serum calcium with radiomorphometric indices of all the groups showed statistically nonsignificant differences. On correlating mean estradiol levels with radiographic indices of patients of Group A and Group B showed statistically nonsignificant differences. On correlating mean estradiol levels with radiographic indices of patients of Group C patients showed statistically significant difference with positive correlation with cortical width (P = 0.04 and AGI (P = 0.02 while statistically nonsignificant correlation with other indices. The statistical tests used for the analysis of the result were one-way ANOVA, multiple comparison Tukey test, Chi-square test, Student's t-test. Conclusion: There is a little evidence of correlation of these indices with serum estradiol and calcium levels, and therefore, detailed further research about this correlation is required.

  18. Calcium imaging in the Drosophila olfactory system with a genetic indicator. (United States)

    Root, Cory M; Wong, Allan M; Flores, Jorge; Wang, Jing W


    Insects show sophisticated odor-mediated behaviors controlled by an olfactory system that is genetically and anatomically simpler than that of vertebrates, providing an attractive system to investigate the mechanistic link between behavior and odor perception. Advances in neuroscience have been facilitated by modern optical imaging technologies--both in instrumentation and in probe design--that permit the visualization of functional neural circuits. Imaging calcium activity in genetically defined populations of neurons provides an important tool for investigating the function of neural circuits. This article describes a two-photon imaging system for monitoring neural activity in the Drosophila antennal lobe. Odor-evoked calcium activity is followed by measuring the specific expression of the calcium-sensitive green fluorescent protein G-CaMP in Drosophila antennae-brain preparations.

  19. Concurrent imaging of synaptic vesicle recycling and calcium dynamics.

    Directory of Open Access Journals (Sweden)

    Haiyan eLi


    Full Text Available Synaptic transmission involves the calcium-dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2, and a presynaptically-localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3 with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Re-acidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real-time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.

  20. Genetically-encoded tools for cAMP probing and modulation in living systems.

    Directory of Open Access Journals (Sweden)

    Valeriy M Paramonov


    Full Text Available Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming - all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells, underpin the ensuing limitations of the conventional cAMP assays: 1 genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; 2 inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control – something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.

  1. Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors.

    Directory of Open Access Journals (Sweden)

    Katrin Gruenwald

    Full Text Available Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.

  2. In vivo optoacoustic monitoring of calcium activity in the brain (Conference Presentation) (United States)

    Deán-Ben, Xose Luís.; Gottschalk, Sven; Sela, Gali; Lauri, Antonella; Kneipp, Moritz; Ntziachristos, Vasilis; Westmeyer, Gil G.; Shoham, Shy; Razansky, Daniel


    Non-invasive observation of spatio-temporal neural activity of large neural populations distributed over the entire brain of complex organisms is a longstanding goal of neuroscience [1,2]. Recently, genetically encoded calcium indicators (GECIs) have revolutionized neuroimaging by enabling mapping the activity of entire neuronal populations in vivo [3]. Visualization of these powerful sensors with fluorescence microscopy has however been limited to superficial regions while deep brain areas have so far remained unreachable [4]. We have developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains [5]. The developed methodology can render 100 volumetric frames per second across scalable fields of view ranging between 50-1000 mm3 with respective spatial resolution of 35-150µm. Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically-encoded calcium indicator GCaMP5G demonstrated, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the depth barrier of optical imaging in scattering brains [6]. It was further possible to monitor calcium transients in a scattering brain of a living adult transgenic zebrafish expressing GCaMP5G calcium indicator [7]. Fast changes in optoacoustic traces associated to GCaMP5G activity were detectable in the presence of other strongly absorbing endogenous chromophores, such as hemoglobin. The results indicate that the optoacoustic signal traces generally follow the GCaMP5G fluorescence dynamics and further enable overcoming the longstanding optical-diffusion penetration barrier associated to scattering in biological tissues [6]. The new functional optoacoustic neuroimaging method can visualize neural activity at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques. Thus, in addition to the well

  3. Engineering of a Genetically Encodable Fluorescent Voltage Sensor Exploiting Fast Ci-VSP Voltage-Sensing Movements


    Alicia Lundby; Hiroki Mutoh; Dimitar Dimitrov; Walther Akemann; Thomas Knöpfel


    Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence re...

  4. Technical Note: Calcium and carbon stable isotope ratios as paleodietary indicators. (United States)

    Melin, Amanda D; Crowley, Brooke E; Brown, Shaun T; Wheatley, Patrick V; Moritz, Gillian L; Yit Yu, Fred Tuh; Bernard, Henry; DePaolo, Donald J; Jacobson, Andrew D; Dominy, Nathaniel J


    Calcium stable isotope ratios are hypothesized to vary as a function of trophic level. This premise raises the possibility of using calcium stable isotope ratios to study the dietary behaviors of fossil taxa and to test competing hypotheses on the adaptive origins of euprimates. To explore this concept, we measured the stable isotope composition of contemporary mammals in northern Borneo and northwestern Costa Rica, two communities with functional or phylogenetic relevance to primate origins. We found that bone collagen δ(13) C and δ(15) N values could differentiate trophic levels in each assemblage, a result that justifies the use of these systems to test the predicted inverse relationship between bioapatite δ(13) C and δ(44) Ca values. As expected, taxonomic carnivores (felids) showed a combination of high δ(13) C and low δ(44) Ca values; however, the δ(44) Ca values of other faunivores were indistinguishable from those of primary consumers. We suggest that the trophic insensitivity of most bioapatite δ(44) Ca values is attributable to the negligible calcium content of arthropod prey. Although the present results are inconclusive, the tandem analysis of δ(44) Ca and δ(13) C values in fossils continues to hold promise for informing paleodietary studies and we highlight this potential by drawing attention to the stable isotope composition of the Early Eocene primate Cantius.

  5. Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells. (United States)

    Araki, Nobukazu; Ikeda, Yuka; Kato, Takuma; Kawai, Katsuhisa; Egami, Youhei; Miyake, Katsuya; Tsurumaki, Nobuhide; Yamaguchi, Mitsunari


    Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.

  6. Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC. (United States)

    Hyrc, K L; Bownik, J M; Goldberg, M P


    Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.

  7. Complexometric titration with potenciometric indicator to determination of calcium and magnesium in soil extracts¹

    Directory of Open Access Journals (Sweden)

    Claudia Mara Pereira


    Full Text Available This study proposes a method of direct and simultaneous determination of the amount of Ca2+ and Mg2+ present in soil extracts using a Calcium Ion-Selective Electrode and by Complexometric Titration (ISE-CT. The results were compared to those obtained by conventional analytical techniques of Complexometric Titration (CT and Flame Atomic Absorption Spectrometry (FAAS. There were no significant differences in the determination of Ca2+ and Mg2+ in comparison with CT and FAAS, at a 95 % confidence level. Additionally, results of this method were more precise and accurate than of the Interlaboratorial Control (IC.

  8. Direct estimates of natural selection in Iberia indicate calcium absorption was not the only driver of lactase persistence in Europe. (United States)

    Sverrisdóttir, Oddny Ósk; Timpson, Adrian; Toombs, Jamie; Lecoeur, Cecile; Froguel, Philippe; Carretero, Jose Miguel; Arsuaga Ferreras, Juan Luis; Götherström, Anders; Thomas, Mark G


    Lactase persistence (LP) is a genetically determined trait whereby the enzyme lactase is expressed throughout adult life. Lactase is necessary for the digestion of lactose--the main carbohydrate in milk--and its production is downregulated after the weaning period in most humans and all other mammals studied. Several sources of evidence indicate that LP has evolved independently, in different parts of the world over the last 10,000 years, and has been subject to strong natural selection in dairying populations. In Europeans, LP is strongly associated with, and probably caused by, a single C to T mutation 13,910 bp upstream of the lactase (LCT) gene (-13,910*T). Despite a considerable body of research, the reasons why LP should provide such a strong selective advantage remain poorly understood. In this study, we examine one of the most widely cited hypotheses for selection on LP--that fresh milk consumption supplemented the poor vitamin D and calcium status of northern Europe's early farmers (the calcium assimilation hypothesis). We do this by testing for natural selection on -13,910*T using ancient DNA data from the skeletal remains of eight late Neolithic Iberian individuals, whom we would not expect to have poor vitamin D and calcium status because of relatively high incident UVB light levels. None of the eight samples successfully typed in the study had the derived T-allele. In addition, we reanalyze published data from French Neolithic remains to both test for population continuity and further examine the evolution of LP in the region. Using simulations that accommodate genetic drift, natural selection, uncertainty in calibrated radiocarbon dates, and sampling error, we find that natural selection is still required to explain the observed increase in allele frequency. We conclude that the calcium assimilation hypothesis is insufficient to explain the spread of LP in Europe.

  9. Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging. (United States)

    Collot, Mayeul; Loukou, Christina; Yakovlev, Aleksey V; Wilms, Christian D; Li, Dongdong; Evrard, Alexis; Zamaleeva, Alsu; Bourdieu, Laurent; Léger, Jean-François; Ropert, Nicole; Eilers, Jens; Oheim, Martin; Feltz, Anne; Mallet, Jean-Maurice


    We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

  10. Physiological Adaptive Indicators in Fasted Neonate Broiler Chicks in Response to Calcium Gluconate Injection

    Directory of Open Access Journals (Sweden)

    Khosravinia H


    Full Text Available Four hundred and eighty mixed-sex broiler chicks aged 3 hrs after hatching were allotted according to a completely random design in a 6 × 2 × 2 factorial schedule into 2 groups of 12 replications of 20 chicks each. The main experimental factors were fasting for 0, 6, 12, 24, 36, and 48 hrs after chick placement, calcium gluconate (Ca-glu injection (0 and 0.6 mL and sex (male and female. Independent of sex, live body weight (BW of chicks decreased linearly (Y=43.36-0.109BW0h, r2=0.876 as neonatal fasting extended. Injection of 0.6 mL Ca-glu at 3 hrs post hatching did not affect weight loss of chicks. Yolk residuals (YR utilized linearly (Y=5.75-0.062YR, r2=0.956 by 0.062 g/hr in neonate fasted chicks showing no effect for Ca-glu injection. Neonatal fasting periods longer than 12 hrs increased liver weight (P. The mean absolute and proportional (% of BW0h breast and leg weight were reduced linearly as neonatal fasting extended (P. Serum glucose concentration in both sexes increased up to 6 hrs fasting, then reduced linearly to 150 mg/dL after 48 hrs feed withdrawal. The Ca-glu treatment influenced serum glucose level for a short period up to 6 hrs of fasting. Serum Ca concentration sharply increased up to three-fold in the birds received Ca-glu injection resulting in acute hypercalcemia, then decreased to the initial level after 24 hrs feed withdrawal. The mean serum level for creatinine, uric acid, cholesterol, HDL, albumins and total proteins significantly increased during the fasting periods of 6 to 48 hrs and significantly elevated in the birds received 0.6 mL Ca-glu injection compared with the non treated chicks. It was concluded that subcutaneous administration of 0.6 mL Ca-glu in the chick's neck did not suitably support the increased metabolic demands for glucose and calcium in feed deprived neonate chicks.

  11. Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements

    DEFF Research Database (Denmark)

    Lundby, Alicia; Mutoh, Hiroki; Dimitrov, Dimitar


    Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a g...... of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs....

  12. In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin. (United States)

    Lark, Arianna R; Kitamoto, Toshihiro; Martin, Jean-René


    Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca(2+)-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and - with the addition of its cofactor coelenterazine - emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca(2+)-transients and Ca(2+)-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca(2+)-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

  13. Lung adenocarcinoma with Lambert–Eaton myasthenic syndrome indicated by voltage-gated calcium channel: a case report

    Directory of Open Access Journals (Sweden)

    Arai Hiromasa


    Full Text Available Abstract Introduction Lambert–Eaton myasthenic syndrome is a rare disorder and it is known as a paraneoplastic neurological syndrome. Small cell lung cancer often accompanies this syndrome. Lambert–Eaton myasthenic syndrome associated with lung adenocarcinoma is extremely rare; there are only a few reported cases worldwide. Case presentation A 75-year-old Japanese man with a past history of chronic rheumatoid arthritis and Sjögren syndrome was diagnosed with Lambert–Eaton myasthenic syndrome by electromyography and serum anti-P/Q-type voltage-gated calcium channel antibody level preceding the diagnosis of lung cancer. A chest computed tomography to screen for malignant lesions revealed an abnormal shadow in the lung. Although a histopathological examination by bronchoscopic study could not reveal the malignancy, lung cancer was mostly suspected after the results of a chest computed tomography and [18F]-fluorodeoxyglucose positron emission tomography. An intraoperative diagnosis based on the frozen section obtained by tumor biopsy was adenocarcinoma so the patient underwent a lobectomy of the right lower lobe and lymph node dissection with video-assisted thoracoscopic surgery. The permanent pathological examination was the same as the frozen diagnosis (pT2aN1M0: Stage IIa: TNM staging 7th edition. Immunohistochemistry revealed that most of the cancer cells were positive for P/Q-type voltage-gated calcium channel. Conclusions Our case is a rare combination of Lambert–Eaton myasthenic syndrome associated with lung adenocarcinoma, rheumatoid arthritis and Sjögren syndrome, and to the best of our knowledge it is the first report that indicates the presence of voltage-gated calcium channel in lung adenocarcinoma by immunostaining.

  14. Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging. (United States)

    Broillet, M; Randin, O; Chatton, J


    The fluorescent indicator of nitric oxide (NO), 4,5-diaminofluorescein (DAF-2), and its membrane-permeable derivative (DAF-2 diacetate) have been recently developed to perform real-time biological imaging of NO. In this study, we show that DAF-2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF-2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application.

  15. A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic Reticulum Calcium Store. (United States)

    Henderson, Mark J; Baldwin, Heather A; Werley, Christopher A; Boccardo, Stefano; Whitaker, Leslie R; Yan, Xiaokang; Holt, Graham T; Schreiter, Eric R; Looger, Loren L; Cohen, Adam E; Kim, Douglas S; Harvey, Brandon K


    Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

  16. A Low Affinity GCaMP3 Variant (GCaMPer for Imaging the Endoplasmic Reticulum Calcium Store.

    Directory of Open Access Journals (Sweden)

    Mark J Henderson

    Full Text Available Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19. A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19 fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19 has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

  17. Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Kamran Honarnejad

    Full Text Available Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER. Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.

  18. Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo. (United States)

    Bedbrook, Claire N; Kato, Mihoko; Ravindra Kumar, Sripriya; Lakshmanan, Anupama; Nath, Ravi D; Sun, Fei; Sternberg, Paul W; Arnold, Frances H; Gradinaru, Viviana


    Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

  19. Proteomic analysis of renal calculi indicates an important role for inflammatory processes in calcium stone formation. (United States)

    Merchant, Michael L; Cummins, Timothy D; Wilkey, Daniel W; Salyer, Sarah A; Powell, David W; Klein, Jon B; Lederer, Eleanor D


    Even though renal stones/calculi occur in approximately 10% of individuals, they are an enormous economic burden to the entire US health system. While the relative metabolic composition of renal calculi is generally known, there is no clear understanding of the genetics of renal stone formation, nor are there clear prognostic indicators of renal stone formation. The application of proteomics to the analysis of renal calculi axiomatically holds that insight into renal stone pathobiology can be gained by a more comprehensive understanding of renal calculus protein composition. We analyzed isolated renal stone matrix proteins with mass spectrometric and immunohistochemical methods identifying 158 proteins with high confidence, including 28 common proteins. The abundant proteins included those identified previously in stones and proteins identified here for the first time, such as myeloid lineage-specific, integral membrane and lipid regulatory proteins. Pathway analyses of all proteins identified suggested that a significant fraction of the most abundant matrix proteins participate in inflammatory processes. These proteomic results support the hypothesis that stone formation induces a cellular inflammatory response and the protein components of this response contribute to the abundant stone matrix proteome.

  20. Calcium and vitamin D supplementation through fortified dairy products counterbalances seasonal variations of bone metabolism indices: the Postmenopausal Health Study. (United States)

    Tenta, Roxane; Moschonis, George; Koutsilieris, Michael; Manios, Yannis


    To assess the effectiveness of a dietary intervention combined with fortified dairy products on bone metabolism and bone mass indices in postmenopausal women. Forty postmenopausal women (55-65 years old) were equally randomized into a dietary group (DG), receiving daily and for 30 months, 1,200 mg of calcium and 7.5 μg of vitamin D(3) for the first 12 months that increased to 22.5 μg for the remaining 18 months of intervention through fortified dairy products; and a control group (CG). Differences in the changes of bone metabolism and bone mass indices were examined with repeated measures ANOVA. A significant increase was observed for PTH levels only in the CG during the first six winter months of intervention (p = 0.049). After 30 months of intervention, during winter, serum 25(OH)D significantly decreased in the CG while remained in the same high levels as in the summer period in the DG. Serum RANKL levels decreased significantly in the DG compared with the increase in the CG during the 30-month intervention period (p = 0.005). Serum CTx decreased significantly in the DG after six (-0.08; -0.12 to -0.03) and 12 (-0.03; -0.08 to -0.02) months of intervention. Finally, the DG had more favorable changes in total body BMD than the CG (p vitamin D in osteopenic postmenopausal women appears to be effective in producing favorable changes in several bone metabolism and bone mass indices and in counterbalancing seasonal variations in hormonal and biochemical molecules.

  1. Filamin and phospholipase C-ε are required for calcium signaling in the Caenorhabditis elegans spermatheca.

    Directory of Open Access Journals (Sweden)

    Ismar Kovacevic


    Full Text Available The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue.

  2. Loading and localization of Fluo-3 and Fluo-3/AM calcium indicators in sinapis alba root tissue. (United States)

    Tretyn, A; Kado, R T; Kendrick, R E


    Stimulus-induced changes in free cytosolic Ca2+ in different types of plant cells have been monitored with the aid of fluorescent calcium indicator dyes. However, there is no simple and convenient method for introducing these dyes into the plant cell cytoplasm. This paper reports tests of different procedures for loading either free fluorescent dyes or their acetoxymethyl esters (Fluo-3 and Fluo-3/AM, respectively) into Sinapis alba root tissue. Loading of Fluo-3 was pH and temperature dependent. Moreover, in the presence of beta-escin (saponin) in the loading medium very high fluorescent signals in root tissues were observed. The highest signals were recorded when tissue was loaded in a medium containing 0.1% beta-escin, at pH 5.0 and 30 degrees C. Only very weak fluorescence signals were found in mustard roots loaded with Fluo-3/AM. Acidity and temperature of the medium had no significant effect on the process. However, addition of eserine, a cholinesterase inhibitor led to a dramatic increase in fluorescence in the root cells. On the basis of these observations rapid and efficient methods of loading both Fluo-3 and Fluo-3/AM into mustard root tissues are proposed.

  3. A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells.

    Directory of Open Access Journals (Sweden)

    Alejandro San Martín

    Full Text Available Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3-5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg

  4. Redesign of genetically encoded biosensors for monitoring mitochondrial redox status in a broad range of model eukaryotes. (United States)

    Albrecht, Simone C; Sobotta, Mirko C; Bausewein, Daniela; Aller, Isabel; Hell, Rüdiger; Dick, Tobias P; Meyer, Andreas J


    The development of genetically encoded redox biosensors has paved the way toward chemically specific, quantitative, dynamic, and compartment-specific redox measurements in cells and organisms. In particular, redox-sensitive green fluorescent proteins (roGFPs) have attracted major interest as tools to monitor biological redox changes in real time and in vivo. Most recently, the engineering of a redox relay that combines glutaredoxin (Grx) with roGFP2 as a translational fusion (Grx1-roGFP2) led to a biosensor for the glutathione redox potential (EGSH ). The expression of this probe in mitochondria is of particular interest as mitochondria are the major source of oxidants, and their redox status is closely connected to cell fate decisions. While Grx1-roGFP2 can be expressed in mammalian mitochondria, it fails to enter mitochondria in various nonmammalian model organisms. Here we report that inversion of domain order from Grx1-roGFP2 to roGFP2-Grx1 yields a biosensor with perfect mitochondrial targeting while fully maintaining its biosensor capabilities. The redesigned probe thus allows extending in vivo observations of mitochondrial redox homeostasis to important nonmammalian model organisms, particularly plants and insects.

  5. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

    Directory of Open Access Journals (Sweden)

    Alexander S. Goryashchenko


    Full Text Available This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds, pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

  6. A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.

    Directory of Open Access Journals (Sweden)

    Jianjie Mi

    Full Text Available Filament bundles (rods of cofilin and actin (1:1 form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30-60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.

  7. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

    Directory of Open Access Journals (Sweden)

    Potzkei Janko


    Full Text Available Abstract Background Molecular oxygen (O2 is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results We developed a genetically encoded Förster resonance energy transfer (FRET-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen consists of the yellow fluorescent protein (YFP that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP. Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (pathophysiological processes in living cells.

  8. Stability indicating method development and validation for simultaneous estimation of atorvastatin calcium and celecoxib in bulk and niosomal formulation by RP-HPLC

    Directory of Open Access Journals (Sweden)

    Priyanka S. Jadhav


    Full Text Available The present work describes development and validation of a specific, sensitive, precise and stability-indicating high-performance liquid chromatographic method of analysis of atorvastatin calcium and celecoxib, both as a bulk drug and in niosomal formulation. The analysis has been performed by using Cosmosil-C18 column (4.6 mm´250 mm, 5 m at 25 °C using acetonitrile: ammonium acetate buffer pH 5.0: methanol (50:25:25 v/v/v as mobile phase. The detection was carried out at 277nm with a flow rate of 1.0mL/min. The retention times of Atorvastatin calcium and Celecoxib were 6.195 and 3.989min, respectively. The method was validated according to ICH guidelines, for specificity, precision, linearity, accuracy and robustness. Atorvastatin calcium and Celecoxib were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation was observed in oxidation and acid hydrolysis. The linearity for atorvastatin calcium and celecoxib were in the range of 100-500 µg/mL. The recovery study of atorvastatin and celecoxib were found to be in the range of 98.96 - 99.92% and 98.90-100%, respectively. The proposed method was validated and successfully applied to the estimation of Atorvastatin calcium and Celecoxib in combined in-house niosomal formulation.

  9. Visualization of Plasticity in Fear-Evoked Calcium Signals in Midbrain Dopamine Neurons (United States)

    Gore, Bryan B.; Soden, Marta E.; Zweifel, Larry S.


    Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium…

  10. Visualization of Plasticity in Fear-Evoked Calcium Signals in Midbrain Dopamine Neurons (United States)

    Gore, Bryan B.; Soden, Marta E.; Zweifel, Larry S.


    Dopamine is broadly implicated in fear-related processes, yet we know very little about signaling dynamics in these neurons during active fear conditioning. We describe the direct imaging of calcium signals of dopamine neurons during Pavlovian fear conditioning using fiber-optic confocal microscopy coupled with the genetically encoded calcium…

  11. Hierarchy of neural organization in the embryonic spinal cord: Granger-causality graph analysis of in vivo calcium imaging data. (United States)

    Fallani, Fabrizio De Vico; Corazzol, Martina; Sternberg, Jenna R; Wyart, Claire; Chavez, Mario


    The recent development of genetically encoded calcium indicators enables monitoring in vivo the activity of neuronal populations. Most analysis of these calcium transients relies on linear regression analysis based on the sensory stimulus applied or the behavior observed. To estimate the basic properties of the functional neural circuitry, we propose a network approach to calcium imaging recorded at single cell resolution. Differently from previous analysis based on cross-correlation, we used Granger-causality estimates to infer information propagation between the activities of different neurons. The resulting functional network was then modeled as a directed graph and characterized in terms of connectivity and node centralities. We applied our approach to calcium transients recorded at low frequency (4 Hz) in ventral neurons of the zebrafish spinal cord at the embryonic stage when spontaneous coiling of the tail occurs. Our analysis on population calcium imaging data revealed a strong ipsilateral connectivity and a characteristic hierarchical organization of the network hubs that supported established propagation of activity from rostral to caudal spinal cord. Our method could be used for detecting functional defects in neuronal circuitry during development and pathological conditions.

  12. Sensing Cardiac Electrical Activity With a Cardiac Myocyte--Targeted Optogenetic Voltage Indicator

    NARCIS (Netherlands)

    Chang Liao, Mei-Ling; de Boer, Teun P; Mutoh, Hiroki; Raad, Nour; Richter, Claudia; Wagner, Eva; Downie, Bryan R; Unsöld, Bernhard; Arooj, Iqra; Streckfuss-Bömeke, Katrin; Döker, Stephan; Luther, Stefan; Guan, Kaomei; Wagner, Stefan; Lehnart, Stephan E; Maier, Lars S; Stühmer, Walter; Wettwer, Erich; van Veen, Toon; Morlock, Michael M; Knöpfel, Thomas; Zimmermann, Wolfram-Hubertus


    RATIONALE: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac


    Khripach, L V; Mikhaylova, R I; Koganova, Z I; Knyazeva, T D; Alekseeva, A V; Savostikova, O N; Ryzhova, I N; Kruglova, E V; Revzova, T L


    There are discussed the changes of an array of indices of the oxidative status in chronic administration of colloidal calcium carbonate preparation with faucet and low-mineralized drinking water to rats. Slight differences between significant effects of administration of 3 and 30 mg/L of preparation permit to suggest that the process of its incoming delivery into organism of rats has a bottleneck in the nature of total capacity of macrophages of intestinal lymphoid tissue to absorption of particles.

  14. Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

    Energy Technology Data Exchange (ETDEWEB)

    Akerboom, Jasper; Velez Rivera, Jonathan D.; Rodriguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Hernandez, Hector H.; Tian, Lin; Hires, S. Andrew; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.; (MIT); (Puerto Rico); (HHMI)


    The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca{sup 2+}-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

  15. Recombinant γT305A fibrinogen indicates severely impaired fibrin polymerization due to the aberrant function of hole 'A' and calcium binding sites. (United States)

    Ikeda, Minami; Kobayashi, Tamaki; Arai, Shinpei; Mukai, Saki; Takezawa, Yuka; Terasawa, Fumiko; Okumura, Nobuo


    We examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio=0.295), suggesting hypodysfibrinogenemia. DNA sequence analysis was performed, and γT305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant γN308K fibrinogen. DNA sequence analysis revealed a heterozygous γT305A substitution (mature protein residue number). The γT305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1mM calcium ion compared with that of γN308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob 'A') and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole 'a' and D:D interaction sites were all markedly impaired, whereas γN308Kwas impaired at the latter two sites. Molecular modeling demonstrated that γT305 is localized at a shorter distance than γN308 from the high affinity calcium binding site and hole 'a'. Our findings suggest that γT305 might be important for construction of the overall structure of the γ module of fibrinogen. Substitution of γT305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant γT305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Concurrent Imaging of Receptor Trafficking and Calcium Dynamics by Spinning Disk Confocal Microscopy. (United States)

    Larsen, DeLaine D; Choy, Regina Wai-Yan; Park, Minjong


    Synaptic activity is modulated by the activation of neuromodulator receptors present in dendrites of neurons. The majority of neuromodulator receptors are G protein coupled receptors (GPCRs), in which membrane trafficking regulates their activities. Membrane trafficking of neuromodulator receptors and their signaling occurs on a rapid time scale and emerging studies indicate that neuromodulator receptors function not just from the plasma membrane but also from the endocytic compartments. Here, we describe a live cell imaging approach using spinning disk confocal microscopy to investigate the effect of neuromodulator receptor activation on synaptic activity by measuring calcium dynamics in primary rat striatal neurons. The advantages of spinning disk confocal microscopy and recent improvements in the genetically encoded calcium sensor, GCaMP6, provide an imaging approach to image both the receptor membrane trafficking to endocytic compartments, and calcium dynamics at a high spatial and temporal resolution. We believe this approach of imaging both the neuromodulator receptor membrane trafficking and synaptic activity using GCaMP6 is a powerful tool to address many questions regarding possible roles of membrane trafficking of neuromodulator receptors in synaptic activity.

  17. A confocal study of mechanism of 5-hydroxytryptaminoinduced intracellular calcium dynamics in cultured ratstomach fundus smooth muscle cells with a new Ca2+indicator STDIn-AM

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Xiaoling; (张小玲); YAN; Hongtao; (阎宏涛)


    A new fluorescent Ca2+ indicator STDIn-AM for detecting i transients in cultured smooth muscle cells is presented. By making a comparison, the difference between STDIn and fluo-3 is discussed in detail. Using the new Ca2+ indicator, the mechanism of 5-hydroxytryptamino (5-HT) induced intracellular calcium dynamics in stomach fundus smooth muscle cells (SFSMC) of rats is investigated. It is shown that in contrast with fluo-3, STDIn is uniformly distributed in the cytosolic compartment but excluded from the nucleus, when it is transfected into cells. This feature makes it a real cytosol Ca2+ indicator and can reflect changes of cytosol more accurately than that of fluo-3. In addition, STDIn responds to the i transients more sensitive and faster than fluo-3. The results also show that, the L-type Ca2+ channel inhibitor Mn9202 and the PLC inhibitor Compound 48/80 can significantly inhibit the i elevation induced by 5-HT, while the PKC inhibitor D-Sphingosine can enhance the effect of 5-HT. The results suggest that 5-HT acts by the way of 5-HT2 receptors on SFSMC, then through 5-HT2 receptors coupled IP3/Ca2+ and GC/PKC double signal transduction pathways to make Ca2+ release from intracellular Ca2+ stores and Ca2+ influx possibly through L-type calcium channels.

  18. Lamivudine, Entecavir, or Tenofovir Treatment of Hepatitis B Infection: Effects on Calcium, Phosphate, FGF23 and Indicators of Bone Metabolism. (United States)

    Saeedi, Ramesh; Mojebi-Mogharar, Ali; Sandhu, Supna K; Dubland, Joshua A; Ford, Jo-Ann; Yousefi, Masoud; Pudek, Morris; Holmes, Daniel T; Erb, Siegfried R; Peter Kwan, Wing; Kendler, David L; Yoshida, Eric M


    Patients with chronic hepatitis B virus (HBV) are often treated with nucleoside/nucleotide antiviral agents and metabolic bone toxicity is a possible concern. To determine the relationships between fibroblast growth factor 23 (FGF23), a phosphaturic hormone, bone mineral density (BMD), and bone biochemical abnormalities in these patients. This is a cross-sectional observational study comparing HBV-infected subjects treated for at least one year with tenofovir (TDF), lamuvidine (LVD), entacavir (ETV), or not treated (CON). Patients with abnormalities in either calcium (Ca), phosphate (PO4), intact parathyroid hormone (iPTH) or FGF23 were further evaluated with BMD by DXA. No difference in liver enzymes or renal function seen among groups, but hypophosphatemia was seen in all groups with the highest incidence with TDF-treatment (14%). FGF 23 levels were found to be elevated in 11.1% of TDF patients, 2.77% amongst controls. No elevations were found in the LVD or ETV groups. Among a subset of subjects (FGF23, PO4, and/or Ca abnormalities) who underwent further evaluation, 67% had insufficient 25-OH vitamin D, and 30% had elevated 24 h urinary Ca or PO4 excretion. No patients with FGF23 abnormalities had urine abnormalities. 40% had low DXA Z-score (<-2) at spine or hip but there was no difference between control and antiviral treatment groups and the mean FRAX score was 2.33% for major osteoporotic fractures and 0.29% for hip fracture. Abnormalities in bone metabolism, particularly involving vitamin D insufficiency, in HBV-treated subjects were observed with a small increased likelihood in TDF treated patients.

  19. Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model.

    Directory of Open Access Journals (Sweden)

    Marina Shirmanova

    Full Text Available The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm and pulsed laser (584 nm, 10 Hz, 18 ns modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

  20. Calcium imaging of odor-evoked responses in the Drosophila antennal lobe. (United States)

    Silbering, Ana F; Bell, Rati; Galizia, C Giovanni; Benton, Richard


    The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions. Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP and Cameleon, the bioluminescent calcium indicator GFP-aequorin, or a reporter of synaptic transmission, synapto-pHluorin has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS, LexA/LexAop, Q system) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed. Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP. The animal preparation is minimally invasive and can be adapted to imaging using wide

  1. Haploinsufficiency of the 22q11.2-microdeletion gene Mrpl40 disrupts short-term synaptic plasticity and working memory through dysregulation of mitochondrial calcium (United States)

    Devaraju, Prakash; Yu, Jing; Eddins, Donnie; Mellado-Lagarde, Marcia M.; Earls, Laurie R.; Westmoreland, Joby J.; Quarato, Giovanni; Green, Douglas R.; Zakharenko, Stanislav S.


    Hemizygous deletion of a 1.5- to 3-megabase region on chromosome 22 causes 22q11.2 deletion syndrome (22q11DS), which constitutes one of the strongest genetic risks for schizophrenia. Mouse models of 22q11DS have abnormal short-term synaptic plasticity (STP) that contributes to working memory deficiencies similar to those in schizophrenia. We screened mutant mice carrying hemizygous deletions of 22q11DS genes and identified haploinsufficiency of Mrpl40 (mitochondrial large ribosomal subunit protein 40) as a contributor to abnormal STP. Two-photon imaging of the genetically encoded fluorescent calcium indicator GCaMP6, expressed in presynaptic cytosol or mitochondria, showed that Mrpl40 haploinsufficiency deregulates STP via impaired calcium extrusion from the mitochondrial matrix through the mitochondrial permeability transition pore. This led to abnormally high cytosolic calcium transients in presynaptic terminals and deficient working memory but did not affect long-term spatial memory. Thus, we propose that mitochondrial calcium deregulation is a novel pathogenic mechanism of cognitive deficiencies in schizophrenia. PMID:27184122

  2. Real-time and high-throughput analysis of mitochondrial metabolic states in living cells using genetically encoded NAD(+)/NADH sensors. (United States)

    Zhao, Yuzheng; Yang, Yi


    Mitochondria are central organelles that regulate cellular bioenergetics, biosynthesis, and signaling processes. NADH, a key player in cell metabolism, is often considered as a marker of mitochondrial function. However, traditional methods for NADH measurements are either destructive or unable to distinguish between NADH and NADPH. In contrast to traditional methods, genetically encoded NADH sensors can be used for the real-time tracking and quantitative measurement of subcellular NADH levels in living cells. Therefore, these sensors provide innovative tools and address the limitations of current techniques. We herein summarize the properties of different types of recently developed NADH biosensors, discuss their advantages and disadvantages, and focus on the high-throughput analysis of mitochondrial function by using highly responsive NAD(+)/NADH sensors.

  3. [Calcium suppletion for patients who use gastric acid inhibitors: calcium citrate or calcium carbonate?].

    NARCIS (Netherlands)

    Jonge, H.J. de; Gans, R.O.; Huls, G.A.


    Various calcium supplements are available for patients who have an indication for calcium suppletion. American guidelines and UpToDate recommend prescribing calcium citrate to patients who use antacids The rationale for this advice is that water-insoluble calcium carbonate needs acid for adequate ab

  4. [Calcium suppletion for patients who use gastric acid inhibitors: calcium citrate or calcium carbonate?].

    NARCIS (Netherlands)

    Jonge, H.J. de; Gans, R.O.; Huls, G.A.


    Various calcium supplements are available for patients who have an indication for calcium suppletion. American guidelines and UpToDate recommend prescribing calcium citrate to patients who use antacids The rationale for this advice is that water-insoluble calcium carbonate needs acid for adequate ab

  5. [Calcium suppletion for patients who use gastric acid inhibitors: calcium citrate or calcium carbonate?].

    NARCIS (Netherlands)

    Jonge, H.J. de; Gans, R.O.; Huls, G.A.


    Various calcium supplements are available for patients who have an indication for calcium suppletion. American guidelines and UpToDate recommend prescribing calcium citrate to patients who use antacids The rationale for this advice is that water-insoluble calcium carbonate needs acid for adequate

  6. Stress Degradation Behavior of Atorvastatin Calcium and Development of a Suitable Stability-Indicating LC Method for the Determination of Atorvastatin, its Related Impurities, and its Degradation Products. (United States)

    Vukkum, Pallavi; Moses Babu, J; Muralikrishna, R


    A rapid, reversed-phase liquid chromatographic method was developed for the quantitative determination of Atorvastatin calcium, its related substances (12 impurities), and degradation impurities in bulk drugs. The chromatographic separation was achieved on a Zorbax Bonus-RP column by employing a gradient elution with water-acetonitrile-trifluoroacetic acid as the mobile phase in a shorter run time of 25 min. The flow rate was 1.0 mL/min and the detection wavelength was 245 nm. The drug substance was subjected to stress studies such as hydrolysis, oxidation, photolysis, and thermal degradation, and considerable degradation was observed in acidic hydrolysis, oxidative, thermal, and photolytic stress conditions. The formed degradation products were reported and were well-resolved from the Atorvastatin and its related substances. The stressed samples were quantified against a qualified reference standard and the mass balance was found to be close to 99.5% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. Atorvastatin), which demonstrates the stability-indicating capability of the method. The method was validated in agreement with ICH requirements. The method developed here was single and shorter (25 min method for the determination of all 12 related impurities of Atorvastatin and its degradation products), with clearly better resolution and higher sensitivity than the European (85 min method for the determination of six impurities) and United States pharmacopeia (115 min and 55 min, two different methods for the determination of six related substances).

  7. Engineering a genetically encoded competitive inhibitor of the KEAP1-NRF2 interaction via structure-based design and phage display. (United States)

    Guntas, Gurkan; Lewis, Steven M; Mulvaney, Kathleen M; Cloer, Erica W; Tripathy, Ashutosh; Lane, Thomas R; Major, Michael B; Kuhlman, Brian


    In its basal state, KEAP1 binds the transcription factor NRF2 (Kd = 5 nM) and promotes its degradation by ubiquitylation. Changes in the redox environment lead to modification of key cysteines within KEAP1, resulting in NRF2 protein accumulation and the transcription of genes important for restoring the cellular redox state. Using phage display and a computational loop grafting protocol, we engineered a monobody (R1) that is a potent competitive inhibitor of the KEAP1-NRF2 interaction. R1 bound to KEAP1 with a Kd of 300 pM and in human cells freed NRF2 from KEAP1 resulting in activation of the NRF2 promoter. Unlike cysteine-reactive small molecules that lack protein specificity, R1 is a genetically encoded, reversible inhibitor designed specifically for KEAP1. R1 should prove useful for studying the role of the KEAP1-NRF2 interaction in several disease states. The structure-based phage display strategy employed here is a general approach for engineering high-affinity binders that compete with naturally occurring interactions.

  8. Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential. (United States)

    Piao, Hong Hua; Rajakumar, Dhanarajan; Kang, Bok Eum; Kim, Eun Ha; Baker, Bradley J


    ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al., 2012; Han et al., 2013). Sequence alignment of voltage-sensing phosphatases from different species revealed conserved polar and charged residues at 7 aa intervals in the S1-S3 transmembrane segments of the voltage-sensing domain, suggesting potential coil-coil interactions. The contribution of these residues to the voltage-induced optical signal was tested using a cassette mutagenesis screen by flanking each transmembrane segment with unique restriction sites to allow for the testing of individual mutations in each transmembrane segment, as well as combinations in all four transmembrane segments. Addition of a counter charge in S2 improved the kinetics of the optical response. A double mutation in the S4 domain dramatically reduced the slow component of the optical signal seen in ArcLight. Combining that double S4 mutant with the mutation in the S2 domain yielded a probe with kinetics voltage-sensing domain could potentially lead to fluorescent sensors capable of optically resolving neuronal inhibition and subthreshold synaptic activity.

  9. Monitoring cytosolic and ER Zn2+ in stimulated breast cancer cells using genetically encoded FRET sensors† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5mt00257e Click here for additional data file. (United States)

    Hessels, Anne M.; Taylor, Kathryn M.


    The Zn2+-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn2+ from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn2+ release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn2+ concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn2+, eZinCh-2 (K d = 1 nM at pH 7.1) and eCALWY-4 (K d = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn2+ and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn2+ in both cytosol and ER, suggesting that Zn2+ was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn2+ levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn2+, nor in experiments in which cytosolic and ER Zn2+ were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF–ionomycin treatment does not result in significant changes in cytosolic Zn2+ levels as a result from Zn2+ release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn2+ dyes. PMID:26739447

  10. A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells (United States)

    Lock, Jeffrey T.; Parker, Ian


    Localized subcellular changes in Ca2+ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca2+ signaling have been greatly facilitated by the availability of fluorescent Ca2+ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca2+ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca2+ signals is lacking. Here, we evaluated several fluorescent Ca2+ indicators in the context of local Ca2+ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca2+ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca2+-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca2+ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca2+ signals. PMID:26572560

  11. Biosynthesis of the 22nd Genetically Encoded Amino Acid Pyrrolysine: Structure and Reaction Mechanism of PylC at 1.5Å Resolution

    KAUST Repository

    Quitterer, Felix


    The second step in the biosynthesis of the 22nd genetically encoded amino acid pyrrolysine (Pyl) is catalyzed by PylC that forms the pseudopeptide l-lysine-Nε-3R-methyl-d-ornithine. Here, we present six crystal structures of the monomeric active ligase in complex with substrates, reaction intermediates, and products including ATP, the non-hydrolyzable ATP analogue 5′-adenylyl-β-γ-imidodiphosphate, ADP, d-ornithine (d-Orn), l-lysine (Lys), phosphorylated d-Orn, l-lysine-Nε-d-ornithine, inorganic phosphate, carbonate, and Mg2 +. The overall structure of PylC reveals similarities to the superfamily of ATP-grasp enzymes; however, there exist unique structural and functional features for a topological control of successive substrate entry and product release. Furthermore, the presented high-resolution structures provide detailed insights into the reaction mechanism of isopeptide bond formation starting with phosphorylation of d-Orn by transfer of a phosphate moiety from activated ATP. The binding of Lys to the enzyme complex is then followed by an SN2 reaction resulting in l-lysine-Nε-d-ornithine and inorganic phosphate. Surprisingly, PylC harbors two adenine nucleotides bound at the active site, what has not been observed in any ATP-grasp protein analyzed to date. Whereas one ATP molecule is involved in catalysis, the second adenine nucleotide functions as a selective anchor for the C- and N-terminus of the Lys substrate and is responsible for protein stability as shown by mutagenesis. © 2012 Elsevier Ltd.

  12. Application of a genetically encoded biosensor for live cell imaging of L-valine production in pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum strains. (United States)

    Mustafi, Nurije; Grünberger, Alexander; Mahr, Regina; Helfrich, Stefan; Nöh, Katharina; Blombach, Bastian; Kohlheyer, Dietrich; Frunzke, Julia


    The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.

  13. Structural analysis of the PSD-95 cluster by electron tomography and CEMOVIS: a proposal for the application of the genetically encoded metallothionein tag. (United States)

    Hirabayashi, Ai; Fukunaga, Yuko; Miyazawa, Atsuo


    Postsynaptic density-95 (PSD-95) accumulates at excitatory postsynapses and plays important roles in the clustering and anchoring of numerous proteins at the PSD. However, a detailed ultrastructural analysis of clusters exclusively consisting of PSD-95 has never been performed. Here, we employed a genetically encoded tag, three tandem repeats of metallothionein (3MT), to study the structure of PSD-95 clusters in cells by electron tomography and cryo-electron microscopy of vitreous sections. We also performed conventional transmission electron microscopy (TEM). Cultured hippocampal neurons expressing a fusion protein of PSD-95 coupled to 3MT (PDS-95-3MT) were incubated with CdCl2 to result in the formation of Cd-bound PSD-95-3MT. Two types of electron-dense deposits composed of Cd-bound PSD-95-3MT were observed in these cells by TEM, as reported previously. Electron tomography revealed the presence of membrane-shaped structures representing PSD-95 clusters at the PSD and an ellipsoidal structure located in the non-synaptic cytoplasm. By TEM, the PSD-95 clusters appeared to be composed of a number of dense cores. In frozen hydrated sections, these dense cores were also found beneath the postsynaptic membrane. Taken together, our findings suggest that dense cores of PSD-95 aggregate to form the larger clusters present in the PSD and the non-synaptic cytoplasm. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail:

  14. The use of the indicator fluo-5N to measure sarcoplasmic reticulum calcium in single muscle fibres of the cane toad. (United States)

    Kabbara, A A; Allen, D G


    1. Single fibres from the lumbrical muscles of the cane toad (Bufo marinus) were incubated in fluo-5N AM for 2 h at 35 degrees C in order to load the indicator into the sarcoplasmic reticulum. Fluo-5N is a low-affinity calcium indicator (K(Ca) 90 microM). Successful sarcoplasmic reticulum (SR) loading was indicated by a fluorescence signal that declined during contraction. 2. Confocal microscopy showed that the dye loaded principally in lines perpendicular to the long axis of the fibre that repeated each sarcomere. This is consistent with much of the dye residing in the SR. 3. To establish the site of loading, fibres were exposed to 30 mM caffeine in the presence of 20 microM 2,5-di(tert-butyl)1,4-hydroquinone (TBQ, an SR pump inhibitor) which should release most Ca(2+) from the SR; this procedure reduced the fluorescence to 46 +/- 4 % of the control value. To determine how much indicator was in the myoplasm, fibres were exposed to 100 microg ml(-1) saponin which permeabilizes the surface membrane; saponin treatment reduced the fluorescence to 51 +/- 2 % of the control value. 4. During maximally activated tetani (100 Hz stimulation rate, 22 degrees C) the component of signal from the SR declined by 33 +/- 4 %. During relaxation the SR signal recovered in two phases with time constants of 0.38 +/- 0.14 s and 10.1 +/- 1.7 s. Partially activated tetani (30 Hz stimulation rate) showed a smaller SR signal. Application of the SR Ca(2+) pump inhibitor TBQ slowed the rate of recovery of the SR signal. 5. Muscle fatigue was produced by repeated short tetani until tension was reduced to 50 %. The SR signal during the periods between tetani declined steadily and the SR Ca(2+) signal was eventually reduced to 71 +/- 8 % of the control signal. This signal recovered in two phases when the muscle was rested. An initial phase had a time constant of 1.7 +/- 0.2 s so that by 20 s of recovery the SR Ca(2+) signal was 86 +/- 7 % of control; the second phase was slower and by 5 min the

  15. Calcium signaling in neurodegeneration

    Directory of Open Access Journals (Sweden)

    Dreses-Werringloer Ute


    Full Text Available Abstract Calcium is a key signaling ion involved in many different intracellular and extracellular processes ranging from synaptic activity to cell-cell communication and adhesion. The exact definition at the molecular level of the versatility of this ion has made overwhelming progress in the past several years and has been extensively reviewed. In the brain, calcium is fundamental in the control of synaptic activity and memory formation, a process that leads to the activation of specific calcium-dependent signal transduction pathways and implicates key protein effectors, such as CaMKs, MAPK/ERKs, and CREB. Properly controlled homeostasis of calcium signaling not only supports normal brain physiology but also maintains neuronal integrity and long-term cell survival. Emerging knowledge indicates that calcium homeostasis is not only critical for cell physiology and health, but also, when deregulated, can lead to neurodegeneration via complex and diverse mechanisms involved in selective neuronal impairments and death. The identification of several modulators of calcium homeostasis, such as presenilins and CALHM1, as potential factors involved in the pathogenesis of Alzheimer's disease, provides strong support for a role of calcium in neurodegeneration. These observations represent an important step towards understanding the molecular mechanisms of calcium signaling disturbances observed in different brain diseases such as Alzheimer's, Parkinson's, and Huntington's diseases.

  16. Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals. (United States)

    Marland, Jamie Roslin Keynes; Hasel, Philip; Bonnycastle, Katherine; Cousin, Michael Alan


    Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

  17. Long-Term Two-Photon Calcium Imaging of Neuronal Populations with Subcellular Resolution in Adult Non-human Primates. (United States)

    Sadakane, Osamu; Masamizu, Yoshito; Watakabe, Akiya; Terada, Shin-Ichiro; Ohtsuka, Masanari; Takaji, Masafumi; Mizukami, Hiroaki; Ozawa, Keiya; Kawasaki, Hiroshi; Matsuzaki, Masanori; Yamamori, Tetsuo


    Two-photon imaging with genetically encoded calcium indicators (GECIs) enables long-term observation of neuronal activity in vivo. However, there are very few studies of GECIs in primates. Here, we report a method for long-term imaging of a GECI, GCaMP6f, expressed from adeno-associated virus vectors in cortical neurons of the adult common marmoset (Callithrix jacchus), a small New World primate. We used a tetracycline-inducible expression system to robustly amplify neuronal GCaMP6f expression and up- and downregulate it for more than 100 days. We succeeded in monitoring spontaneous activity not only from hundreds of neurons three-dimensionally distributed in layers 2 and 3 but also from single dendrites and axons in layer 1. Furthermore, we detected selective activities from somata, dendrites, and axons in the somatosensory cortex responding to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits at subcellular resolution in non-human primates.

  18. Effect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate

    Directory of Open Access Journals (Sweden)

    Michael Ho


    Full Text Available Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP at alternative sites to produce Aβ and the APP intracellular domain (AICD. Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro  γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg2+ stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca2+ and Mg2+ stabilize γ-secretase and enhance its activity.

  19. In vivo neuronal calcium imaging in C. elegans. (United States)

    Chung, Samuel H; Sun, Lin; Gabel, Christopher V


    The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon and GCaMP allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

  20. Calcium in diet (United States)

    ... D is needed to help your body use calcium. Milk is fortified with vitamin D for this reason. ... of calcium dietary supplements include calcium citrate and calcium carbonate. Calcium citrate is the more expensive form of ...

  1. Calcium supplements (United States)

    ... Related Bone Diseases National Resource Center. Calcium and vitamin D: Important at every age. website. . Updated May 2015. Accessed March ...

  2. Calcium Electroporation

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha;


    BACKGROUND: Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill...... efficacy-and normal cell sensitivity. METHODS: Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780......), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). RESULTS: The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p

  3. Effect of 1,25-dihydroxyvitamin D3 on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term imaging. (United States)

    Murata, Kaori; Takahashi, Ayumi; Morita, Takao; Nezu, Akihiro; Fukumoto, Satoshi; Saitoh, Masato; Tanimura, Akihiro


    Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca(2+) release from intracellular stores and subsequent Ca(2+) entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 μM ATP. We applied long-term imaging (for ~10 h) to monitor Ca(2+) responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca(2+) concentration in the presence of 100 nM 1,25-dihydroxyvitamin D3. Many of these Ca(2+) responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca(2+) responses that occur only 3-10 times/h/100 cells. This method could be useful to study the effects of Ca(2+) responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.

  4. The Integrated Calcium II Triplet as a Metallicity Indicator: Comparisons with High Resolution [Fe/H] in M31 Globular Clusters

    CERN Document Server

    Sakari, Charli M


    Medium resolution (R=4,000 to 9,000) spectra of the near infrared Ca II lines (at 8498, 8542, and 8662 A) in M31 globular cluster integrated light spectra are presented. In individual stars the Ca II triplet (CaT) traces stellar metallicity; this paper compares integrated CaT strengths to well determined, high precision [Fe/H] values from high resolution integrated light spectra. The target globular clusters cover a wide range in metallicity (from [Fe/H] = -2.1 to -0.2). While most are older than 10 Gyr, some may be of intermediate age (2-6 Gyr). A handful (3-6) have detailed abundances (e.g. low [Ca/Fe]) that indicate they may have been accreted from dwarf galaxies. Using various measurements and definitions of CaT strength, it is confirmed that for GCs with [Fe/H] < -0.4 and older than 2 Gyr the integrated CaT traces cluster [Fe/H] to within about 0.2 dex, independent of age. CaT lines in metal rich GCs are very sensitive to nearby atomic lines (and TiO molecular lines in the most metal rich GCs), largel...

  5. Effects of Phytase on Tibia Indicators, Calcium and Phosphorus Utilization in Broilers%植酸酶对肉鸡胫骨指标和钙磷利用率的影响

    Institute of Scientific and Technical Information of China (English)

    张旭; 蒋桂韬; 王向荣; 李昊帮; 戴求仲


    :通过对胫骨指标的测定和代谢试验,研究了微生物植酸酶在两个不同磷酸氢钙添加水平下对肉仔鸡胫骨指标和钙、磷利用率的影响试验结果表明,与正常磷酸氢钙添加水平(1-10%)的对照组相比,降低磷酸氢钙用量并添加植酸酶,可以提高24日龄肉仔鸡的胫骨长度,提高24和42日龄肉仔鸡的胫骨重量/体积;0.44%磷酸氢钙水平添加植酸酶组的24日龄肉鸡的脱脂干骨重和粗灰分含量较对照组显著降低(P〈0.05);降低磷酸氢钙水平并添加植酸酶不会对肉鸡胫骨的钙、磷含量造成影响(P〉0.05);降低磷酸氢钙水平并添加植酸酶能够显著提高磷的消化利用率(P〈0.05),极显著提高24日龄肉仔鸡的钙表观消化率(P〈O.01),提高42日龄肉仔鸡的钙表观消化率,但差异不显著(P〉O.05),%To study the impacts of microbial phytase added in two different CaHP04 addition levels of the broiler tibia indicators and calcium and phosphorus utilization through determination of tibia indicators and broiler metabolic test. The results showed that, compared with the control group of normal CaHP04 addition level(1.10%), reduce CaHP04 dosage and phytase supplementation increased the length of the 24-day-old broiler tibia, improved the tib- ia weight / volume ratio of 24-day-old and 42-day-old broilers; nonfat dry bone weight and crude ash content of 24-day-old broilers at the CaHP04 level of 0.44% phytase group were lower than the control group(P〈0.05); re- duce CaHPO~ levels and adding phytase had no impact on calcium and phosphorus content of broiler tibia(P〉0.05); reduce the CaHP04 level and add phytase c ouht significantly improved the digestibility of apparent phosphorus(P〈 0.05), and a very significant increase in the 24 days broilers'calcium apparent digestibility(P〈0.01), and increased in the 42 days broilers calcium apparent digestibility(P〉0.05).

  6. Comparative analysis of MAMP-induced calcium influx in Arabidopsis seedlings and protoplasts. (United States)

    Maintz, Jens; Cavdar, Meltem; Tamborski, Janina; Kwaaitaal, Mark; Huisman, Rik; Meesters, Christian; Kombrink, Erich; Panstruga, Ralph


    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.

  7. Decalcification of calcium polycarbophil in rats. (United States)

    Yamada, T; Saito, T; Takahara, E; Nagata, O; Tamai, I; Tsuji, A


    The in vivo decalcification of calcium polycarbophil was examined. The decalcification ratio of [45Ca]calcium polycarbophil in the stomach after oral dosing to rats was more than 70% at each designated time and quite closely followed in the in vitro decalcification curve, indicating that the greater part of the calcium ion is released from calcium polycarbophil under normal gastric acidic conditions. The residual radioactivity in rat gastrointestine was nearly equal to that after oral administration of either [45Ca]calcium chloride + polycarbophil. The serum level of radioactivity was nearly equal to that after oral dosing of [45Ca]calcium lactate. These results indicate that the greater part of orally administered calcium polycarbophil released calcium ions to produce polycarbophil in vivo.

  8. Lactulose stimulates calcium absorption in postmenopausal women

    NARCIS (Netherlands)

    Heuvel, E.G.H.M. van den; Muijs, T.; Dokkum, W. van; Schaafsma, G.


    Animal studies have indicated that calcium absorption is increased by lactulose, a synthetic disaccharide. Therefore, the influence of lactulose on calcium absorption was measured in postmenopausal women who may benefit from the possible enhancing effect of lactulose on calcium absorption. Twelve

  9. Lactulose stimulates calcium absorption in postmenopausal women

    NARCIS (Netherlands)

    Heuvel, E.G.H.M. van den; Muijs, T.; Dokkum, W. van; Schaafsma, G.


    Animal studies have indicated that calcium absorption is increased by lactulose, a synthetic disaccharide. Therefore, the influence of lactulose on calcium absorption was measured in postmenopausal women who may benefit from the possible enhancing effect of lactulose on calcium absorption. Twelve po

  10. Calcium Forms,Subcelluar Distribution and Ultrastructure of Pulp Cells as Influenced by Calcium Deficiency in Apple (Malus pumila) Fruits

    Institute of Scientific and Technical Information of China (English)

    CHEN Jian-hui; ZHOU Wei


    Calcium in Red Fuji and Starkrimson apples during storage were fractionated by sequent extracting. Localization and distribution of calcium and influence of calcium nutrition on cell ultrastructure were observed by transmission electron microscopy combined with in situ precipitation of calcium with an improved method of potassium pyroantimonate technique. Results indicated that spraying calcium solution on surface of young fruits increased contents of calcium in all forms. During storage, contents of soluble calcium and pectic calcium declined and thosein calcium phosphate, calcium oxalate and calcium silicate increased. Calcium contents of Red Fuji in all forms were higher than those of Starkrimson, indicating that calcium accumulating capability of Red Fuji fruits preceded that of Starkrimson. Under transmission electron microscopy, calcium antimonite precipitates (CaAP) was mainly distributed in cell wall, tonoplast, nuclear membrane and nucleoplasm,much more CaAP deposited in vacuole. Calcium deficiency during storage leads to decrease of CaAP in locations mentioned above, disappearance of compartmentation, and entrance of CaAP to cytoplasm. Transformation from soluble calcium and pectic calcium to calcium phosphate,oxalate and damages of biomembranes structuraly and functionally resulted from calcium deficiency during storage were the crucial causation of physiological disorder.

  11. Calcium and bones (United States)

    Bone strength and calcium ... calcium (as well as phosphorus) to make healthy bones. Bones are the main storage site of calcium in ... your body does not absorb enough calcium, your bones can get weak or will not grow properly. ...

  12. Calcium Carbonate (United States)

    ... doctor if you have or have ever had kidney disease or stomach conditions.tell your doctor if you are pregnant, plan to become pregnant, or are breast-feeding. If you become pregnant while taking calcium carbonate, call your doctor.

  13. Calcium Test (United States)

    ... if a person has symptoms of a parathyroid disorder , malabsorption , or an overactive thyroid. A total calcium level is often measured as part of a routine health screening. It is included in the comprehensive metabolic panel (CMP) and the basic metabolic panel (BMP) , ...

  14. The effect of variable calcium and very low calcium diets on human calcium metabolism. Ph.D. Thesis. Final Report (United States)

    Chu, J.


    The effects of a very low calcium diet, with variable high and low protein intake, on the dynamics of calcium metabolism and the mechanism of calciuretics, are examined. The experiment, using male subjects, was designed to study the role of intestinal calcium absorption on urinary calcium excretion, and the rate of production of endogeneously secreted calcium in the gastrointestinal tract. The study showed an average of 70% fractional absorption rate during very low calcium intake, and that a decrease in renal tubular reabsorption of calcium is responsible for calciuretic effects of high protein intake. The study also indicates that there is a tendency to develop osteoporosis after long periods of low calcium intake, especially with a concurrent high protein intake.

  15. 新型钙离子指示剂GCaMP的发展与应用%GCaMPs: promising tools for in vivo calcium imaging

    Institute of Scientific and Technical Information of China (English)

    董斐斐; 安丽娜; 王国坤; 秦永文


    新型钙离子指示剂GCaMPs是以单个荧光蛋白为基础构建而成,可以定位到特定的细胞亚型或细胞器,对细胞或细胞器内钙离子敏感,荧光强度与钙离子浓度成正比,可用以观察钙离子介导的信号传递过程,研究机体发育、疾病、药物作用等过程中细胞内钙信号的产生、传递和时空特点,近年来被广泛应用于生理学、细胞生物学和临床研究等领域,具有独特的优势和广阔的前景.本文通过对GCaMPs的原理、发展和应用进行综述,旨在为生命科学工作者介绍一种新的钙离子检测工具,以利于更好地探索生理和病理状态下细胞的行为和功能,揭示其内在的病理生理机制.%GCaMPs are new genetically encoded calcium indicators based on a single fluorescent protein molecule with high calcium affinity.The fluorescence intensity of GCaMPs is proportional to intracellular calcium concentration.GCaMPs can be easily targeted and expressed in specific cells and cellular compartments,so as to make it possible for observing calciummediated signal transduction.GCaMPs can provide long-term in vivo imaging of calcium signals during development,disease,and drug therapy,making them promising tools for physiological,cytobiological and clinical studies.This paper summarized the principle,progression and application of GCaMPs,hoping to introduce a new calcium detection tool for further exploring the behavior and functions of ceils under physiological and pathological states.

  16. Dissection of local Ca(2+) signals inside cytosol by ER-targeted Ca(2+) indicator. (United States)

    Niwa, Fumihiro; Sakuragi, Shigeo; Kobayashi, Ayana; Takagi, Shin; Oda, Yoichi; Bannai, Hiroko; Mikoshiba, Katsuhiko


    Calcium (Ca(2+)) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca(2+) signals, generated by the coordination of Ca(2+) influx from the extracellular space and Ca(2+) release from the intracellular Ca(2+) store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca(2+) signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca(2+) signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca(2+) indicator (GECI) targeted to the outer ER membrane, can monitor Ca(2+) release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca(2+) imaging of C. elegans. We also found that the spontaneous Ca(2+) elevation in cultured astrocytes reported by OER-GCaMP6f showed a distinct spatiotemporal pattern from that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f); less frequent Ca(2+) signal was detected by OER-GCaMP6f, in spite of the fact that Ca(2+) release from the ER plays important roles in astrocytes. These findings suggest that targeting of GECIs to the ER outer membrane enables sensitive detection of Ca(2+) release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca(2+). Our results indicate that Ca(2+) imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca(2+) signals, by enabling dissection of Ca(2+) signals at subcellular resolution.

  17. Heat transfer analysis of unsteady graphene oxide nanofluid flow using a fuzzy identifier evolved by genetically encoded mutable smart bee algorithm

    Directory of Open Access Journals (Sweden)

    Mohammadreza Azimi


    indicate that the gradient of Nu has a direct nonlinear relation with the values of ϕ and Ec. It is also observed that an increase in the value of S decreases the value of Nu.

  18. Calcium paradox and calcium entry blockers

    NARCIS (Netherlands)

    Ruigrok, T.J.C.; Slade, A.M.; Nayler, W.G.; Meijler, F.L.


    Reperfusion of isolated hearts with calcium-containing solution after a short period of calcium-free perfusion results in irreversible cell damage (calcium paradox). This phenomenon is characterized by an excessive influx of calcium into the cells, the rapid onset of myocardial contracture, exhausti

  19. Protein intake and calcium absorption – Potential role of the calcium sensor receptor (United States)

    Dietary protein induces calcium excretion but the source of this calcium is unclear. Evidence from short-term studies indicates that protein promotes bone resorption, but many epidemiologic studies do not corroborate this. Evidence is also mixed on weather protein promotes calcium absorption. Stud...

  20. Calcium signals in olfactory neurons. (United States)

    Tareilus, E; Noé, J; Breer, H


    Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

  1. Genetically encoded fluorescent coumarin amino acids (United States)

    Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.


    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

  2. Genetically encoded fluorescent coumarin amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiangyun [San Diego, CA; Xie, Jianming [San Diego, CA; Schultz, Peter G [La Jolla, CA


    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

  3. Calcium regulation in endosymbiotic organelles of plants. (United States)

    Bussemer, Johanna; Vothknecht, Ute C; Chigri, Fatima


    In plant cells calcium-dependent signaling pathways are involved in a large array of biological processes in response to hormones, biotic/abiotic stress signals and a variety of developmental cues. This is generally achieved through binding of calcium to diverse calcium-sensing proteins, which subsequently control downstream events by activating or inhibiting biochemical reactions. Regulation by calcium is considered as a eukaryotic trait and has not been described for prokaryotes. Nevertheless, there is increasing evidence indicating that organelles of prokaryotic origin, such as chloroplasts and mitochondria, are integrated into the calcium-signaling network of the cell. An important transducer of calcium in these organelles appears to be calmodulin. In this review we want to give an overview over present data showing that endosymbiotic organelles harbour calcium-dependent biological processes with a focus on calmodulin-regulation.

  4. Calcium D-saccharate

    DEFF Research Database (Denmark)

    Garcia, André Castilho; Hedegaard, Martina Vavrusova; Skibsted, Leif Horsfelt


    K-1. Equilibria in supersaturated solutions of calcium d-saccharate seem only to adjust slowly, as seen from calcium activity measurements in calcium d-saccharate solutions made supersaturated by cooling. Solutions formed by isothermal dissolution of calcium d-gluconate in aqueous potassium d......-saccharate becomes spontaneously supersaturated with both d-gluconate and d-saccharate calcium salts, from which only calcium d-saccharate slowly precipitates. Calcium d-saccharate is suggested to act as a stabilizer of supersaturated solutions of other calcium hydroxycarboxylates with endothermic complex formation...

  5. Imaging the nanomolar range of nitric oxide with an amplifier-coupled fluorescent indicator in living cells (United States)

    Sato, Moritoshi; Hida, Naoki; Umezawa, Yoshio


    Nitric oxide (NO) is a small uncharged free radical that is involved in diverse physiological and pathophysiological mechanisms. NO is generated by three isoforms of NO synthase, endothelial, neuronal, and inducible ones. When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation, in particular. Here, we describe an amplifier-coupled fluorescent indicator for NO to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM). This genetically encoded high-sensitive indicator revealed that 1 nM of NO, which is enough to relax blood vessels, is generated in vascular endothelial cells even in the absence of shear stress. The nanomolar range of basal endothelial NO thus revealed appears to be fundamental to vascular homeostasis. fluorescence resonance energy transfer | genetic encoding

  6. Calcium antagonists and vasospasm. (United States)

    Meyer, F B


    A critical review of the clinical data supports the conclusion that nimodipine decreases the severity of neurologic deficits and improves outcome after subarachnoid hemorrhage. The mechanisms by which mortality and morbidity are reduced are still controversial. First, the frequency of vasospasm is not altered (Figs. 5 and 6). Second, the consistent reversal of vasospasm once present has not been demonstrated either angiographically or by noninvasive cerebral blood flow studies. These observations suggest that there is either modification of microcirculatory flow (i.e., dilation of pial conducting vessels or decreased platelet aggregation) or a direct neuronal protective effect. As suggested previously, support for either mechanism is not resolute, and further investigation is necessary. Currently, nimodipine has been the most thoroughly investigated calcium antagonist both from an experimental and clinical perspective. Oral administration has had few reported complications. Therefore, the benefit/risk ratio clearly supports the prophylactic use of this calcium antagonist in patients of all clinical grades after subarachnoid hemorrhage. Evidence also indicates that starting nimodipine after the onset of delayed ischemic deficits is of benefit. Finally, it can be predicted that in the future additional calcium antagonists with more selective vascular or neuronal effects will be developed for use in neurologic disorders.

  7. Calcium sensing in exocytosis

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Wu, Bingbing; Han, Weiping


    an increase in intracellular calcium levels. Besides the triggering role, calcium signaling modulates the precise amount and kinetics of vesicle release. Thus, it is a central question to understand the molecular machineries responsible for calcium sensing in exocytosis. Here we provide an overview of our...... current understanding of calcium sensing in neurotransmitter release and hormone secretion....

  8. Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy.

    Directory of Open Access Journals (Sweden)

    Alex Costa

    Full Text Available Selective Plane Illumination Microscopy (SPIM is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET. Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+ signal percolation, predicted in previous studies, has been directly visualized.

  9. Role of calcium in gravity perception of plant roots (United States)

    Evans, Michael L.


    Calcium ions may play a key role in linking graviperception by the root cap to the asymmetric growth which occurs in the elongation zone of gravistimulated roots. Application of calcium-chelating agents to the root cap inhibits gravitropic curvature without affecting growth. Asymmetric application of calcium to one side of the root cap induces curvature toward the calcium source, and gravistimulation induces polar movement of applied (Ca-45)(2+) across the root cap toward the lower side. The action of calcium may be linked to auxin movement in roots since: (1) auxin transport inhibitors interfere both with gravitropic curvature and graviinduced polar calcium movement and (2) asymmetric application of calcium enhances auxin movement across the elongation zone of gravistimulated roots. Indirect evidence indicates that the calcium-modulated regulator protein, calmodulin, may be involved in either the transport or action of calcium in the gravitropic response mechanism of roots.

  10. Effects of Adding Chymosin to Milk on Calcium Homeostasis

    DEFF Research Database (Denmark)

    Møller, Ulla Kristine; Jensen, Lars Thorbjørn; Mosekilde, Leif


    Calcium intake and absorption is important for bone health. In a randomized double-blind cross-over trial, we investigated effects of adding chymosin to milk on the intestinal calcium absorption as measured by renal calcium excretion and indices of calcium homeostasis. The primary outcome...... of the study was 24-h renal calcium excretion that is considered a proxy measure of the amount of calcium absorbed from the intestine. We studied 125 healthy men and women, aged 34 (25-45) years on two separate days. On each day, a light breakfast was served together with 500 ml of semi-skimmed milk to which...

  11. Lipid Storage Disorders Block Lysosomal Trafficking By Inhibiting TRP Channel and Calcium Release



    Lysosomal lipid accumulation, defects in membrane trafficking, and altered Ca2+ homeostasis are common features in many lysosomal storage diseases. Mucolipin TRP channel 1 (TRPML1) is the principle Ca2+ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells....

  12. Contracture of Slow Striated Muscle during Calcium Deprivation (United States)

    Irwin, Richard L.; Hein, Manfred M.


    When deprived of calcium the slow striated muscle fibers of the frog develop reversible contractures in either hypertonic or isotonic solutions. While calcium deprivation continues because of a flowing calcium-free solution the muscles relax slowly and completely. Restoration of calcium during contracture relaxes the muscle promptly to initial tension. When relaxed during calcium lack the return of calcium does not change tension and the muscle stays relaxed. When contractures are induced by solutions containing small amounts of calcium relaxation does not occur or requires several hours. The rate of tension development depends upon the rate at which calcium moves outward since the contractures develop slower in low concentrations of calcium and are absent or greatly slowed in a stagnant calcium-free solution. Withdrawal of calcium prevents the contractile responses to ACh, KCl, or electrical stimulation through the nerve. Muscles return to their original excitability after calcium is restored. Origin of the contractures is unrelated to nerve activity since they are maximal during transmission failure from calcium lack, occur in denervated muscles, and are not blocked by high concentrations of d-tubocurarine, procaine, or atropine. The experiments also indicate that the contractures do not originate from repetitive activity of muscle membranes. The findings are most simply explained by relating the outward movement of calcium as a link for initiating contraction in slow type striated muscle. PMID:14065284

  13. Apoptosis induction-related cytosolic calcium responses revealed by the dual FRET imaging of calcium signals and caspase-3 activation in a single cell. (United States)

    Miyamoto, Akitoshi; Miyauchi, Hiroshi; Kogure, Takako; Miyawaki, Atsushi; Michikawa, Takayuki; Mikoshiba, Katsuhiko


    Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction.

  14. Integumentary loss of calcium. (United States)

    Chu, J Y; Margen, S; Calloway, D H; Costa, F M


    Integumentary calcium loss was studied in 16 healthy young men. The daily loss by the 16 ambulatory but relatively sedentary young men in 52 determinations of 6-day periods each was 8.7 +/- 1.9 mg/m2 per day (average 15.8 mg/man per day). The amount lost was not influenced by calcium intake (0.1 to 2.3 g/day). In contrast to urinary calcium excretion, which is directly related to protein intake, there was no significant change in integumentary calcium loss with varying protein intakes (1 to 96 g nitrogen per day). No compensatory relationship between urinary and integumentary calcium excretion was noted. During strenuous exercise calcium loss increased to an average of 25 mg in 40 min. There was no compensatory decrease in urinary excretion on the day of strenuous exercise. It was also noted that integumentary calcium loss was not affected by general calcium balance.

  15. Calcium and Mitosis (United States)

    Hepler, P.


    Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

  16. Calcium and Mitosis (United States)

    Hepler, P.


    Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.

  17. Calcium - Function and effects

    NARCIS (Netherlands)

    Liang, Jianfen; He, Yifan; Gao, Qian; Wang, Xuan; Nout, M.J.R.


    Rice is the primary food source for more than half of the world population. Levels of calcium contents and inhibitor - phytic acid are summarized in this chapter. Phytic acid has a very strong chelating ability and it is the main inhibit factor for calcium in rice products. Calcium contents in br

  18. Calcium en cardioplegie

    NARCIS (Netherlands)

    Ruigrok, T.J.C.; Meijler, F.L.


    Coronary perfusion with a calcium-free solution, followed by reperfusion with a calcium containing solution, may result in acute myocardial cell death and in irreversible loss of the e1ectrical and mechanical activity of the heart. This phenomenon is known as the calcium paradox. A number of cardiop

  19. Relationship of calcium absorption with 25(OH)D and calcium intake in children with rickets. (United States)

    Thacher, Tom D; Abrams, Steven A


    Nutritional rickets has long been considered a disease caused by vitamin D deficiency, but recent data indicate that inadequate dietary calcium intake is an important cause of rickets, particularly in tropical countries. Children with rickets due to calcium deficiency do not have very low 25(OH)D concentrations, and serum 1,25(OH)(2) D values are markedly elevated. Studies of Nigerian children with rickets demonstrated they have high fractional calcium absorption. A high-phytate diet was demonstrated to increase calcium absorption compared with the fasting state, and enzymatic dephytinization did not significantly improve calcium absorption. When given vitamin D, children with rickets have a marked increase in 1,25(OH)(2) D concentrations without any change in fractional calcium absorption. No positive relationship was found between fractional calcium absorption and serum 25(OH)D concentrations in children on low-calcium diets. More research is needed to understand the interaction between calcium and vitamin D and the role of vitamin D in calcium absorption.

  20. Hydrolytic conversion of amorphous calcium phosphate into apatite accompanied by sustained calcium and orthophosphate ions release. (United States)

    Niu, Xufeng; Chen, Siqian; Tian, Feng; Wang, Lizhen; Feng, Qingling; Fan, Yubo


    The aim of this study is to investigate the calcium and orthophosphate ions release during the transformation of amorphous calcium phosphate (ACP) to hydroxyapatite (HA) in aqueous solution. The ACP is prepared by a wet chemical method and further immersed in the distilled water for various time points till 14d. The release of calcium and orthophosphate ions is measured with calcium and phosphate colorimetric assay kits, respectively. The transition of ACP towards HA is detected by x-ray diffraction (XRD), transmission electron microscopy (TEM), and fourier transform infrared spectroscopy (FTIR). The results indicate that the morphological conversion of ACP to HA occurs within the first 9h, whereas the calcium and orthophosphate ions releases last for over 7d. Such sustained calcium and orthophosphate ions release is very useful for ACP as a candidate material for hard tissue regeneration.

  1. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. (United States)

    Wang, Wenjing; Wildes, Craig P; Pattarabanjird, Tanyaporn; Sanchez, Mateo I; Glober, Gordon F; Matthews, Gillian A; Tye, Kay M; Ting, Alice Y


    Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

  2. Calcium signaling in pluripotent stem cells. (United States)

    Apáti, Ágota; Pászty, Katalin; Erdei, Zsuzsa; Szebényi, Kornélia; Homolya, László; Sarkadi, Balázs


    Pluripotent stem cells represent a new source of biological material allowing the exploration of signaling phenomena during normal cell development and differentiation. Still, the calcium signaling pathways and intracellular calcium responses to various ligands or stress conditions have not been sufficiently explored as yet in embryonic or induced pluripotent stem cells and in their differentiated offspring. This is partly due to the special culturing conditions of these cell types, the rapid morphological and functional changes in heterogeneous cell populations during early differentiation, and methodological problems in cellular calcium measurements. In this paper, we review the currently available data in the literature on calcium signaling in pluripotent stem cells and discuss the potential shortcomings of these studies. Various assay methods are surveyed for obtaining reliable data both in undifferentiated embryonic stem cells and in specific, stem cell-derived human tissues. In this paper, we present the modulation of calcium signaling in human embryonic stem cells (hESC) and in their derivates; mesenchymal stem cell like (MSCl) cells and cardiac tissues using the fluorescent calcium indicator Fluo-4 and confocal microscopy. LPA, trypsin and angiotensin II were effective in inducing calcium signals both in HUES9 and MSCl cells. Histamine and thrombin induced calcium signal exclusively in the MSCl cells, while ATP was effective only in HUES9 cells. There was no calcium signal evoked by GABA, even at relatively high concentrations. In stem cell-derived cardiomyocytes a rapid increase in the beating rate and an increase of the calcium signal peaks could be observed after the addition of adrenaline, while verapamil led to a strong decrease in cellular calcium and stopped spontaneous contractions in a relaxed state.

  3. Presynaptic calcium signalling in cerebellar mossy fibres

    Directory of Open Access Journals (Sweden)

    Louiza B Thomsen


    Full Text Available Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A TTX-sensitive fast Na+ spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers. Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none tetrodotoxin (TTX -sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon appeared to be isolated from one another in terms of calcium signalling. CGP55845 application showed that GABA B receptors mediated presynaptic inhibition of the calcium signal over the entire firing frequency range of mossy fibres. A paired-pulse depression of the calcium signal lasting more than one second affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists. While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells. This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette.

  4. Calcium absorption from fortified ice cream formulations compared with calcium absorption from milk. (United States)

    van der Hee, Regine M; Miret, Silvia; Slettenaar, Marieke; Duchateau, Guus S M J E; Rietveld, Anton G; Wilkinson, Joy E; Quail, Patricia J; Berry, Mark J; Dainty, Jack R; Teucher, Birgit; Fairweather-Tait, Susan J


    Optimal bone mass in early adulthood is achieved through appropriate diet and lifestyle, thereby protecting against osteoporosis and risk of bone fracture in later life. Calcium and vitamin D are essential to build adequate bones, but calcium intakes of many population groups do not meet dietary reference values. In addition, changes in dietary patterns are exacerbating the problem, thereby emphasizing the important role of calcium-rich food products. We have designed a calcium-fortified ice cream formulation that is lower in fat than regular ice cream and could provide a useful source of additional dietary calcium. Calcium absorption from two different ice cream formulations was determined in young adults and compared with milk. Sixteen healthy volunteers (25 to 45 years of age), recruited from the general public of The Netherlands, participated in a randomized, reference-controlled, double-blind cross-over study in which two test products and milk were consumed with a light standard breakfast on three separate occasions: a standard portion of ice cream (60 g) fortified with milk minerals and containing a low level (3%) of butter fat, ice cream (60 g) fortified with milk minerals and containing a typical level (9%) of coconut oil, and reduced-fat milk (1.7% milk fat) (200 mL). Calcium absorption was measured by the dual-label stable isotope technique. Effects on calcium absorption were evaluated by analysis of variance. Fractional absorption of calcium from the 3% butterfat ice cream, 9% coconut oil ice cream, and milk was 26%+/-8%, 28%+/-5%, and 31%+/-9%, respectively, and did not differ significantly (P=0.159). Results indicate that calcium bioavailability in the two calcium-fortified ice cream formulations used in this study is as high as milk, indicating that ice cream may be a good vehicle for delivery of calcium.

  5. Calcium signaling and epilepsy. (United States)

    Steinlein, Ortrud K


    Calcium signaling is involved in a multitude of physiological and pathophysiological mechanisms. Over the last decade, it has been increasingly recognized as an important factor in epileptogenesis, and it is becoming obvious that the excess synchronization of neurons that is characteristic for seizures can be linked to various calcium signaling pathways. These include immediate effects on membrane excitability by calcium influx through ion channels as well as delayed mechanisms that act through G-protein coupled pathways. Calcium signaling is able to cause hyperexcitability either by direct modulation of neuronal activity or indirectly through calcium-dependent gliotransmission. Furthermore, feedback mechanisms between mitochondrial calcium signaling and reactive oxygen species are able to cause neuronal cell death and seizures. Unravelling the complexity of calcium signaling in epileptogenesis is a daunting task, but it includes the promise to uncover formerly unknown targets for the development of new antiepileptic drugs.

  6. Role of Calcium Hydroxide in Endodontics: A Review


    Arun A; Sangameshwar Sajjanshetty; Deepak Jain; Saujanya KP; Mohammed Mustafa; Laxmi Uppin; Mahnoor Kadri


    Calcium hydroxide is a multipurpose agent, and there have been an increasing number of indications for its use in endodontics. Some of its indications include inter-appointment intracanal medicaments, endodontic sealers, pulp capping agents, apexification, pulpotomy and weeping canals. The purpose of this article is to review the properties, advantages, disadvantages and various indications for the use of calcium hydroxide in endodontics.

  7. 饲粮钙水平对7~11周龄临武鸭生长性能、胫骨发育及血清生化指标的影响%Effects of Dietary Calcium Level on Growth Performance, Tibia Development and Serum Biochemical Indices of Linwu Ducks Aged from 7 to 11 Weeks

    Institute of Scientific and Technical Information of China (English)

    李闯; 黄璇; 蒋桂韬; 王向荣; 张旭; 戴求仲


    本试验旨在研究饲粮钙水平对7~11周龄临武鸭生长性能、胫骨发育及血清生化指标的影响. 选择48日龄健康、体重相近的临武鸭母鸭270只,随机分成5组,每组6个重复,每个重复9只鸭. 饲粮以石粉为钙源,分别设置0.60%、0.70%、0.80%、0.90%和1.00% 5个钙水平.试验期28 d. 结果表明:1)随着饲粮钙水平增加,试验鸭平均日增重表现出先增加后降低的趋势,料重比先降低后升高;0.90%钙水平组平均日增重最大,料重比最低. 饲粮钙水平对试验鸭生长性能无显著影响(P>0.05). 2)饲粮钙水平对试验鸭胫骨指标均无显著影响(P>0.05), 0.70%钙水平组胫骨灰分含量最高,0.80%钙水平组胫骨折断力最大. 3)饲粮钙水平对试验鸭血清钙、磷含量无显著影响( P>0.05) ,但血清碱性磷酸酶活性随着饲粮钙水平的升高而显著降低( P0.05) . 2) Dietary calcium level had no effect on all the tibia inde-xes ( P>0.05) , and the ash content of tibia was the highest in 0.70% calcium level group, and the maximum of tibia broking strength in 0.80% calcium level group was the highest. 3) Dietary calcium level had no effect on serum calcium and phosphorus content of ducks ( P>0.05) . Increasing dietary calcium level significantly re-duced serum alkaline phosphatase activity ( P<0.05) . According to the growth performance and tibia indices, 0.60% calcium level can meet the growth needs of Linwu ducks aged from 7 to 11 weeks. With serum alkaline phosphatase activity as the basis for evaluation, through the regression analysis and derivation to get the lowest serum alkaline phosphatase activity and the corresponding calcium requirement is 0.996%.

  8. Activation of the calcium sensing receptor with cinacalcet increases serum gastrin levels in healthy older subjects (United States)

    Gastric acidity is postulated to enhance calcium absorption since calcium is better dissolved at low pH. Extracellular calcium stimulates gastrin and gastric acid secretion in humans. Ex vivo studies indicate that the calcium sensing receptor (CaR), which is expressed on the surface of human G cells...

  9. Calcium imaging of cortical neurons using Fura-2 AM. (United States)

    Barreto-Chang, Odmara L; Dolmetsch, Ricardo E


    Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

  10. Coronary artery calcium score: current status (United States)

    Neves, Priscilla Ornellas; Andrade, Joalbo; Monção, Henry


    The coronary artery calcium score plays an Important role In cardiovascular risk stratification, showing a significant association with the medium- or long-term occurrence of major cardiovascular events. Here, we discuss the following: protocols for the acquisition and quantification of the coronary artery calcium score by multidetector computed tomography; the role of the coronary artery calcium score in coronary risk stratification and its comparison with other clinical scores; its indications, interpretation, and prognosis in asymptomatic patients; and its use in patients who are symptomatic or have diabetes. PMID:28670030

  11. Calcium Intake in Elderly Australian Women Is Inadequate

    Directory of Open Access Journals (Sweden)

    Colin W. Binns


    Full Text Available The role of calcium in the prevention of bone loss in later life has been well established but little data exist on the adequacy of calcium intakes in elderly Australian women. The aim of this study was to compare the dietary intake including calcium of elderly Australian women with the Australian dietary recommendation, and to investigate the prevalence of calcium supplement use in this population. Community-dwelling women aged 70–80 years were randomly recruited using the Electoral Roll for a 2-year protein intervention study in Western Australia. Dietary intake was assessed at baseline by a 3-day weighed food record and analysed for energy, calcium and other nutrients. A total of 218 women were included in the analysis. Mean energy intake was 7,140 ± 1,518 kJ/day and protein provided 19 ± 4% of energy. Mean dietary calcium intake was 852 ± 298 mg/day, which is below Australian recommendations. Less than one quarter of women reported taking calcium supplements and only 3% reported taking vitamin D supplements. Calcium supplements by average provided calcium 122 ± 427 mg/day and when this was taken into account, total calcium intake increased to 955 ± 504 mg/day, which remained 13% lower than the Estimated Average Requirement (EAR, 1,100 mg/day for women of this age group. The women taking calcium supplements had a higher calcium intake (1501 ± 573 mg compared with the women on diet alone (813 ± 347 mg. The results of this study indicate that the majority of elderly women were not meeting their calcium requirements from diet alone. In order to achieve the recommended dietary calcium intake, better strategies for promoting increased calcium, from both diet and calcium supplements appears to be needed.

  12. Calcium channel blocker poisoning

    Directory of Open Access Journals (Sweden)

    Miran Brvar


    Full Text Available Background: Calcium channel blockers act at L-type calcium channels in cardiac and vascular smooth muscles by preventing calcium influx into cells with resultant decrease in vascular tone and cardiac inotropy, chronotropy and dromotropy. Poisoning with calcium channel blockers results in reduced cardiac output, bradycardia, atrioventricular block, hypotension and shock. The findings of hypotension and bradycardia should suggest poisoning with calcium channel blockers.Conclusions: Treatment includes immediate gastric lavage and whole-bowel irrigation in case of ingestion of sustainedrelease products. All patients should receive an activated charcoal orally. Specific treatment includes calcium, glucagone and insulin, which proved especially useful in shocked patients. Supportive care including the use of catecholamines is not always effective. In the setting of failure of pharmacological therapy transvenous pacing, balloon pump and cardiopulmonary by-pass may be necessary.

  13. Calcium is important forus.

    Institute of Scientific and Technical Information of China (English)



    Calcium is important for our health.We must have it in our diet to stay well.A good place to get it is from dairy products like milk, cheese and ice cream.One pound of cheese has fifty times the calcium we should have every day.Other foods have less.For example,a pound of beans also has calcium.But it has only three times the amount we ought to have daily.

  14. Apatite Formation from Amorphous Calcium Phosphate and Mixed Amorphous Calcium Phosphate/Amorphous Calcium Carbonate. (United States)

    Ibsen, Casper J S; Chernyshov, Dmitry; Birkedal, Henrik


    Crystallization from amorphous phases is an emerging pathway for making advanced materials. Biology has made use of amorphous precursor phases for eons and used them to produce structures with remarkable properties. Herein, we show how the design of the amorphous phase greatly influences the nanocrystals formed therefrom. We investigate the transformation of mixed amorphous calcium phosphate/amorphous calcium carbonate phases into bone-like nanocrystalline apatite using in situ synchrotron X-ray diffraction and IR spectroscopy. The speciation of phosphate was controlled by pH to favor HPO4 (2-) . In a carbonate free system, the reaction produces anisotropic apatite crystallites with large aspect ratios. The first formed crystallites are highly calcium deficient and hydrogen phosphate rich, consistent with thin octacalcium phosphate (OCP)-like needles. During growth, the crystallites become increasingly stoichiometric, which indicates that the crystallites grow through addition of near-stoichiometric apatite to the OCP-like initial crystals through a process that involves either crystallite fusion/aggregation or Ostwald ripening. The mixed amorphous phases were found to be more stable against phase transformations, hence, the crystallization was inhibited. The resulting crystallites were smaller and less anisotropic. This is rationalized by the idea that a local phosphate-depletion zone formed around the growing crystal until it was surrounded by amorphous calcium carbonate, which stopped the crystallization.

  15. Vitamin D, calcium homeostasis and aging (United States)

    Veldurthy, Vaishali; Wei, Ran; Oz, Leyla; Dhawan, Puneet; Jeon, Yong Heui; Christakos, Sylvia


    Osteoporosis is characterized by low bone mass and microarchitecture deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk. Evidence is accumulating for an important role of calcium deficiency as the process of aging is associated with disturbed calcium balance. Vitamin D is the principal factor that maintains calcium homeostasis. Increasing evidence indicates that the reason for disturbed calcium balance with age is inadequate vitamin D levels in the elderly. In this article, an overview of our current understanding of vitamin D, its metabolism, and mechanisms involved in vitamin D-mediated maintenance of calcium homeostasis is presented. In addition, mechanisms involved in age-related dysregulation of 1,25(OH)2D3 action, recommended daily doses of vitamin D and calcium, and the use of vitamin D analogs for the treatment of osteoporosis (which remains controversial) are reviewed. Elucidation of the molecular pathways of vitamin D action and modifications that occur with aging will be an active area of future research that has the potential to reveal new therapeutic strategies to maintain calcium balance. PMID:27790378

  16. Normal and Malignant Cells Exhibit Differential Responses to Calcium Electroporation

    DEFF Research Database (Denmark)

    Frandsen, Stine K; Krüger, Mie B; Mangalanathan, Uma M


    tissue after calcium electroporation but decreased in skin tissue 4 hours after treatment to levels comparable with untreated controls, whereas calcium content endured at high levels in tumor tissue. Mechanistic experiments in vitro indicated that calcium influx was similar in fibroblasts and cancer...... necrosis, with a range of sensitivities observed (36%-88%) 2 days after treatment. Necrosis was induced using calcium concentrations of 100-500 mmol/L and injection volumes 20%-80% of tumor volume. Notably, only limited effects were seen in normal tissue. Calcium content increased >7-fold in tumor and skin......Calcium electroporation may offer a simple general tool for anticancer therapy. Transient permeabilization of cancer cell membranes created by applying short, high-voltage pulses in tumors enables high calcium influxes that trigger cell death. In this study, we compared the relative sensitivity...

  17. Fluorescent observations of calcium ion activity in living benthic foraminifera (United States)

    Toyofuku, T.; de Nooijer, L. J.; Kitazato, H.


    Foraminifera are one of the main sources of marine biogenic carbonate and are commonly used to reconstruct paleoenvironments. However, little is known about the intracellular control on elements. In particular, knowledge on calcium ion activities in living foraminiferal cells is of great interest, since it may have implications for many studies in paleoceanography. Recently, fluorescent calcium indicators have been developed that can be used to observe calcium ion activities within a living foraminiferal cell directly. In this study, we applied the fluorescent calcium indicator Fluo-3 AM to observe intracellular calcium ion mobility within one species of a shallow water benthic foraminifers. We show that with this fluorescent calcium indicator is possible to 1) perform real time calcium observations, and 2) study intracellular calcium ion distribution of foraminifera during calcification. We incubate living foraminiferal specimens under two conditions, one under Fluo-3 AM solution in normal filtrated seawater and the other Fluo-3 AM solution in calcium-free artificial seawater. Fluorescence was seen all over foraminiferal cell in specimens incubated in Fluo-3 AM/normal seawater, while there are no fluorescence was observed in individuals that were incubated with Fluo-3 AM in calcium-free artificial seawater, though the specimens extend their pseudopodia actively under both conditions. Therefore the observed fluorescence should be indicated the calcium ion existence. This method may allow us detailed real-time observation of in-vivo calcium activities in foraminiferal cell. It may be over the many limitations of the existing methods to trace calcium uptake of foraminifera.

  18. Acidosis and Urinary Calcium Excretion

    DEFF Research Database (Denmark)

    Alexander, R Todd; Cordat, Emmanuelle; Chambrey, Régine


    Metabolic acidosis is associated with increased urinary calcium excretion and related sequelae, including nephrocalcinosis and nephrolithiasis. The increased urinary calcium excretion induced by metabolic acidosis predominantly results from increased mobilization of calcium out of bone and inhibi...

  19. Recording of calcium transient and analysis of calcium removal mechanisms in cardiac myocytes from rats and ground squirrels

    Institute of Scientific and Technical Information of China (English)

    王世强; 周曾铨; 钱洪


    With confocal microscopy, we recorded calcium transients and analyzed calcium removal rate at different temperatures in cardiac myocytes from the rat, a non-hibernator, and the ground squirrel, a hibernator. The results showed a remarkable increase of the diastolic level of calcium transients in the rat but no detectable change in the ground squirrel. Calcium transient of the ground squirrel, compared with that of the rat at the same temperature, had a shorter duration and showed a faster calcium removal. As indicated by the pharmacological effect of cyclopiazonic acid, calcium uptake by sarcoplasmic reticulum (SR) was the major mechanism of calcium removal, and was faster in the ground squirrel than in the rat. Our results confirmed the essential role of SR in hypothermia-tolerant adaptation, and negated the importance of Na-Ca exchange. We postulated the possibility to improve hypothermia-tolerance of the cardiac tissue of non-hibernating mammals.

  20. Effects of Exterior Abscisic Acid on Calcium Distribution of Mesophyll Cells and Calcium Concentration of Guard Cells in Maize Seedlings

    Institute of Scientific and Technical Information of China (English)

    GUO Xiu-lin; MA Yuan-yuan; LIU Zi-hui; LIU Bin-hui


    In this study, the direct effects of exterior abscisic acid (ABA) on both calcium distribution of mesophyll cells and cytosolic calcium concentration of guard cells were examined. The distribution of Ca2+ localization were observed with calcium antimonate precipitate-electromicroscopic-cyto-chemical methods after treated with ABA and pretreated with ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), verapamil (Vp), and trifluoperazine (TFP). The laser scanning confocal microscopy was used to measure the cytosolic calcium concentrations of guard cells under different treatments. The results showed that the cytosolic Ca2+ concentration of mesophyll cells was induced to increase by ABA, but to decrease in both outside cell and the vacuoles within 10 min after treatments. The cytosolic calcium concentration of guard cells was increased gradually with the lag in treatment time. However, both EGTA and TFP could inverse those effects, indicating that the increase of cytosolic calcium induced by exterior ABA was mainly caused by calcium influx. The results also showed that calmodulin could influence both the calcium distribution of mesophyll cells and calcium concentration of guard cells. It shows that calmodulin participates in the process of ABA signal transduction, but the mechanism is not known as yet. The changes both calcium distribution of mesophyll cells and calcium concentration of guard cells further proved that the variations of cytosolic Ca2+ concentration induced by ABA were involved in the stomatal movements of maize seedlings.

  1. Dysbalance of astrocyte calcium under hyperammonemic conditions.

    Directory of Open Access Journals (Sweden)

    Nicole Haack

    Full Text Available Increased brain ammonium (NH4(+/NH3 plays a central role in the manifestation of hepatic encephalopathy (HE, a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH4(+/NH3 on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH4(+/NH3-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH4(+/NH3 for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼ 90% experienced a persistent calcium increase by ∼ 50 nM. This persistent increase was already evoked at concentrations of 1-2 mM NH4(+/NH3, developed within 10-20 minutes and was maintained as long as the NH4(+/NH3 was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH4(+/NH3, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC, the reverse mode of sodium/calcium exchange (NCX, AMPA- or mGluR5-receptors did not dampen NH4(+/NH3-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH4(+/NH3 causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study

  2. Discussion on the mechanism of the calcium absorption in the human body

    Institute of Scientific and Technical Information of China (English)


    The present article discusses a new mechanism of calcium absorption in the human body. The mechanism is revealed as follows. First, after food is digested in the stomach, calcium ions (Ca2+) are released. The small intestine secretes amino acid or short peptide chain with small molecniar weight automatically, which are called chelating agent; when the calcium ions from the stomach get to the small intestine, the reaction of the chelating agent with the calcium ions occurs, producing the neutral amino acid calcium chelate. Then, this kind of calcium chelate with small molecular weight is absorbed as a whole into the tissues of the small intestine. After being absorbed, in the cell the calcium chelate can break down its chelating bond automatically and decompose into the amino acid and calcium ion again. Finally, the calcium ion goes into blood through portal vein and is transferred to the organs and also deposits on the bone. The reason for the body's calcium insufficiency, which has no linear relation with the calcium intake amount, is the lack of the amino acid secreted by the small intestine. The main barrier that influences the calcium absorption is anion pollution. The calcium absorptivity of the body has nothing to do with the solubility of the calcium source out of the body.A new kind of calcium supplement agent--glycine calcium chelate--is synthesized, whose molecular weight is 206.06(containing a molecular water). If the glycine calcium chelate is used to make calcium supplement agent, about 20 mg calcium element (converted from the glycine calcium chelate,the same below, no longer indicated) per day for one person,50 mg at most, is enough to maintain the positive balance of calcium metabolism.``

  3. CCN3 and calcium signaling

    Directory of Open Access Journals (Sweden)

    Li Chang Long


    Full Text Available Abstract The CCN family of genes consists presently of six members in human (CCN1-6 also known as Cyr61 (Cystein rich 61, CTGF (Connective Tissue Growth Factor, NOV (Nephroblastoma Overexpressed gene, WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins. Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions. In this article, we review the data showing that CCN3 regulates the levels of intracellular calcium and discuss potential models that may account for the biological effects of CCN3.

  4. Effects of nano calcium carbonate and nano calcium citrate on toxicity in ICR mice and on bone mineral density in an ovariectomized mice model (United States)

    Huang, Sherry; Chen, Jin Ching; Hsu, Chin Wei; Chang, Walter H.


    Taking calcium supplements can reduce the risk of developing osteoporosis, but they are not readily absorbed in the gastrointestinal tract. Nanotechnology is expected to resolve this problem. In the present study, we examined whether the bioavailability of calcium carbonate and calcium citrate can be improved by reducing the particle size. The morphology of nano calcium carbonate and nano calcium citrate was characterized by dynamic laser-light scattering (DLS), field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). The measurements obtained from DLS, FE-SEM and TEM were comparable. Acute and sub-chronic toxicity tests were performed to establish the safety of these products after oral administration. The no-observed-adverse-effect levels of nano calcium carbonate and nano calcium citrate were 1.3 and 2.3 g kg-1 body weight, respectively. The results of our in vivo studies indicate that administering nano calcium carbonate and nano calcium citrate can enhance the serum calcium concentration and maintain the whole-body bone mineral density in ovariectomized mice. These data suggest that nano calcium carbonate and nano calcium citrate are more bioavailable than micro calcium carbonate and micro calcium citrate, respectively.

  5. Effects of nano calcium carbonate and nano calcium citrate on toxicity in ICR mice and on bone mineral density in an ovariectomized mice model

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Sherry; Chen, Jin Ching; Hsu, Chin Wei; Chang, Walter H, E-mail: [Center for Nano Bioengineering, Chung Yuan Christian University, Chung Li 32023, Taiwan (China); Department of Biomedical Engineering, Chung Yuan Christian University, Chung Li 32023, Taiwan (China)


    Taking calcium supplements can reduce the risk of developing osteoporosis, but they are not readily absorbed in the gastrointestinal tract. Nanotechnology is expected to resolve this problem. In the present study, we examined whether the bioavailability of calcium carbonate and calcium citrate can be improved by reducing the particle size. The morphology of nano calcium carbonate and nano calcium citrate was characterized by dynamic laser-light scattering (DLS), field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). The measurements obtained from DLS, FE-SEM and TEM were comparable. Acute and sub-chronic toxicity tests were performed to establish the safety of these products after oral administration. The no-observed-adverse-effect levels of nano calcium carbonate and nano calcium citrate were 1.3 and 2.3 g kg{sup -1} body weight, respectively. The results of our in vivo studies indicate that administering nano calcium carbonate and nano calcium citrate can enhance the serum calcium concentration and maintain the whole-body bone mineral density in ovariectomized mice. These data suggest that nano calcium carbonate and nano calcium citrate are more bioavailable than micro calcium carbonate and micro calcium citrate, respectively.

  6. Calcium Imaging of Sonoporation of Mammalian Cells (United States)

    Sabens, David; Aehle, Matthew; Steyer, Grant; Kourennyi, Dmitri; Deng, Cheri X.


    Ultrasound mediated delivery of compounds is a relatively recent development in drug delivery and gene transfection techniques. Due to the lack of methods for real-time monitoring of sonoporation at the cellular level, the efficiency of drug/gene delivery and sonoporation associated side effects, such as the loss of cell viability and enhanced apoptosis, have been studied only through post US exposure analyses, requiring days for cell incubation. Furthermore, because microporation appears to be transient in nature, it was not possible to correlate transfection with microporation on an individual cellular basis. By studying the role of calcium in the cell and using fluorescent calcium imaging to study sonoporation it is possible to quantify both cell porosity and sonoporation side effects. Since both post sonoporation cell survival and delivery efficiency are related to the dynamic process of the cell membrane poration, calcium imaging of sonoporation will provide important knowledge to obtain improved understanding of sonoporation mechanism. Our experimental results demonstrated the feasibility of calcium imaging of sonoporation in Chinese Hamster Ovary (CHO) cells. We have measured the changes in the intracellular calcium concentration using Fura-2, a fluorescent probe, which indicate influx or flow of Calcium across the cell membrane. Analysis of data identified key aspects in the dynamic sonoporation process including the formation of pores in the cell membrane, and the relative temporal duration of the pores and their resealing. These observations are obtained through the analysis of the rate the calcium concentration changes within the cells, making it possible to visualize membrane opening and repair in real-time through such changes in the intracellular calcium concentration.

  7. Calcium and Vitamin D (United States)

    ... Pizza, cheese, frozen 1 serving 115 mg Pudding, chocolate, prepared with 2% milk 4 oz 160 mg ... Treatment Medication and Treatment Adherence Calcium/Vitamin D Nutrition Overall Health Fractures/Fall Prevention Exercise/Safe Movement ...

  8. Stoichiometry of Calcium Medicines (United States)

    Pinto, Gabriel


    The topic of calcium supplement and its effects on human lives is presented in the way of questions to the students. It enables the students to realize the relevance of chemistry outside the classroom surrounding.

  9. Get Enough Calcium (United States)

    ... Resources You may also be interested in: Calcium: Shopping list Menopause: Questions for ... A Federal Government website managed by the U.S. Department of Health and Human Services is ...

  10. Stoichiometry of Calcium Medicines (United States)

    Pinto, Gabriel


    The topic of calcium supplement and its effects on human lives is presented in the way of questions to the students. It enables the students to realize the relevance of chemistry outside the classroom surrounding.

  11. Calcium and Your Child (United States)

    ... for dinner. Create mini-pizzas by topping whole-wheat English muffins or bagels with pizza sauce, low- ... Minerals Do I Need to Drink Milk? Lactose Intolerance Becoming a Vegetarian Soy Foods and Health Calcium ...

  12. 不同增钙模式对蛋鸡生产性能、胫骨质量和血清生化指标的影响%Different Calcium Supplementation Modes Affect Production Performance, Tibia Quality and Serum Biochemical Indices of Laying Hens

    Institute of Scientific and Technical Information of China (English)

    孔路欣; 臧素敏; 刘培培; 李泽茹


    本试验旨在研究不同增钙模式对蛋鸡生产性能、胫骨质量以及血清相关生化指标的影响,以确定蛋鸡生产过程中饲粮钙的增加模式以及钙的添加量。选取18周龄海兰灰商品蛋鸡480只,随机分为4组,每组4个重复,每个重复30只。增钙时间点分别为18周龄以及产蛋率为5%、50%和90%时,各时间点增钙方式分别为:Ⅰ组2.00%、2.20%、2.40%和3.75%;Ⅱ组2.00%、2.50%、3.00%和3.75%;Ⅲ组2.00%、3.00%、3.75%和3.75%;Ⅳ组2.00%、3.75%、3.75%和3.75%。试验期9周。结果表明:1)Ⅲ组蛋鸡产蛋率显著高于Ⅰ组和Ⅳ组(P0.05);2)Ⅲ组胫骨强度、胫骨重、胫骨钙含量显著高于Ⅰ组和Ⅱ组( P0.05);3)蛋鸡产蛋率达50%时,Ⅰ组和Ⅱ组血钙含量显著高于Ⅲ组和Ⅳ组(P0.05)。结果提示:当蛋鸡产蛋率达5%、50%和90%时,饲粮钙水平分别为3.00%、3.75%和3.75%有助于提高蛋鸡产蛋率,改善体况和稳定骨骼质量,同时不造成钙源浪费。%This study was conducted to investigate the effects of different calcium supplementation modes on production performance, tibia quality and serum biochemical indices of laying hens, with an aim to determine the optimal supplementation mode and amount of calcium in production. Four hundred and eighty 18-week-old Hy-Line Gray commercial layers were divided into 4 groups with 4 replicates each and 30 hens in each repli-cate. The calcium was supplemented at 18 weeks of age and when the laying rate was 5%, 50% and 90%, re-spectively. The calcium supplementation modes were: 2. 00%, 2. 20%, 2. 40% and 3. 75% for group Ⅰ;2.00%, 2.50%, 3.00% and 3.75% for groupⅡ;2.00%, 3.00%, 3.75% and 3.75% for groupⅢ;2.00%, 3.75%, 3.75% and 3.75% for group Ⅳ. The experiment lasted for 9 weeks. The results showed as follows:1) the laying rate of hens in group Ⅲ was significantly higher than that in group Ⅰ and group Ⅳ ( P0.05) . 2) The tibia strength, weight and calcium content in

  13. Calcium and Calcium-Base Alloys (United States)


    should be satisfactory, because the electrolytic process for •(!>: A. H. Everts and G. D. Baglev’, " Physical «nrt m<„.+„4 i «_ of Calcium«, Electrochem...Rev. Metalurgie , 3j2, (1), 129 (1935). 10 ^sm^mssss^ma^^ extension between two known loads, is preferable to the value of 3,700,000 p.B.i. obtained

  14. Diagram of Calcium Movement in the Human Body (United States)


    This diagram shows the normal pathways of calcium movement in the body and indicates changes (green arrows) seen during preliminary space flight experiments. Calcium plays a central role because 1) it gives strength and structure to bone and 2) all types of cells require it to function normally. To better understand how and why weightlessness induces bone loss, astronauts have participated in a study of calcium kinetics -- that is, the movement of calcium through the body, including absorption from food, and its role in the formation and breakdown of bone.

  15. Roscovitine increases intracellular calcium release and capacitative calcium entry in PC12 cells. (United States)

    Choi, Ho Sook; Chung, Sul-Hee


    Cyclin-dependent kinase 5 (Cdk5), which is activated by the non-cyclin regulator p35 or p39, is a proline-directed serine/threonine kinase that is implicated in many physiological and pathological processes. Here, we studied calcium signaling using the fluorescent cytosolic calcium indicator, Fura-4, in NGF-differentiated PC12 cells treated with roscovitine, a Cdk5 inhibitor. As compared to the control cells, the roscovitine-treated cells significantly potentiated intracellular calcium release by membrane depolarization (high K(+)) or through thapsigargin. In addition, roscovitine increased the magnitude of capacitative calcium entry (CCE), i.e., a calcium influx mechanism triggered by the depletion of intracellular calcium stores. Notably, roscovitine markedly slowed the rate of Ca(2+) removal from the plasma membrane. These results suggest that Cdk5 regulates intracellular calcium homeostasis and that the dysregulation of Cdk5 may contribute to disease pathogenesis by perturbing cellular Ca(2+) signaling. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

  16. Calcium and calcium isotope changes during carbon cycle perturbations at the end-Permian (United States)

    Komar, Nemanja; Zeebe, Richard


    Negative carbon and calcium isotope excursions, as well as climate shifts, took place during the most severe mass extinction event in Earth's history, the end-Permian (˜252 Ma). Investigating the connection between carbon and calcium cycles during transient carbon cycle perturbation events, such as the end-Permian, may help resolve the intricacies between the coupled calcium-carbon cycles, as well as provide a tool for constraining the causes of mass extinction. Here, we identify the deficiencies of a simplified calcium model employed in several previous studies and we demonstrate the importance of a fully coupled carbon-cycle model when investigating the dynamics of carbon and calcium cycling. Simulations with a modified version of the LOSCAR model, which includes a fully coupled carbon-calcium cycle, indicate that increased weathering rates and ocean acidification (potentially caused by Siberian Trap volcanism) are not capable of producing trends observed in the record, as previously claimed. Our model results suggest that combined effects of carbon input via Siberian Trap volcanism (12,000 Pg C), the cessation of biological carbon export, and variable calcium isotope fractionation (due to a change in the seawater carbonate ion concentration) represents a more plausible scenario. This scenario successfully reconciles δ13C and δ44Ca trends observed in the sediment record, as well as the proposed warming of >6oC.

  17. Resveratrol Interferes with Fura-2 Intracellular Calcium Measurements. (United States)

    Kopp, Richard F; Leech, Colin A; Roe, Michael W


    Resveratrol, a naturally occurring polyphenol found in some fruits and especially in grapes, has been reported to provide diverse health benefits. Resveratrol's mechanism of action is the subject of many investigations, and some studies using the ratiometric calcium indicator Fura-2 suggest that it modulates cellular calcium responses. In the current study, contradictory cellular calcium responses to resveratrol applied at concentrations exceeding 10 μM were observed during in vitro imaging studies depending on the calcium indicator used, with Fura-2 indicating an increase in intracellular calcium while Fluo-4 and the calcium biosensor YC3.60 indicated no response. When cells loaded with Fura-2 were treated with 100 μM resveratrol, excitation at 340 nm resulted in a large intensity increase at 510 nm, but the expected concurrent decline with 380 nm excitation was not observed. Pre-treatment of cells with the calcium chelator BAPTA-AM did not prevent a rise in the 340/380 ratio when resveratrol was present, but it did prevent an increase in 340/380 when ATP was applied, suggesting that the resveratrol response was an artifact. Cautious data interpretation is recommended from imaging experiments using Fura-2 concurrently with resveratrol in calcium imaging experiments.

  18. Magnesium: Origin and role in calcium-treated inclusions

    CSIR Research Space (South Africa)

    Pistorius, CP


    Full Text Available -magnesia-lime phase diagram. Inclusions with higher CaO contents generally had lower MgO contents, indicating that the calcium wire is not the origin of the magnesium in the inclusions; this was also confirmed by wet chemical analysis of the calcium wire. Instead...

  19. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau


    Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

  20. Elemental calcium intake associated with calcium acetate/calcium carbonate in the treatment of hyperphosphatemia


    Wilson, Rosamund J; Copley, J Brian


    Background Calcium-based and non-calcium-based phosphate binders have similar efficacy in the treatment of hyperphosphatemia; however, calcium-based binders may be associated with hypercalcemia, vascular calcification, and adynamic bone disease. Scope A post hoc analysis was carried out of data from a 16-week, Phase IV study of patients with end-stage renal disease (ESRD) who switched to lanthanum carbonate monotherapy from baseline calcium acetate/calcium carbonate monotherapy. Of the intent...

  1. Role of Calcium Hydroxide in Endodontics: A Review

    Directory of Open Access Journals (Sweden)

    Arun A


    Full Text Available Calcium hydroxide is a multipurpose agent, and there have been an increasing number of indications for its use in endodontics. Some of its indications include inter-appointment intracanal medicaments, endodontic sealers, pulp capping agents, apexification, pulpotomy and weeping canals. The purpose of this article is to review the properties, advantages, disadvantages and various indications for the use of calcium hydroxide in endodontics.

  2. Calcium and vitamin D supplementation increases spinal BMD in healthy, postmenopausal women

    DEFF Research Database (Denmark)

    Baeksgaard, L; Andersen, K P; Hyldstrup, Lars


    We undertook a double-masked, randomized, placebo-controlled trial to evaluate the effect of a calcium and vitamin D supplement and a calcium supplement plus multivitamins on bone loss at the hip, spine and forearm. The study was performed in 240 healthy women, 58-67 years of age. Duration...... of treatment was 2 years. Bone mineral density (BMD) was measured at the lumbar spine, hip and forearm. A dietary questionnaire was administered twice during the study and revealed a fairly good calcium and vitamin D intake (919 mg calcium/day; 3.8 micrograms vitamin D/day). An increase in lumbar spine BMD....... Together with significant changes in serum calcium and serum parathyroid hormone, this indicates that a long-term calcium and vitamin supplement of 1 g elementary calcium (calcium carbonate) and 14 micrograms vitamin D3 increases intestinal calcium absorption. A positive effect on BMD was demonstrated...

  3. [Microbial geochemical calcium cycle]. (United States)

    Zavarzin, G A


    The participation of microorganisms in the geochemical calcium cycle is the most important factor maintaining neutral conditions on the Earth. This cycle has profound influence on the fate of inorganic carbon, and, thereby, on the removal of CO2 from the atmosphere. The major part of calcium deposits was formed in the Precambrian, when prokaryotic biosphere predominated. After that, calcium recycling based on biogenic deposition by skeletal organisms became the main process. Among prokaryotes, only a few representatives, e.g., cyanobacteria, exhibit a special calcium function. The geochemical calcium cycle is made possible by the universal features of bacteria involved in biologically mediated reactions and is determined by the activities of microbial communities. In the prokaryotic system, the calcium cycle begins with the leaching of igneous rock predominantly through the action of the community of organotrophic organisms. The release of carbon dioxide to the soil air by organotrophic aerobes leads to leaching with carbonic acid and soda salinization. Under anoxic conditions, of major importance is the organic acid production by primary anaerobes (fermentative microorganisms). Calcium carbonate is precipitated by secondary anaerobes (sulfate reducers) and to a smaller degree by methanogens. The role of the cyanobacterial community in carbonate deposition is exposed by stromatolites, which are the most common organo-sedimentary Precambrian structures. Deposition of carbonates in cyanobacterial mats as a consequence of photoassimilation of CO2 does not appear to be a significant process. It is argued that carbonates were deposited at the boundary between the "soda continent", which emerged as a result of subaerial leaching with carbonic acid, and the ocean containing Ca2+. Such ecotones provided favorable conditions for the development of the benthic cyanobacterial community, which was a precursor of stromatolites.

  4. Waste indicators

    Energy Technology Data Exchange (ETDEWEB)

    Dall, O.; Lassen, C.; Hansen, E. [Cowi A/S, Lyngby (Denmark)


    The Waste Indicator Project focuses on methods to evaluate the efficiency of waste management. The project proposes the use of three indicators for resource consumption, primary energy and landfill requirements, based on the life-cycle principles applied in the EDIP Project. Trial runs are made With the indicators on paper, glass packaging and aluminium, and two models are identified for mapping the Danish waste management, of which the least extensive focuses on real and potential savings. (au)

  5. Quality indicators

    DEFF Research Database (Denmark)

    Hjorth-Andersen, Christian


    In recent literature it has been suggested that consumers need have no knowledge of product quality as a number of quality indicators (or signals) may be used as substitutes. Very little attention has been paid to the empirical verification of these studies. The present paper is devoted...... to the issue of how well these indicators perform, using market data provided by consumer magazines from 3 countries. The results strongly indicate that price is a poor quality indicator. The paper also presents some evidence which suggests that seller reputation and easily observable characteristics are also...


    Alterations in calcium ion activity by ELF and RF electromagnetic fieldsIntroductionCalcium ions play many important roles in biological systems. For example, calcium ion activity can be used as an indicator of second-messenger signal-transduction processe...


    Alterations in calcium ion activity by ELF and RF electromagnetic fieldsIntroductionCalcium ions play many important roles in biological systems. For example, calcium ion activity can be used as an indicator of second-messenger signal-transduction processe...

  8. Inositol trisphosphate and calcium signalling (United States)

    Berridge, Michael J.


    Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

  9. Calcium supplementation does not augment bone gain in young women consuming diets moderately low in calcium. (United States)

    Barger-Lux, M Janet; Davies, K Michael; Heaney, Robert P


    In earlier observational work, the dietary calcium:protein ratio was directly related to bone accrual in healthy postadolescent women. In this study, we sought to test the hypothesis that augmented calcium intake would increase postadolescent skeletal consolidation, using a double-blind, randomized, placebo-controlled design. We recruited 152 healthy young women (age 23.1 +/- 2.7 y, BMI 22.5 +/- 3.0 kg/m2); their usual diets, as assessed by 7-d food diaries, were low in calcium (605 +/- 181 mg/d; 15.1 +/- 4.5 mmol/d) and in the calcium:protein ratio (10.1 +/- 2.0 mg/g). The subjects were randomly assigned to supplemental calcium [500 mg calcium (12.5 mmol) as the carbonate, 3 times/d, with meals] or placebo capsules identical in appearance; all participants also took a daily multivitamin, and they were followed for up to 36 mo with bone densitometry (dual energy X-ray absorptiometry; DXA) at 6-mo intervals. A total of 121 subjects remained in the study for at least 12 mo (median time in the study, 35 mo), with a mean compliance level (observed/expected tablet consumption) of 87.7%. DXA data for these 121 subjects indicated modest but significant mean rates of increase (i.e., 0.24 to 1.10%/y) in bone mineral content (BMC; total body, total hip, and lumbar spine) and in lumbar spine bone mineral density (BMD) but no change in total hip BMD. None of these rates of change differed by group, i.e., calcium supplementation did not have any measurable effect on bone mass accrual. By midstudy, the calcium content of the subjects' usual diets for both groups had risen by approximately 15%. The combined effect of improved intakes of dietary calcium and the small amount of calcium added by the multivitamin tablets resulted in a mean calcium intake for the control group > 800 mg (20 mmol)/d, possibly at or near the threshold beyond which additional calcium has no further effect on bone accrual.

  10. Calcium and Calcium Supplements: Achieving the Right Balance (United States)

    ... bone mass, which is a risk factor for osteoporosis. Many Americans don't get enough calcium in their diets. Children and adolescent girls are at particular risk, but so are adults age 50 and older. How much calcium you ...

  11. Gravimetric Determination of Calcium as Calcium Carbonate Hydrate. (United States)

    Henrickson, Charles H.; Robinson, Paul R.


    The gravimetric determination of calcium as calcium carbonate is described. This experiment is suitable for undergraduate quantitative analysis laboratories. It is less expensive than determination of chloride as silver chloride. (BB)

  12. A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

    Directory of Open Access Journals (Sweden)

    Christine Dorothee Schmeitz


    Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

  13. Serum calcium levels are not associated with coronary heart disease

    Directory of Open Access Journals (Sweden)

    Jin Y


    Full Text Available Yuelong Jin,* Lianping He,* Quanhai Wang, Yan Chen, Xiaohua Ren, Hui Tang, Xiuli Song, Lingling Ding, Qin Qi, Zhiwei Huang, Jiegen Yu, Yingshui Yao Department of Preventive Medicine, Wannan Medical College, Wuhu, People's Republic of China *These authors contributed equally to this work Background: Numerous studies have reported that low calcium intake is related to a higher prevalence of cardiovascular disease. However, the relationship between serum calcium and coronary heart disease is unclear. The purpose of this study was to compare serum calcium levels in patients with coronary heart disease and those in healthy individuals. Methods: This retrospective, case-control study conducted in the People's Republic of China comprised 380 cases and 379 controls. Serum calcium levels, blood lipids, and anthropometric measurements were measured in both groups. The Student's unpaired t-test or Chi-square test was used to compare differences between cases and controls. Pearson's partial correlation coefficient was used to determine the association between serum calcium, blood lipids, and blood pressure in both groups. Results: Our results indicate that the average level of serum calcium in cases was higher than in controls. Serum calcium levels showed no correlation with any parameter except for triglycerides in either group. Conclusion: Overall, these data suggest that serum calcium has no influence on coronary heart disease or triglyceride levels in the general population. Keywords: serum calcium, hypertension, blood lipids

  14. Solar Indices (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Collection includes a variety of indices related to solar activity contributed by a number of national and private solar observatories located worldwide. This...

  15. Voltage-Gated Calcium Channels in Nociception (United States)

    Yasuda, Takahiro; Adams, David J.

    Voltage-gated calcium channels (VGCCs) are a large and functionally diverse group of membrane ion channels ubiquitously expressed throughout the central and peripheral nervous systems. VGCCs contribute to various physiological processes and transduce electrical activity into other cellular functions. This chapter provides an overview of biophysical properties of VGCCs, including regulation by auxiliary subunits, and their physiological role in neuronal functions. Subsequently, then we focus on N-type calcium (Cav2.2) channels, in particular their diversity and specific antagonists. We also discuss the role of N-type calcium channels in nociception and pain transmission through primary sensory dorsal root ganglion neurons (nociceptors). It has been shown that these channels are expressed predominantly in nerve terminals of the nociceptors and that they control neurotransmitter release. To date, important roles of N-type calcium channels in pain sensation have been elucidated genetically and pharmacologically, indicating that specific N-type calcium channel antagonists or modulators are particularly useful as therapeutic drugs targeting chronic and neuropathic pain.

  16. Calcium, vitamin D and bone


    Borg, Andrew A.


    Calcium, protein and vitamin D are the main nutrients relevant to bone health. This short article discusses the importance of vitamin D and its relation to calcium homeostasis. The various causes, clinical manifestations and treatment are outlined.

  17. Mechanical Properties of a Calcium Dietary Supplement, Calcium Fumarate Trihydrate. (United States)

    Sun, Shijing; Henke, Sebastian; Wharmby, Michael T; Yeung, Hamish H-M; Li, Wei; Cheetham, Anthony K


    The mechanical properties of calcium fumarate trihydrate, a 1D coordination polymer considered for use as a calcium source for food and beverage enrichment, have been determined via nanoindentation and high-pressure X-ray diffraction with single crystals. The nanoindentation studies reveal that the elastic modulus (16.7-33.4 GPa, depending on crystallographic orientation), hardness (1.05-1.36 GPa), yield stress (0.70-0.90 GPa), and creep behavior (0.8-5.8 nm/s) can be rationalized in view of the anisotropic crystal structure; factors include the directionality of the inorganic Ca-O-Ca chain and hydrogen bonding, as well as the orientation of the fumarate ligands. High-pressure single-crystal X-ray diffraction studies show a bulk modulus of ∼ 20 GPa, which is indicative of elastic recovery intermediate between small molecule drug crystals and inorganic pharmaceutical ingredients. The combined use of nanoindentation and high-pressure X-ray diffraction techniques provides a complementary experimental approach for probing the critical mechanical properties related to tableting of these dietary supplements.

  18. Calcium signaling and T-type calcium channels in cancer cell cycling

    Institute of Scientific and Technical Information of China (English)

    James T Taylor; Xiang-Bin Zeng; Jonathan E Pottle; Kevin Lee; Alun R Wang; Stephenie G Yi; Jennifer A S Scruggs; Suresh S Sikka; Ming Li


    Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells,free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear;however, there is a growing body of evidence that suggests the T-type Ca2+ channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca2+ channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel isminimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca2+ channels are not expressed in epithelial cells, selective T-type Ca2+ channel blockers may be useful in the treatment of certain types of cancers.

  19. Calcium ion channel and epilepsy

    Institute of Scientific and Technical Information of China (English)

    Yudan Lü; Weihong Lin; Dihui Ma


    OBJECTIVE: To review the relationship between calcium ion channel and epilepsy for well investigating the pathogenesis of epilepsy and probing into the new therapeutic pathway of epilepsy.DATA SOURCES: A computer-based online research Calcium ion channel and epilepsy related articles published between January 1994 and December 2006 in the CKNI and Wanfang database with the key words of "calcium influxion, epilepsy, calcium-channel blocker". The language was limited to Chinese. At the same time,related articles published between January 1993 and December 2006 in Pubmed were searched for on online with the key words of "calcium influxion, epilepsy" in English.STUDY SELECTION: The materials were selected firstly. Inclusive criteria: ① Studies related to calcium ion channel and the pat1hogenesis of epilepsy. ② Studies on the application of calcium ion channel blocker in the treatment of epilepsy. Exclusive criteria: repetitive or irrelated studies.DATA EXTRACTION: According to the criteria, 123 articles were retrieved and 93 were excluded due to repetitive or irrelated studies. Altogether 30 articles met the inclusive criteria, 11 of them were about the structure and characters of calcium ion channel, 10 about calcium ion channel and the pathogenesis of epilepsy and 9 about calcium blocker and the treatment of epilepsy.DATA SYNTHESIS: Calcium ion channels mainly consist of voltage dependent calcium channel and receptor operated calcium channel. Depolarization caused by voltage gating channel-induced influxion is the pathological basis of epileptic attack, and it is found in many studies that many anti-epileptic drugs have potential and direct effect to rivalizing voltage-dependent calcium ion channel.CONCLUSION: Calcium influxion plays an important role in the seizure of epilepsy. Some calcium antagonists seen commonly are being tried in the clinical therapy of epilepsy that is being explored, not applied in clinical practice. If there are enough evidences to

  20. High Blood Calcium (Hypercalcemia) (United States)

    ... as well as kidney function and levels of calcium in your urine. Your provider may do other tests to further assess your condition, such as checking your blood levels of phosphorus (a mineral). Imaging studies also may be helpful, such as bone ...

  1. Calcium carbonate overdose (United States)

    Calcium carbonate is not very poisonous. Recovery is quite likely. But, long-term overuse is more serious than a single overdose, because it can cause kidney damage. Few people die from an antacid overdose. Keep all medicines in child-proof bottles and out ...

  2. Solar Imagery - Chromosphere - Calcium (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This dataset consists of full-disk images of the sun in Calcium (Ca) II K wavelength (393.4 nm). Ca II K imagery reveal magnetic structures of the sun from about 500...

  3. Extracellular Calcium and Magnesium

    African Journals Online (AJOL)

    i cellular and neuronal metabolism and functions. The objective of ... as having preeclampsia or eclampsia, in the same age range. ... Booking status Number (n) ("/o) Number (n) (%). Booked 7 ... is influx of calcium ions into the cell leacling to.

  4. Calcium aluminate in alumina (United States)

    Altay, Arzu

    The properties of ceramic materials are determined not only by the composition and structure of the phases present, but also by the distribution of impurities, intergranular films and second phases. The phase distribution and microstructure both depend on the fabrication techniques, the raw materials used, the phase-equilibrium relations, grain growth and sintering processes. In this dissertation research, various approaches have been employed to understand fundamental phenomena such as grain growth, impurity segregation, second-phase formation and crystallization. The materials system chosen was alumina intentionally doped with calcium. Atomic-scale structural analyses of grain boundaries in alumina were carried on the processed samples. It was found that above certain calcium concentrations, CA6 precipitated as a second phase at all sintering temperatures. The results also showed that abnormal grain growth can occur after precipitation and it is not only related to the calcium level, but it is also temperature dependent. In order to understand the formation mechanism of CA6 precipitates in calcium doped alumina samples, several studies have been carried out using either bulk materials or thin films The crystallization of CA2 and CA6 powders has been studied. Chemical processing techniques were used to synthesize the powders. It was observed that CA2 powders crystallized directly, however CA6 powders crystallized through gamma-Al 2O3 solid solution. The results of energy-loss near-edge spectrometry confirmed that gamma-Al2O3 can dissolve calcium. Calcium aluminate/alumina reaction couples have also been investigated. All reaction couples were heat treated following deposition. It was found that gamma-Al2O3 was formed at the interface as a result of the interfacial reaction between the film and the substrate. gamma-Al 2O3 at the interface was stable at much higher temperatures compared to the bulk gamma-Al2O3 formed prior to the CA6 crystallization. In order to

  5. Antenatal calcium intake in Malaysia. (United States)

    Mahdy, Zaleha Abdullah; Basri, Hashimah; Md Isa, Zaleha; Ahmad, Shuhaila; Shamsuddin, Khadijah; Mohd Amin, Rahmah


    To determine the adequacy of antenatal calcium intake in Malaysia, and the influencing factors. A cross-sectional study was conducted among postnatal women who delivered in two tertiary hospitals. Data were collected from antenatal cards, hospital documents and diet recall on daily milk and calcium intake during pregnancy. SPSS version 19.0 was used for statistical analyses. A total of 150 women were studied. The total daily calcium intake was 834 ± 43 mg (mean ± standard error of the mean), but the calcium intake distribution curve was skewed to the right with a median intake of 725 mg daily. When calcium intake from milk and calcium supplements was excluded, the daily dietary calcium intake was only 478 ± 25 mg. Even with inclusion of milk and calcium supplements, more than a third (n=55 or 36.7%) of the women consumed less than 600 mg calcium in their daily diet. The adequacy of daily calcium intake was not influenced by maternal age, ethnicity, income or maternal job or educational status as well as parity. The daily dietary calcium intake of the Malaysian antenatal population is far from adequate without the addition of calcium supplements and milk. © 2013 The Authors. Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology.

  6. Calcium addition in straw gasification

    DEFF Research Database (Denmark)

    Risnes, H.; Fjellerup, Jan Søren; Henriksen, Ulrik Birk


    The present work focuses on the influence of calcium addition in gasification. The inorganic¿organic element interaction as well as the detailed inorganic¿inorganic elements interaction has been studied. The effect of calcium addition as calcium sugar/molasses solutions to straw significantly...

  7. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion. (United States)

    Nishitani, Wagner Shin; Alencar, Adriano Mesquita; Wang, Yingxiao


    A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs) in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7) expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount) and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment) and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.

  8. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.

    Directory of Open Access Journals (Sweden)

    Wagner Shin Nishitani

    Full Text Available A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7 expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.

  9. Bioceramics of calcium orthophosphates. (United States)

    Dorozhkin, Sergey V


    A strong interest in use of ceramics for biomedical applications appeared in the late 1960's. Used initially as alternatives to metals in order to increase a biocompatibility of implants, bioceramics have become a diverse class of biomaterials, presently including three basic types: relatively bioinert ceramics, bioactive (or surface reactive) and bioresorbable ones. Furthermore, any type of bioceramics could be porous to provide tissue ingrowth. This review is devoted to bioceramics prepared from calcium orthophosphates, which belong to the categories of bioresorbable and bioactive compounds. During the past 30-40 years, there have been a number of major advances in this field. Namely, after the initial work on development of bioceramics that was tolerated in the physiological environment, emphasis was shifted towards the use of bioceramics that interacted with bones by forming a direct chemical bond. By the structural and compositional control, it became possible to choose whether the bioceramics of calcium orthophosphates was biologically stable once incorporated within the skeletal structure or whether it was resorbed over time. At the turn of the millennium, a new concept of calcium orthophosphate bioceramics, which is able to regenerate bone tissues, has been developed. Current biomedical applications of calcium orthophosphate bioceramics include replacements for hips, knees, teeth, tendons and ligaments, as well as repair for periodontal disease, maxillofacial reconstruction, augmentation and stabilization of the jawbone, spinal fusion and bone fillers after tumor surgery. Potential future applications of calcium orthophosphate bioceramics will include drug-delivery systems, as well as they will become effective carriers of growth factors, bioactive peptides and/or various types of cells for tissue engineering purposes.

  10. Calcium in Gluten-Free Life: Health-Related and Nutritional Implications

    Directory of Open Access Journals (Sweden)

    Urszula Krupa-Kozak


    Full Text Available Calcium deficiency and metabolic bone diseases are a frequent co-morbidity of coeliac disease (CD. Gluten-free diet (GFD is the only effective treatment of CD. However, CD patients on the strict GFD consume less than the recommended amounts of calcium. In this review, the main etiological factors responsible for calcium deficiency in CD were presented. Additionally, the research on the application of calcium supplements in the gluten-free breadmaking was reviewed, and its effect on the technological and sensory properties of baked products was indicated. Calcium-fortified gluten-free products could increase the calcium content in the diet of CD patients, supplying the amount of calcium they need for prophylactic or therapeutic use. Apart from this, the consumption of the naturally GF products as well as functional ingredients beneficially affecting calcium absorption need to be encouraged.

  11. Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons. (United States)

    Pivovarova, Natalia B; Stanika, Ruslan I; Watts, Charlotte A; Brantner, Christine A; Smith, Carolyn L; Andrews, S Brian


    In central neurons, over-stimulation of NMDA receptors leads to excessive mitochondrial calcium accumulation and damage, which is a critical step in excitotoxic death. This raises the possibility that low susceptibility to calcium overload-induced mitochondrial damage might characterize excitotoxicity-resistant neurons. In this study, we have exploited two complementary models of preconditioning-induced excitotoxicity resistance to demonstrate reduced calcium-dependent mitochondrial damage in NMDA-tolerant hippocampal neurons. We have further identified adaptations in mitochondrial calcium handling that account for enhanced mitochondrial integrity. In both models, enhanced tolerance was associated with improved preservation of mitochondrial membrane potential and structure. In the first model, which exhibited modest neuroprotection, mitochondria-dependent calcium deregulation was delayed, even though cytosolic and mitochondrial calcium loads were quantitatively unchanged, indicating that enhanced mitochondrial calcium capacity accounts for reduced injury. In contrast, the second model, which exhibited strong neuroprotection, displayed further delayed calcium deregulation and reduced mitochondrial damage because downregulation of NMDA receptor surface expression depressed calcium loading. Reducing calcium entry also modified the chemical composition of the calcium-buffering precipitates that form in calcium-loaded mitochondria. It thus appears that reduced mitochondrial calcium loading is a major factor underlying the robust neuroprotection seen in highly tolerant cells.

  12. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips (United States)

    Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.


    Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

  13. Calcium dependent magnesium uptake in myocardium. (United States)

    Bianchi, C P; Liu, D


    The frog myocardium maintains magnesium content at a steady state level when stimulated at 0.4Hz while being perfused with Ringer's solution containing 1 x 10(-3) M Ca2+ and 5 x 10(-7) M magnesium. When calcium is removed 43% of tissue magnesium is lost within 30 seconds or 12 beats. Restoration of calcium to the perfusion solution causes reaccumulation of magnesium from a solution containing 5 x 10(-7) M magnesium. The reaccumulation of magnesium indicates a highly selective transport system for magnesium which is dependent upon the presence of calcium. Calcium appears to reduce the leak of magnesium from the myocardium and enhances the transport of magnesium into the myocardial cell. Intracellular magnesium is a necessary cofactor for hundreds of enzymes, and is essential for protein synthesis and as an extracellular divalent cation helps to stabilize excitable membranes in conjunction with calcium. The concentration of ionized magnesium in the sarcoplasm of myocardial muscle has an average value of 1.45 mM +/- 1.37 (standard deviation), N = 19) with a range of 0.5 to 3.6 mM (1). The heart with its numerous mitochondria and high enzymatic activity is vulnerable to myocardial damage due to magnesium loss. The isolated frog ventricle conserves intracellular magnesium when perfused with Ringer's solution containing no added magnesium and maintains function for hours. The ability to conserve magnesium suggests a low permeability of the sarcolemma to magnesium and an extremely efficient inward transport system. Removal of calcium as well as magnesium from the perfusion solution causes a rapid loss of tension in the electrically driven frog ventricle (0.4) Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Calcium signaling in taste cells. (United States)

    Medler, Kathryn F


    The sense of taste is a common ability shared by all organisms and is used to detect nutrients as well as potentially harmful compounds. Thus taste is critical to survival. Despite its importance, surprisingly little is known about the mechanisms generating and regulating responses to taste stimuli. All taste responses depend on calcium signals to generate appropriate responses which are relayed to the brain. Some taste cells have conventional synapses and rely on calcium influx through voltage-gated calcium channels. Other taste cells lack these synapses and depend on calcium release to formulate an output signal through a hemichannel. Beyond establishing these characteristics, few studies have focused on understanding how these calcium signals are formed. We identified multiple calcium clearance mechanisms that regulate calcium levels in taste cells as well as a calcium influx that contributes to maintaining appropriate calcium homeostasis in these cells. Multiple factors regulate the evoked taste signals with varying roles in different cell populations. Clearly, calcium signaling is a dynamic process in taste cells and is more complex than has previously been appreciated. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

  15. Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients

    Directory of Open Access Journals (Sweden)

    García Juan F


    Full Text Available Abstract Background Asthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia. Methods Human ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4–5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2. Results Treatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia. Conclusion Our results suggest

  16. Fruit Calcium: Transport and Physiology

    Directory of Open Access Journals (Sweden)

    Bradleigh eHocking


    Full Text Available Calcium has well-documented roles in plant signaling, water relations and cell wall interactions. Significant research into how calcium impacts these individual processes in various tissues has been carried out; however, the influence of calcium on fruit ripening has not been thoroughly explored. Here, we review the current state of knowledge on how calcium may impact fruit development, physical traits and disease susceptibility through facilitating developmental and stress response signaling, stabilizing membranes, influencing water relations and modifying cell wall properties through cross-linking of de-esterified pectins. We explore the involvement of calcium in hormone signaling integral to ripening and the physiological mechanisms behind common disorders that have been associated with fruit calcium deficiency (e.g. blossom end rot in tomatoes or bitter pit in apples. This review works towards an improved understanding of how the many roles of calcium interact to influence fruit ripening, and proposes future research directions to fill knowledge gaps. Specifically, we focus mostly on grapes and present a model that integrates existing knowledge around these various functions of calcium in fruit, which provides a basis for understanding the physiological impacts of sub-optimal calcium nutrition in grapes. Calcium accumulation and distribution in fruit is shown to be highly dependent on water delivery and cell wall interactions in the apoplasm. Localized calcium deficiencies observed in particular species or varieties can result from differences in xylem morphology, fruit water relations and pectin composition, and can cause leaky membranes, irregular cell wall softening, impaired hormonal signaling and aberrant fruit development. We propose that the role of apoplasmic calcium-pectin crosslinking, particularly in the xylem, is an understudied area that may have a key influence on fruit water relations. Furthermore, we believe that improved

  17. Social indicators. (United States)

    Sheldon, E B; Parke, R


    The notions of social indicators and social accounting, expressed by analogy with the national economic accounts, generated excitement in the 1960's, and the interest continues to grow if we may judge from governmental activity and the publication of programmatic and research papers. But the concepts which focused much of the early enthusiasm gave exaggerated promise of policy applications and provided an unproductive basis for research. The essential theoretical prerequisites for developing a system of social accounts-defining the variables and the interrelationships among them-are missing. It is now realized that evaluation research, particularly experimentation, must be relied on for evaluation of government programs. Through the development and analysis of descriptive time series and the modeling of social processes, we will be able to describe the state of the society and its dynamics and thus improve immensely our ability to state problems in a productive fashion, obtain clues as to promising lines of endeavor, and ask good questions. But these activities cannot measure program effectiveness. Finally, we must be skeptical about definitions of the social indicators enterprise which confine it to social engineering efforts. The issue is not whether social indicators are useful for policy but, rather, how this usefulness comes about. The interest in social indicators has stimulated a revival of interest in quantitative, comparative, social analysis (60), in the analysis of social change, in conceptual and measurement work on such topics as prejudice, crime, and learning, and in the development of models of social processes. The fruit of these efforts will be more directly a contribution to the policy-maker's cognition than to his decisions. Decision emerges from a mosaic of inputs, including valuational and political, as well as technical components. The work we have described deals with only one type of input; it is a contribution to the intellectual mapping

  18. Genetically encoded biosensors based on engineered fluorescent proteins. (United States)

    Frommer, Wolf B; Davidson, Michael W; Campbell, Robert E


    Fluorescent proteins have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. While the most common applications of fluorescent proteins are to image expression, localization, and dynamics of protein chimeras, there is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. This tutorial review provides an overview of the progress made in the development of fluorescent protein-based biosensors to date.

  19. A genetically encoded fluorescent probe in mammalian cells. (United States)

    Chatterjee, Abhishek; Guo, Jiantao; Lee, Hyun Soo; Schultz, Peter G


    Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biology.

  20. A genetically encoded biosensor for visualising hypoxia responses in vivo (United States)

    Misra, Tvisha; Baccino-Calace, Martin; Meyenhofer, Felix; Rodriguez-Crespo, David; Akarsu, Hatice; Armenta-Calderón, Ricardo; Gorr, Thomas A.; Frei, Christian; Cantera, Rafael; Egger, Boris


    ABSTRACT Cells experience different oxygen concentrations depending on location, organismal developmental stage, and physiological or pathological conditions. Responses to reduced oxygen levels (hypoxia) rely on the conserved hypoxia-inducible factor 1 (HIF-1). Understanding the developmental and tissue-specific responses to changing oxygen levels has been limited by the lack of adequate tools for monitoring HIF-1 in vivo. To visualise and analyse HIF-1 dynamics in Drosophila, we used a hypoxia biosensor consisting of GFP fused to the oxygen-dependent degradation domain (ODD) of the HIF-1 homologue Sima. GFP-ODD responds to changing oxygen levels and to genetic manipulations of the hypoxia pathway, reflecting oxygen-dependent regulation of HIF-1 at the single-cell level. Ratiometric imaging of GFP-ODD and a red-fluorescent reference protein reveals tissue-specific differences in the cellular hypoxic status at ambient normoxia. Strikingly, cells in the larval brain show distinct hypoxic states that correlate with the distribution and relative densities of respiratory tubes. We present a set of genetic and image analysis tools that enable new approaches to map hypoxic microenvironments, to probe effects of perturbations on hypoxic signalling, and to identify new regulators of the hypoxia response. PMID:28011628

  1. Synthesis of calcium superoxide (United States)

    Rewick, R. T.; Blucher, W. G.; Estacio, P. L.


    Efforts to prepare Ca(O2) sub 2 from reactions of calcium compounds with 100% O3 and with O(D-1) atoms generated by photolysis of O3 at 2537 A are described. Samples of Ca(OH) sub 2, CaO, CaO2, Ca metal, and mixtures containing suspected impurities to promote reaction have been treated with excess O3 under static and flow conditions in the presence and absence of UV irradiation. Studies with KO2 suggest that the superoxide anion is stable to radiation at 2537 A but reacts with oxygen atoms generated by the photolysis of O3 to form KO3. Calcium superoxide is expected to behave in an analogous.


    Barton, J.


    This invention relates to an improvement in the process for the purification of caicium or magnesium containing an alkali metal as impurity, which comprises distiiling a batch of the mixture in two stages, the first stage distillation being carried out in the presence of an inert gas at an absolute pressure substantially greater than the vapor pressure of calcium or maguesium at the temperature of distillation, but less than the vaper pressure at that temperature of the alkali metal impurity so that only the alkali metal is vaporized and condensed on a condensing surface. A second stage distilso that substantially only the calcium or magnesium distills under its own vapor pressure only and condenses in solid form on a lower condensing surface.

  3. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability. (United States)

    Tang, Ning; Skibsted, Leif H


    Deprotonation of amino acids as occurs during transfer from stomach to intestines during food digestion was found by comparison of complex formation constants as determined electrochemically for increasing pH to increase calcium binding (i) by a factor of around 6 for the neutral amino acids, (ii) by a factor of around 4 for anions of the acidic amino acids aspartic and glutamic acid, and (iii) by a factor of around 5.5 for basic amino acids. Optimized structures of the 1:1 complexes and ΔHbinding for calcium binding as calculated by density functional theory (DFT) confirmed in all complexes a stronger calcium binding and shorter calcium-oxygen bond length in the deprotonated form. In addition, the stronger calcium binding was also accompanied by a binding site shift from carboxylate binding to chelation by α-amino group and carboxylate oxygen for leucine, aspartate, glutamate, alanine, and asparagine. For binary amino acid mixtures, the calcium-binding constant was close to the predicted geometric mean of the individual amino acid binding constants indicating separate binding of calcium to two amino acids when present together in solution. At high pH, corresponding to conditions for calcium absorption, the binding affinity increased in the order Lys amino acid mixtures.

  4. Calcium, essential for health (United States)

    Martínez de Victoria, Emilio


    Calcium (Ca) is the most abundant mineral element in our body. It accounts for about 2% of body weight. The functions of calcium are: a) functions skeletal and b) regulatory functions. Bone consists of a protein matrix that mineralizes mainly with calcium (the most abundant), phosphate and magnesium, for it is essential an adequate dietary intake of Ca, phosphorus and vitamin D. The ionic Ca (Ca2+) is essential to maintain and / or perform different specialized functions of, virtually, all body cells cellular. Because of its important functions Ca2+ must be closely regulated, keeping plasma concentrations within narrow ranges. For this reason there is an accurate response against hypocalcemia or hypercalcemia in which the parathormone, calcitriol, calcitonin and vitamin K are involved. Ca intakes in the Spanish population are low in a significant percentage of the older adult’s population, especially in women. The main source of Ca in the diet is milk and milk derivatives. Green leafy vegetables, fruits and legumes can be important sources of Ca in a Mediterranean dietary pattern. The bioavailability of dietary Ca depends on physiological and dietary factors. Physiological include age, physiological status (gestation and lactation) Ca and vitamin D status and disease. Several studies relate Ca intake in the diet and various diseases, such as osteoporosis, cancer, cardiovascular disease and obesity.

  5. Models of calcium signalling

    CERN Document Server

    Dupont, Geneviève; Kirk, Vivien; Sneyd, James


    This book discusses the ways in which mathematical, computational, and modelling methods can be used to help understand the dynamics of intracellular calcium. The concentration of free intracellular calcium is vital for controlling a wide range of cellular processes, and is thus of great physiological importance. However, because of the complex ways in which the calcium concentration varies, it is also of great mathematical interest.This book presents the general modelling theory as well as a large number of specific case examples, to show how mathematical modelling can interact with experimental approaches, in an interdisciplinary and multifaceted approach to the study of an important physiological control mechanism. Geneviève Dupont is FNRS Research Director at the Unit of Theoretical Chronobiology of the Université Libre de Bruxelles;Martin Falcke is head of the Mathematical Cell Physiology group at the Max Delbrück Center for Molecular Medicine, Berlin;Vivien Kirk is an Associate Professor in the Depar...

  6. Intracellular calcium dynamics in cortical microglia responding to focal laser injury in the PC::G5-tdT reporter mouse

    Directory of Open Access Journals (Sweden)

    Amir ePozner


    Full Text Available Microglia, the resident immune cells of the brain parenchyma, are highly responsive to tissue injury. Following cell damage, microglial processes redirect their motility from randomly scouting the extracellular space to specifically reaching toward the compromised tissue. While the cell morphology aspects of this defense mechanism have been characterized, the intracellular events underlying these responses remain largely unknown. Specifically, the role of intracellular Ca2+ dynamics has not been systematically investigated in acutely activated microglia due to technical difficulty. Here we used live two-photon imaging of the mouse cortex ubiquitously expressing the genetically encoded Ca2+ indicator GCaMP5G and fluorescent marker tdTomato in central nervous system microglia. We found that spontaneous Ca2+ transients in microglial somas and processes were generally low (only 4% of all microglia showing transients within 20 min, but baseline activity increased about 8-fold when the animals were treated with LPS 12 h before imaging. When challenged with focal laser injury, an additional surge in Ca2+ activity was observed in the somas and protruding processes. Notably, coherent and simultaneous Ca2+ rises in multiple microglial cells were occasionally detected in LPS-treated animals. We show that Ca2+ transients were predominantly mediated via purinergic receptors. This work demonstrates the usefulness of genetically encoded Ca2+ indicators for investigation of microglial physiology.

  7. Calcium channel blockers in cardiovascular pharmacotherapy. (United States)

    Godfraind, Theophile


    This paper summarizes the pharmacological properties of calcium channel blockers (CCBs), their established therapeutic uses for cardiovascular disorders and the current improvement of their clinical effects through drug combinations. Their identification resulted from study of small molecules including coronary dilators, which were named calcium antagonists. Further experiments showed that they reduced contraction of arteries by inhibiting calcium entry and by interacting with binding sites identified on voltage-dependent calcium channels. This led to the denomination calcium channel blockers. In short-term studies, by decreasing total peripheral resistance, CCBs lower arterial pressure. By unloading the heart and increasing coronary blood flow, CCBs improve myocardial oxygenation. In long-term treatment, the decrease in blood pressure is more pronounced in hypertensive than in normotensive patients. A controversy on the safety of CCBs ended after a large antihypertensive trial (ALLHAT) sponsored by the National Heart, Lung, and Blood Institute. There are two main types of CCBs: dihydopyridine and non-dihydropyridine; the first type is vascular selective. Dihydropyrines are indicated for hypertension, chronic, stable and vasospastic angina. Non-dihydropyridines have the same indications plus antiarrythmic effects in atrial fibrillation or flutter and paroxysmal supraventricular tachycardia. In addition, CCBs reduced newly formed coronary lesions in atherosclerosis. In order to reach recommended blood pressure goals, there is a recent therapeutic move by combination of CCBs with other antihypertensive agents particularly with inhibitors acting at the level of the renin-angiotensin system. They are also combined with statins. Prevention of dementia has been reported in hypertensive patients treated with nitrendipine, opening a way for further studies on CCBs' beneficial effect in cognitive deterioration associated with aging.

  8. Calcium signalling and calcium channels: evolution and general principles. (United States)

    Verkhratsky, Alexei; Parpura, Vladimir


    Calcium as a divalent cation was selected early in evolution as a signaling molecule to be used by both prokaryotes and eukaryotes. Its low cytosolic concentration likely reflects the initial concentration of this ion in the primordial soup/ocean as unicellular organisms were formed. As the concentration of calcium in the ocean subsequently increased, so did the diversity of homeostatic molecules handling calcium. This includes the plasma membrane channels that allowed the calcium entry, as well as extrusion mechanisms, i.e., exchangers and pumps. Further diversification occurred with the evolution of intracellular organelles, in particular the endoplasmic reticulum and mitochondria, which also contain channels, exchanger(s) and pumps to handle the homeostasis of calcium ions. Calcium signalling system, based around coordinated interactions of the above molecular entities, can be activated by the opening of voltage-gated channels, neurotransmitters, second messengers and/or mechanical stimulation, and as such is all-pervading pathway in physiology and pathophysiology of organisms.

  9. Elemental calcium intake associated with calcium acetate/calcium carbonate in the treatment of hyperphosphatemia (United States)

    Wilson, Rosamund J; Copley, J Brian


    Background Calcium-based and non-calcium-based phosphate binders have similar efficacy in the treatment of hyperphosphatemia; however, calcium-based binders may be associated with hypercalcemia, vascular calcification, and adynamic bone disease. Scope A post hoc analysis was carried out of data from a 16-week, Phase IV study of patients with end-stage renal disease (ESRD) who switched to lanthanum carbonate monotherapy from baseline calcium acetate/calcium carbonate monotherapy. Of the intent-to-treat population (N=2520), 752 patients with recorded dose data for calcium acetate (n=551)/calcium carbonate (n=201) at baseline and lanthanum carbonate at week 16 were studied. Elemental calcium intake, serum phosphate, corrected serum calcium, and serum intact parathyroid hormone levels were analyzed. Findings Of the 551 patients with calcium acetate dose data, 271 (49.2%) had an elemental calcium intake of at least 1.5 g/day at baseline, and 142 (25.8%) had an intake of at least 2.0 g/day. Mean (95% confidence interval [CI]) serum phosphate levels were 6.1 (5.89, 6.21) mg/dL at baseline and 6.2 (6.04, 6.38) mg/dL at 16 weeks; mean (95% CI) corrected serum calcium levels were 9.3 (9.16, 9.44) mg/dL and 9.2 (9.06, 9.34) mg/dL, respectively. Of the 201 patients with calcium carbonate dose data, 117 (58.2%) had an elemental calcium intake of at least 1.5 g/day, and 76 (37.8%) had an intake of at least 2.0 g/day. Mean (95% CI) serum phosphate levels were 5.8 (5.52, 6.06) mg/dL at baseline and 5.8 (5.53, 6.05) mg/dL at week 16; mean (95% CI) corrected serum calcium levels were 9.7 (9.15, 10.25) mg/dL and 9.2 (9.06, 9.34) mg/dL, respectively. Conclusion Calcium acetate/calcium carbonate phosphate binders, taken to control serum phosphate levels, may result in high levels of elemental calcium intake. This may lead to complications related to calcium balance. PMID:28182142

  10. Targeting Mitochondrial Calcium Handling and Reactive Oxygen Species in Heart Failure. (United States)

    Dietl, Alexander; Maack, Christoph


    In highly prevalent cardiac diseases, new therapeutic approaches are needed. Since the first description of oxidative stress in heart failure, reactive oxygen species (ROS) have been considered as attractive drug targets. Though clinical trials evaluating antioxidant vitamins as ROS-scavenging agents yielded neutral results in patients at cardiovascular risk, the knowledge of ROS as pathophysiological factors has considerably advanced in the past few years and led to novel treatment approaches. Here, we review recent new insights and current strategies in targeting mitochondrial calcium handling and ROS in heart failure. Mitochondria are an important ROS source, and more recently, drug development focused on targeting mitochondria (e.g. by SS-31 or MitoQ). Important advancement has also been made to decipher how the matching of energy supply and demand through calcium (Ca(2+)) handling impacts on mitochondrial ROS production and elimination. This opens novel opportunities to ameliorate mitochondrial dysfunction in heart failure by targeting cytosolic and mitochondrial ion transporters to improve this matching process. According to this approach, highly specific substances as the preclinical CGP-37157, as well as the clinically used ranolazine and empagliflozin, provide promising results on different levels of evidence. Furthermore, the understanding of redox signalling relays, resembled by catalyst-mediated protein oxidation, is about to change former paradigms of ROS signalling. Novel methods, as redox proteomics, allow to precisely analyse key regulatory thiol switches, which may induce adaptive or maladaptive signalling. Additionally, the generation of genetically encoded probes increased the spatial and temporal resolution of ROS imaging and opened a new methodological window to subtle, formerly obscured processes. These novel insights may broaden our understanding of why previous attempts to target oxidative stress have failed, and at the same time provide us

  11. Novel fluo-4 analogs for fluorescent calcium measurements. (United States)

    Martin, Vladimir V; Beierlein, Michael; Morgan, Josh L; Rothe, Anca; Gee, Kyle R


    We report new fluorescent calcium indicators based on fluo-4. Attachment of a carboxamide or methylenecarboxamide moiety to the BAPTA chelator portion of fluo-4 allowed for the attachment of dextrans, protein-reactive moieties, and biotin. In particular, a high affinity fluo-4 dextran conjugate was prepared and shown to be functional in brain slices. All new probes were characterized spectroscopically and exhibited large fluorescence increases upon calcium-binding. The biotinylated version of fluo-4 formed a persistent streptavidin complex which still responded to increasing calcium concentrations with a large fluorescence increase.

  12. Stability of calcium silicate in basic solution

    Institute of Scientific and Technical Information of China (English)

    刘桂华; 李小斌; 彭志宏; 周秋生


    Mixture of CaO and SiO2 was sintered at 1 200 or 1 400 ℃ according to the mole ratio of CaO/SiO2 of 1 or 2, and then calcium silicate was leached in pure caustic or soda solution. The results indicated that calcium silicate exists much more stably in caustic solution than that in soda solution, and CaO*SiO2 is more stable than β-2CaO*SiO2 whether in caustic solution or in soda solution. The increase of sintering temperature favored the stability of calcium silicate in the leaching process. When β-2CaO*SiO2 was leached in soda solution, the increase of leaching temperature and time resulted in decomposing of more calcium silicate. And when β-2CaO*SiO2 was leached in caustic solution at high temperature, much 2CaO*SiO2*H2O but little CaO*SiO2*H2O appeared in slag.

  13. Cadmium induces transcription independently of intracellular calcium mobilization.

    Directory of Open Access Journals (Sweden)

    Brooke E Tvermoes

    Full Text Available BACKGROUND: Exposure to cadmium is associated with human pathologies and altered gene expression. The molecular mechanisms by which cadmium affects transcription remain unclear. It has been proposed that cadmium activates transcription by altering intracellular calcium concentration ([Ca(2+](i and disrupting calcium-mediated intracellular signaling processes. This hypothesis is based on several studies that may be technically problematic; including the use of BAPTA chelators, BAPTA-based fluorescent sensors, and cytotoxic concentrations of metal. METHODOLOGY/PRINCIPAL FINDING: In the present report, the effects of cadmium on [Ca(2+](i under non-cytotoxic and cytotoxic conditions was monitored using the protein-based calcium sensor yellow cameleon (YC3.60, which was stably expressed in HEK293 cells. In HEK293 constitutively expressing YC3.60, this calcium sensor was found to be insensitive to cadmium. Exposing HEK293::YC3.60 cells to non-cytotoxic cadmium concentrations was sufficient to induce transcription of cadmium-responsive genes but did not affect [Ca(2+](i mobilization or increase steady-state mRNA levels of calcium-responsive genes. In contrast, exposure to cytotoxic concentrations of cadmium significantly reduced intracellular calcium stores and altered calcium-responsive gene expression. CONCLUSIONS/SIGNIFICANCE: These data indicate that at low levels, cadmium induces transcription independently of intracellular calcium mobilization. The results also support a model whereby cytotoxic levels of cadmium activate calcium-responsive transcription as a general response to metal-induced intracellular damage and not via a specific mechanism. Thus, the modulation of intracellular calcium may not be a primary mechanism by which cadmium regulates transcription.

  14. Calcium – how and why?

    Indian Academy of Sciences (India)

    J K Jaiswal


    Calcium is among the most commonly used ions, in a multitude of biological functions, so much so that it is impossible to imagine life without calcium. In this article I have attempted to address the question as to how calcium has achieved this status with a brief mention of the history of calcium research in biology. It appears that during the origin and early evolution of life the Ca2+ ion was given a unique opportunity to be used in several biological processes because of its unusual physical and chemical properties.

  15. Calcium Phosphate Biomaterials: An Update

    Institute of Scientific and Technical Information of China (English)


    Current calcium phosphate (CaP) biomaterials for bone repair, substitution, augmentation and regeneration include hydroxyapatite ( HA ) from synthetic or biologic origin, beta-tricalcium phosphate ( β-TCP ) , biphasic calcium phosphate (BCP), and are available as granules, porous blocks, components of composites (CaP/polymer) cements, and as coatings on orthopedic and dental implants. Experimental calcium phosphate biomaterials include CO3- and F-substituted apatites, Mg-and Zn-substituted β-TCP, calcium phosphate glasses. This paper is a brief review of the different types of CaP biomaterials and their properties such as bioactivity, osteoconductivity, osteoinductivity.

  16. Cardiovascular Effects of Calcium Supplements

    Directory of Open Access Journals (Sweden)

    Ian R. Reid


    Full Text Available Calcium supplements reduce bone turnover and slow the rate of bone loss. However, few studies have demonstrated reduced fracture incidence with calcium supplements, and meta-analyses show only a 10% decrease in fractures, which is of borderline statistical and clinical significance. Trials in normal older women and in patients with renal impairment suggest that calcium supplements increase the risk of cardiovascular disease. To further assess their safety, we recently conducted a meta-analysis of trials of calcium supplements, and found a 27%–31% increase in risk of myocardial infarction, and a 12%–20% increase in risk of stroke. These findings are robust because they are based on pre-specified analyses of randomized, placebo-controlled trials and are consistent across the trials. Co-administration of vitamin D with calcium does not lessen these adverse effects. The increased cardiovascular risk with calcium supplements is consistent with epidemiological data relating higher circulating calcium concentrations to cardiovascular disease in normal populations. There are several possible pathophysiological mechanisms for these effects, including effects on vascular calcification, vascular cells, blood coagulation and calcium-sensing receptors. Thus, the non-skeletal risks of calcium supplements appear to outweigh any skeletal benefits, and are they appear to be unnecessary for the efficacy of other osteoporosis treatments.

  17. Calcium measurement methods

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi


    Full Text Available Rightly stressed by prof. Wolfgang Walz in the Preface to the series Neuromethods series, the “careful application of methods is probably the most important step in the process of scientific inquiry”. Thus, I strongly suggest to all those interested in calcium signaling and especially to the new-comers in the hot topic of neuroscience (which has so much space even in science-society debate for its implications in legal issues and in the judge-decision process to take profit from this so well edited book. I am saying this since prof. Verkhratsky and prof. Petersen......

  18. Ionized calcium and cyclic AMP in plasma and urine. Biochemical evaluation in calcium metabolic disease. (United States)

    Thode, J


    Measurement of ionized calcium and cAMP in plasma and urine are used as sensitive parameters for the evaluation of calcium disorders. Ionized calcium is accepted as the biologically active form of calcium in the extracellular fluid, while urine cAMP provides an in vivo receptor assay for the biologically active parathyroid hormone. When urine is included as part of the calcium metabolic investigation it usually requires 24 h urine collection with a variety of different laboratory tests. Ionized calcium and cAMP are described in the literature in terms of several derived quantities, nomenclatures, and units which are rather unsystematic. The author developed reliable techniques and proposed systematic names and symbols and reference values for these quantities. Due to the lack of guidelines for the collection of urines in calcium metabolic evaluation, the author presented a simplified protocol (4 h standardized urine collection). In clinical investigation plasma and urine cAMP have been used to differentiate idiopathic hypoparathyroidism from pseudohypoparathyroidism (PsHP) based on the results of i.v. injection of parathyroid hormone (PTH). Nephrogenous cAMP has also been used for the detection of primary and secondary hyperparathyroidism with a high nosographic sensitivity (90%) (Broadus). The author showed that measurement of cAMP after i.v. PTH was a reliable and sensitive test to establish the diagnosis of PsHP, and that the urinary cAMP was useful for the diagnosis of secondary hyperparathyroidism in patients with jejunoileal bypass, but could not confirm the high nosographic sensitivity for the diagnosis of primary hyperparathyroidism. Further data are needed for proper conclusion. Although pursued vigorously the research into idiopathic stone formation using different protocols has not prevented stone recurrence nor indicated where further progress might be made. For the evaluation of recurrent calcium disease, the author proposed a simplified 4 h

  19. Extracellular calcium sensing and extracellular calcium signaling (United States)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)


    , localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.

  20. Mechano-activated surface modification of calcium carbonate in wet stirred mill and its properties

    Institute of Scientific and Technical Information of China (English)


    Surface modification of calcium carbonate particles using sodium stearate(SDS) as a modification agent incorporated with the simultaneous wet ultra-fine grinding in the laboratory stirred mill was investigated. The physical properties and application properties of modified calcium carbonate were measured and evaluated. The action mechanism between SDS and calcium carbonate in the modification was studied by infrared spectrometry(IR) and X-ray photoelectron energy spectroscopy(XPS). The results indicate that the crushing mechanic force intensity can obviously influence the modification effect of calcium carbonate because of mechano-chemical effect. The hydrophilic surface of calcium carbonate is turned into hydrophobic after modification. The properties of polyethylene(PE) filled by modified calcium carbonate powder is markedly improved. And the adsorption of SDS could occur by chemical reaction with calcium carbonate surface.

  1. Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Timmermann, D B; Lund, T M; Belhage, B


    using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium...... channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively...... in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q-type calcium channel appears...

  2. 21 CFR 172.330 - Calcium pantothenate, calcium chloride double salt. (United States)


    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium pantothenate, calcium chloride double salt..., calcium chloride double salt. The food additive calcium chloride double salt of calcium pantothenate may... information required by the Act, the following: (1) The name of the additive “calcium chloride double salt of...

  3. Calcium channels and migraine. (United States)

    Pietrobon, Daniela


    Missense mutations in CACNA1A, the gene that encodes the pore-forming α1 subunit of human voltage-gated Ca(V)2.1 (P/Q-type) calcium channels, cause a rare form of migraine with aura (familial hemiplegic migraine type 1: FHM1). Migraine is a common disabling brain disorder whose key manifestations are recurrent attacks of unilateral headache that may be preceded by transient neurological aura symptoms. This review, first, briefly summarizes current understanding of the pathophysiological mechanisms that are believed to underlie migraine headache, migraine aura and the onset of a migraine attack, and briefly describes the localization and function of neuronal Ca(V)2.1 channels in the brain regions that have been implicated in migraine pathogenesis. Then, the review describes and discusses i) the functional consequences of FHM1 mutations on the biophysical properties of recombinant human Ca(V)2.1 channels and native Ca(V)2.1 channels in neurons of knockin mouse models carrying the mild R192Q or severe S218L mutations in the orthologous gene, and ii) the functional consequences of these mutations on neurophysiological processes in the cerebral cortex and trigeminovascular system thought to be involved in the pathophysiology of migraine, and the insights into migraine mechanisms obtained from the functional analysis of these processes in FHM1 knockin mice. This article is part of a Special Issue entitled: Calcium channels. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Calcium electroporation in three cell lines; a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille


    BACKGROUND: Electroporation with calcium (calcium electroporation) can induce ATP depletion-associated cellular death. In the clinical setting, the cytotoxic drug bleomycin is currently used with electroporation (electrochemotherapy) for palliative treatment of tumors. Calcium electroporation...... offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium...

  5. Large-Scale Fluorescence Calcium-Imaging Methods for Studies of Long-Term Memory in Behaving Mammals. (United States)

    Jercog, Pablo; Rogerson, Thomas; Schnitzer, Mark J


    During long-term memory formation, cellular and molecular processes reshape how individual neurons respond to specific patterns of synaptic input. It remains poorly understood how such changes impact information processing across networks of mammalian neurons. To observe how networks encode, store, and retrieve information, neuroscientists must track the dynamics of large ensembles of individual cells in behaving animals, over timescales commensurate with long-term memory. Fluorescence Ca(2+)-imaging techniques can monitor hundreds of neurons in behaving mice, opening exciting avenues for studies of learning and memory at the network level. Genetically encoded Ca(2+) indicators allow neurons to be targeted by genetic type or connectivity. Chronic animal preparations permit repeated imaging of neural Ca(2+) dynamics over multiple weeks. Together, these capabilities should enable unprecedented analyses of how ensemble neural codes evolve throughout memory processing and provide new insights into how memories are organized in the brain. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

  6. Effect of nano-calcium-enriched milk on calcium metabolism in ovariectomized rats. (United States)

    Park, Heung-Sik; Ahn, Joungjwa; Kwak, Hae-Soo


    This study was designed to examine the effect of different kinds of calcium enrichment on serum and urine indices of mineral status in ovariectomized rats. Twenty-four 7-week-old Sprague-Dawley female rats were divided into four groups, ovariectomized, and fed diets containing the following: (1) Control, non-Ca-enriched milk; (2) OVX1, calcium carbonate-enriched milk; (3) OVX2, ionized Ca-enriched milk; and (4) OVX3, nano-Ca-enriched milk. After 18 weeks of feeding, the food efficiency ratio in the nano-Ca-fed group was significantly lower compared with those in the Control and OVX2 groups. There was no difference in serum and fecal Ca among the groups. The bone/total alkaline phosphatase ratio was significantly higher in rats fed milk enriched with nano-Ca (59%) and calcium carbonate (62%) than in control (44%) animals. Urinary Ca was the highest in the nano-Ca-enriched group; however, urinary excretions of deoxypyridinoline and hydroxyproline were significantly decreased in the nano-Ca-enriched group. The present results indicate that consumption of nano-Ca-enriched milk resulted in an increase of urinary excretion of calcium and a decrease in deoxypyridinoline and hydroxyproline in ovariectomized rats.

  7. Effect of Indole Butyric Acid on the Transportation of Stored Calcium in Malus hupehensis Rhed. Seedling

    Institute of Scientific and Technical Information of China (English)

    LI Jia; YANG Hong-qiang; YAN Tian-li; SHU Huai-rui


    Calcium (Ca) plays an important role in the metabolism of higher plants. Recently, research on Ca2+ in plants has been focused especially at the cellular and molecular levels. Uptake, transport, and distribution are also very important for Ca to accomplish its function at the whole-plant level. In this experiment, one-year-old apple seedlings (M. hupehensis Rehd.) were investigated to determine the distribution of stored Ca, the different forms of Ca, and Ca2+-ATPase activity after treatment with indole butyric acid (IBA). The results showed that the total Ca measured in mature leaves and Ca2+-ATPase activity in tender leaves were higher compared with those in the control (CK). Calcium nitrate and calcium chloride (ALe-Ca) and calcium phosphate and calcium carbonate (HAC-Ca) decreased in both mature leaves and shoots,whereas water-soluble calcium (H2O-Ca), calcium pectate (NaCl-Ca), and calcium oxalate (HCl-Ca) increased. The percentage of active calcium, calcium pectate, and water-soluble calcium increased, whereas the percentage of calcium phosphate and calcium carbonate decreased. When treated with IBA, calcium fractions and percentage of the different forms of Ca was enhanced in 40 part per million (ppm) IBA compared with 20 ppm IBA and water. The results indicated that IBA increased the percentage of both active calcium (NaCl-Ca and H2O-Ca) in tender shoots and boosted the transportation of stored Ca in plants. IBA promoted Ca2+-ATPase activity and Ca2+ uptake in tender shoots of M. hupehensis. It can improve the total Ca contents and the relative percentage of Ca.

  8. Glucocorticoids specifically enhance L-type calcium current amplitude and affect calcium channel subunit expression in the mouse hippocampus. (United States)

    Chameau, Pascal; Qin, Yongjun; Spijker, Sabine; Smit, August Benjamin; Smit, Guus; Joëls, Marian


    Previous studies have shown that corticosterone enhances whole cell calcium currents in CA1 pyramidal neurons, through a pathway involving binding of glucocorticoid receptor homodimers to the DNA. We examined whether glucocorticoids show selectivity for L- over N-type of calcium currents. Moreover, we addressed the putative gene targets that eventually lead to the enhanced calcium currents. Electrophysiological recordings were performed in nucleated patches that allow excellent voltage control. Calcium currents in these patches almost exclusively involve N- and L-type channels. We found that L- but not N-type calcium currents were largely enhanced after treatment with a high dose of corticosterone sufficient to activate glucocorticoid receptors. Voltage dependency and kinetic properties of the currents were unaffected by the hormone. Nonstationary noise analysis suggests that the increased current is not caused by a larger unitary conductance, but rather to a doubling of the number of functional channels. Quantitative real-time PCR revealed that transcripts of the Ca(v)1 subunits encoding for the N- or L-type calcium channels are not upregulated in the mouse CA1 area; instead, a strong, direct, and consistent upregulation of the beta4 subunit was observed. This indicates that the corticosteroid-induced increase in number of L-type calcium channels is not caused by a simple transcriptional regulation of the pore-forming subunit of the channels.

  9. Vitamin D, Calcium, and Bone Health (United States)

    ... in Balance › Vitamin D, Calcium, and Bone Health Vitamin D, Calcium, and Bone Health March 2012 Download ... also helps keep your bones strong. Why are vitamin D and calcium important to bone health? Vitamin ...

  10. Calcium, vitamin D, and your bones (United States)

    ... page: // Calcium, vitamin D, and your bones To use the sharing ... and maintain strong bones. How Much Calcium and Vitamin D do I Need? Amounts of calcium are ...

  11. Fish bones--a highly available calcium source for growing pigs. (United States)

    Malde, M K; Graff, I E; Siljander-Rasi, H; Venäläinen, E; Julshamn, K; Pedersen, J I; Valaja, J


    In general, there is a lack of scientific documentation of nutritional value of marine by-products. The bone fraction from fish has been regarded as waste. Due to the high mineral content of fish bones, this material can be well suitable as a natural calcium source. In the present study, apparent calcium absorption of different fish bone sources was tested using growing pigs. The experimental diets consisted of boiled salmon frames, or salmon, saithe or cod bones treated with enzymes. Calcium carbonate (CaCO(3)) was used as control. The experimental diets were formulated to contain 0.7% total calcium of which the added calcium source to be tested contributed about 71% (study 1) and 86% (study 2). Except for the calcium and phosphorus sources, the animals received similar basal diets. Apparent calcium digestibility coefficient was calculated using yttrium as indicator (both studies) and was based on complete collection of faeces and urine (study 2). The experimental design was parallel and cross-over in study 1 and study 2, respectively. In study 1, piglets getting salmon bone treated with enzymes had significantly higher calcium absorption than piglets getting boiled fish bone or calcium carbonate. Therefore, in the second study only enzymatically treated fish bones were included. The higher calcium absorption from enzymatically treated salmon bone was also found in study 2, but this time not significant. Calcium from boiled salmon bones in study I, and from enzymatically treated saithe and cod bones in study II were absorbed as well as the calcium carbonate control. The results indicate that fish bones may be a useful and well absorbed calcium source. Due to the high mineral content of the bone fraction, salmon bones can be well suitable as a natural calcium and phosphorus source in, for example, food, feed or as supplement.

  12. Technological testing of calcium carbonate tablets for use in the treatment of renal osteodystrophy. (United States)

    Dal Zotto, M; Ragazzi, E; Realdon, N; Dalla Fini, G


    Samples of calcium carbonate tablets produced by different manufacturers were subjected to various tests in order to evaluate tablet quality parameters, mostly indicative for calcium availability. Indications about tablet suitability for treatment of renal osteodystrophy in uremic patients were also tested. The disintegration test turned out to be the most useful in evaluating calcium carbonate availability from tablets. Samples from several manufacturers varied in their behaviour to disaggregation. The availability of calcium dissolved in gastric fluid and the extent of phosphorus binding appeared to depend on disintegration behaviour.

  13. Calcium signaling mediates cold sensing in insect tissues. (United States)

    Teets, Nicholas M; Yi, Shu-Xia; Lee, Richard E; Denlinger, David L


    The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

  14. Calcium regulates caveolin-1 expression at the transcriptional level

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Li, Yan [Experimental Animal Center, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Liu, Dan; Zhang, Xue-Cheng [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Sato, Toshinori [Department of Biosciences and Informatics, Keio University, Hiyoshi, Yokohama 223-8522 (Japan); Yamagata, Sadako [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China); Yamagata, Tatsuya, E-mail: [Laboratory of Tumor Biology and Glycobiology, Department of Life Sciences, Shenyang Pharmaceutical University, Shenyang 110016, People' s Republic of China (China)


    Highlights: Black-Right-Pointing-Pointer Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. Black-Right-Pointing-Pointer An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. Black-Right-Pointing-Pointer Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. Black-Right-Pointing-Pointer Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca{sup 2+}/calcineurin/NFAT.

  15. Interactive effects of copper and calcium in Daphnia pulex

    Directory of Open Access Journals (Sweden)

    Andrew Liorti


    Full Text Available Many freshwater habitats around the world suffered dramatic water chemistry changes over the last century mostly due to anthropogenic activities, including an overall reduction in pH due to high sulfur emissions and unsustainable forestry practices. One consequence of this change in water chemistry is a drop in available calcium concentration, which creates problems for aquatic organisms that rely on dissolved calcium to build their exo- or endoskeletons and reinforce their carapace during regular molts. Daphnia populations in shield lakes in northern Ontario are also exposed to other stressors, including copper, which persists at high concentrations in many of these freshwater lakes and ponds due to mining and other human activities. Copper toxicity on animals is influenced by the availability of other competing ions, such as calcium. Using our newly developed high throughput toxicity screening system, we show that mortality of Daphnia pulex increases with exposure to low calcium (0.05 mg L-1 and high copper (300 µg L-1. When these two stressors were combined, we found that copper was less toxic at high calcium concentrations, indicating a protective effect of calcium against copper toxicity. We then established basic calcium uptake kinetics in D. pulex using radioactive tracer 45Ca and provide evidence that copper, at environmentally relevant concentrations, competes with calcium uptake based on Km and Vmax. Our data show that both calcium decline and copper increase in aquatic ecosystems may negatively impact natural Daphnia populations, and that interactions between these two metals may occur in natural environments that result in fitness consequences for zooplankton. 

  16. Mammary-Specific Ablation of the Calcium-Sensing Receptor During Lactation Alters Maternal Calcium Metabolism, Milk Calcium Transport, and Neonatal Calcium Accrual


    Mamillapalli, Ramanaiah; VanHouten, Joshua; Dann, Pamela; Bikle, Daniel; Chang, Wenhan; Brown, Edward; Wysolmerski, John


    To meet the demands for milk calcium, the lactating mother adjusts systemic calcium and bone metabolism by increasing dietary calcium intake, increasing bone resorption, and reducing renal calcium excretion. As part of this adaptation, the lactating mammary gland secretes PTHrP into the maternal circulation to increase bone turnover and mobilize skeletal calcium stores. Previous data have suggested that, during lactation, the breast relies on the calcium-sensing receptor (CaSR) to coordinate ...

  17. Vitamin D and Intestinal Calcium Absorption


    Christakos, Sylvia; Dhawan, Puneet; Porta, Angela; Mady, Leila J.; Seth, Tanya


    The principal function of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. Calcium is absorbed by both an active transcellular pathway, which is energy dependent, and by a passive paracellular pathway through tight junctions. 1,25Dihydroxyvitamin D3 (1,25(OH)2D3) the hormonally active form of vitamin D, through its genomic actions, is the major stimulator of active intestinal calcium absorption which involves calcium influx, translocation of calcium throu...

  18. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs). (United States)

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina


    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response.

  19. Calmodulin activation by calcium transients in the postsynaptic density of dendritic spines.

    Directory of Open Access Journals (Sweden)

    Daniel X Keller

    Full Text Available The entry of calcium into dendritic spines can trigger a sequence of biochemical reactions that begins with the activation of calmodulin (CaM and ends with long-term changes to synaptic strengths. The degree of activation of CaM can depend on highly local elevations in the concentration of calcium and the duration of transient increases in calcium concentration. Accurate measurement of these local changes in calcium is difficult because the spaces are so small and the numbers of molecules are so low. We have therefore developed a Monte Carlo model of intracellular calcium dynamics within the spine that included calcium binding proteins, calcium transporters and ion channels activated by voltage and glutamate binding. The model reproduced optical recordings using calcium indicator dyes and showed that without the dye the free intracellular calcium concentration transient was much higher than predicted from the fluorescent signal. Excitatory postsynaptic potentials induced large, long-lasting calcium gradients across the postsynaptic density, which activated CaM. When glutamate was released at the synapse 10 ms before an action potential occurred, simulating activity patterns that strengthen hippocampal synapses, the calcium gradient and activation of CaM in the postsynaptic density were much greater than when the order was reversed, a condition that decreases synaptic strengths, suggesting a possible mechanism underlying the induction of long-term changes in synaptic strength. The spatial and temporal mechanisms for selectivity in CaM activation demonstrated here could be used in other signaling pathways.

  20. Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields. (United States)

    Golbach, Lieke A; Philippi, John G M; Cuppen, Jan J M; Savelkoul, Huub F J; Verburg-van Kemenade, B M Lidy


    We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30 min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50 Hz sine wave) at three magnetic flux densities (5 μT, 300 μT, and 500 μT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50 Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro.

  1. Association of serum calcium levels with infarct size in acute ischemic stroke: Observations from Northeast India

    Directory of Open Access Journals (Sweden)

    Meghna Borah


    Full Text Available Background: Calcium is known to be major mediator in ischemic neuronal cell death. Recent studies have shown that elevated serum calcium levels at admission in patients with stroke have been associated with less severe clinical deficits and with better outcomes. Aim: The aim of this to determine the correlation between serum calcium (total, corrected, and ionized and infarct size (IS in patients with acute ischemic stroke. Materials and Methods: Data were collected from 61 patients presenting with acute ischemic stroke from May 2015 to April 2016 at a tertiary care institute in Northeast India. Only patients aged ≥40 years and diagnosed as having acute ischemic cerebrovascular stroke with clinical examination and confirmed by a computed tomography scan were included in the study. Serum calcium levels (total, albumin corrected, and ionized were collapsed into quartiles, and these quartile versions were used for calculating correlation. Pearson's correlation coefficient was used for comparing calcium levels with IS. Results: Total calcium, albumin-corrected calcium, and ionized calcium had a statistically significant negative correlation with IS with r = −0.578, −0.5396, and −0.5335, respectively. Total and ionized calcium showed a significant negative correlation with IS across all four quartiles. Albumin-corrected calcium levels showed a significant negative correlation with IS only across the lowest and highest quartiles. Conclusion: The findings in our study suggest that serum calcium can be used as a prognostic indicator in ischemic stroke as its levels directly correlates with the IS.

  2. Calcium Activation Profile In Electrically Stimulated Intact Rat Heart Cells (United States)

    Geerts, Hugo; Nuydens, Rony; Ver Donck, Luc; Nuyens, Roger; De Brabander, Marc; Borgers, Marcel


    Recent advances in fluorescent probe technology and image processing equipment have made available the measurement of calcium in living systems on a real-time basis. We present the use of the calcium indicator Fura-2 in intact normally stimulated rat heart cells for the spatial and dynamic measurement of the calcium excitation profile. After electric stimulation (1 Hz), the activation proceeds from the center of the myocyte toward the periphery. Within two frame times (80 ms), the whole cell is activated. The activation is slightly faster in the center of the cell than in the periphery. The mean recovery time is 200-400 ms. There is no difference along the cell's long axis. The effect of a beta-agonist and of a calcium antagonist is described.

  3. Injectable bioactive calcium-magnesium phosphate cement for bone regeneration. (United States)

    Wu, Fan; Su, Jiacan; Wei, Jie; Guo, Han; Liu, Changsheng


    Novel injectable and degradable calcium-magnesium phosphate cement (CMPC) with rapid-setting characteristic was developed by the introduction of magnesium phosphate cement (MPC) into calcium phosphate cement (CPC). The calcium-magnesium phosphate cement prepared under the optimum P/L ratio exhibited good injectability and desired workability. It could set within 10 min at 37 degrees C in 100% relative humidity and the compressive strength could reach 47 MPa after setting for 48 h, indicating that the prepared cement has relatively high initial mechanical strength. The results of in vitro degradation experiments demonstrated the good degradability of the injectable CMPC, and its degradation rate occurred significantly faster than that of pure CPC in simulated body fluid (SBF) solution. It can be concluded that the novel injectable calcium-magnesium phosphate cement is highly promising for a wide variety of clinical applications, especially for the development of minimally invasive techniques.

  4. Physicochemical properties of calcium polycarbophil, a water-absorbing polymer. (United States)

    Yamada, T; Kitayama, M; Yamazaki, M; Nagata, O; Tamaj, I; Tsuji, A


    The physicochemical properties of calcium polycarbophil were examined. Calcium polycarbophil was decalcified rapidly under acidic conditions, affording polycarbophil. Polycarbophil absorbed about 10 times its own weight of water under acidic conditions, but the swelling ratio markedly increased at above pH 4.0 and reached 70 times the initial weight under neutral conditions. The swelling of polycarbophil was not affected by non-ionic osmolarity, but was affected by ionic strength, showing a decrease with increase of ionic strength. Monovalent metal ions such as sodium and potassium ions in gastrointestinal fluid did not reduce the equilibrium swelling of polycarbophil, but divalent ions such as calcium and magnesium ions did. However, calcium ion only slightly reduced the equilibrium swelling under sodium-rich conditions. The viscosity (as an indicator of fluidity) of polycarbophil was larger than that of CMC-Na at every shear rate and polymer content examined.

  5. Allopregnanolone-induced rise in intracellular calcium in embryonic hippocampal neurons parallels their proliferative potential

    Directory of Open Access Journals (Sweden)

    Brinton Roberta


    Full Text Available Abstract Background Factors that regulate intracellular calcium concentration are known to play a critical role in brain function and neural development, including neural plasticity and neurogenesis. We previously demonstrated that the neurosteroid allopregnanolone (APα; 5α-pregnan-3α-ol-20-one promotes neural progenitor proliferation in vitro in cultures of rodent hippocampal and human cortical neural progenitors, and in vivo in triple transgenic Alzheimer's disease mice dentate gyrus. We also found that APα-induced proliferation of neural progenitors is abolished by a calcium channel blocker, nifedipine, indicating a calcium dependent mechanism for the proliferation. Methods In the present study, we investigated the effect of APα on the regulation of intracellular calcium concentration in E18 rat hippocampal neurons using ratiometric Fura2-AM imaging. Results Results indicate that APα rapidly increased intracellular calcium concentration in a dose-dependent and developmentally regulated manner, with an EC50 of 110 ± 15 nM and a maximal response occurring at three days in vitro. The stereoisomers 3β-hydroxy-5α-hydroxy-pregnan-20-one, and 3β-hydroxy-5β-hydroxy-pregnan-20-one, as well as progesterone, were without significant effect. APα-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker La3+, or the L-type calcium channel blocker nifedipine. Furthermore, the GABAA receptor blockers bicuculline and picrotoxin abolished APα-induced intracellular calcium concentration rise. Conclusion Collectively, these data indicate that APα promotes a rapid, dose-dependent, stereo-specific, and developmentally regulated increase of intracellular calcium concentration in rat embryonic hippocampal neurons via a mechanism that requires both the GABAA receptor and L-type calcium channel. These data suggest that AP

  6. 21 CFR 184.1191 - Calcium carbonate. (United States)


    ... HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of... soda process”; (2) By precipitation of calcium carbonate from calcium hydroxide in the...

  7. Evolution of the Calcium Paradigm: The Relation between Vitamin D, Serum Calcium and Calcium Absorption

    Directory of Open Access Journals (Sweden)

    Borje E. Christopher Nordin


    Full Text Available Osteoporosis is the index disease for calcium deficiency, just as rickets/osteomalacia is the index disease for vitamin D deficiency, but there is considerable overlap between them. The common explanation for this overlap is that hypovitaminosis D causes malabsorption of calcium which then causes secondary hyperparathyroidism and is effectively the same thing as calcium deficiency. This paradigm is incorrect. Hypovitaminosis D causes secondary hyperparathyroidism at serum calcidiol levels lower than 60 nmol/L long before it causes malabsorption of calcium because serum calcitriol (which controls calcium absorption is maintained until serum calcidiol falls below 20 nmol/L. This secondary hyperparathyroidism, probably due to loss of a “calcaemic” action of vitamin D on bone first described in 1957, destroys bone and explains why vitamin D insufficiency is a risk factor for osteoporosis. Vitamin D thus plays a central role in the maintenance of the serum (ionised calcium, which is more important to the organism than the preservation of the skeleton. Bone is sacrificed when absorbed dietary calcium does not match excretion through the skin, kidneys and bowel which is why calcium deficiency causes osteoporosis in experimental animals and, by implication, in humans.

  8. Limestone reaction in calcium aluminate cement–calcium sulfate systems

    Energy Technology Data Exchange (ETDEWEB)

    Bizzozero, Julien, E-mail:; Scrivener, Karen L.


    This paper reports a study of ternary blends composed of calcium aluminate cement, calcium sulfate hemihydrate and limestone. Compressive strength tests and hydration kinetics were studied as a function of limestone and calcium sulfate content. The phase evolution and the total porosity were followed and compared to thermodynamic simulation to understand the reactions involved and the effect of limestone on these binders. The reaction of limestone leads to the formation of hemicarboaluminate and monocarboaluminate. Increasing the ratio between sulfate and aluminate decreases the extent of limestone reaction.

  9. Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging (United States)

    Oscar, Breland G.; Liu, Weimin; Zhao, Yongxin; Tang, Longteng; Wang, Yanli; Campbell, Robert E.; Fang, Chong


    Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca2+) sensing. This study reveals that, in the absence of Ca2+, the dominant skeletal motion is a ∼170 cm−1 phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ∼30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca2+ binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca2+ in physiologically relevant environments. PMID:24987121

  10. LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

    Directory of Open Access Journals (Sweden)

    Seok-Kyu Kwon


    Full Text Available Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance.

  11. LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons (United States)

    Kwon, Seok-Kyu; Sando, Richard; Maximov, Anton; Polleux, Franck


    Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU)-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance. PMID:27429220

  12. Pressure effects on the interactions of the sarcoplasmic reticulum calcium transport enzyme with calcium and dinitrophenyl phosphate. (United States)

    Hasselbach, W


    The effect of hydrostatic pressure on the calcium-dependent hydrolysis of dinitrophenyl phosphate by the sarcoplasmic calcium transport enzyme has been studied. The magnesium dinitrophenyl phosphate complex is the true substrate of the enzyme (K = 7000 M-1) by which it is hydrolyzed at 20 degrees C with a turnover rate of 4 s-1. Activation by calcium ions occurs between 0.1 and 1 microM as observed for ATP hydrolysis. The activation volume of the enzyme saturated with both ligands exhibits pronounced pressure-dependence, rising from 25 ml/mol at atmospheric pressure to 80 ml/mol at 100 MPa. The apparent binding volumes for magnesium dinitrophenyl phosphate and calcium are likewise pressure-dependent. The volume changes connected with the binding of magnesium dinitrophenyl phosphate is quite small approaching zero at 100 MPa. The apparent binding volume for calcium greatly increases with pressure from 35 ml/mol at atmospheric pressure to 150 ml/mol at 70 MPa. A nearly constant binding volume of approximately 40 ml/mol results if the effect of pressure on the respective rate constants that contribute to the apparent binding constant, is taken into account. The pressure-dependence of enzyme activity at subsaturating calcium concentrations yields an activation volume of 250 ml/mol related to the rate of calcium binding indicating the occurrence of a transient large volume expansion of the enzyme complex. The volume changes observed for the calcium-dependent interaction of the enzyme with magnesium dinitrophenyl phosphate well agree with that found for magnesium p-nitrophenyl phosphate (W. Hasselbach and L. Stephan,Z. Naturforsch. 42 c, 641-652 (1987)) indicating that the found volume changes are intrinsic properties of the transport enzyme, independent of the respective energy donor.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

    Institute of Scientific and Technical Information of China (English)

    Nasrin NFJATBAKHSH; Zhong-ping FENG


    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

  14. Hypercalcaemia of malignancy: evidence for a nonparathyroid humoral agent with an effect on renal tubular handling of calcium. (United States)

    Ralston, S H; Fogelman, I; Gardner, M D; Dryburgh, F J; Cowan, R A; Boyle, I T


    The renal handling of calcium was examined in 31 patients with hypercalcaemia of malignancy. Results were compared with those from patients with primary hyperparathyroidism, and normal controls rendered hypercalcaemic by calcium infusion. On relating the urinary calcium excretion indices to serum calcium values, inappropriately low rates of urinary calcium excretion were generally found in patients with malignancy associated hypercalcaemia. Further, the pattern of urinary calcium excretion in these subjects was similar to that found in patients with primary hyperparathyroidism. These observations suggest that, in many solid tumours, the development of hypercalcaemia may be attributable to a humoral mediator with a parathyroid hormone-like effect on renal tubular calcium reabsorption. The relatively frequent occurrence of hypercalcaemia in malignant disease thus may be partially explained by the presence of this humoral agent, which may impair the renal excretion of an increase in filtered calcium load, whether due to bone metastases, or humorally mediated osteolysis.

  15. Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells. (United States)

    Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S


    TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner.

  16. Comparative impact of voltage-gated calcium channels and NMDA receptors on mitochondria-mediated neuronal injury. (United States)

    Stanika, Ruslan I; Villanueva, Idalis; Kazanina, Galina; Andrews, S Brian; Pivovarova, Natalia B


    Glutamate excitotoxicity, a major component of many neurodegenerative disorders, is characterized by excessive calcium influx selectively through NMDARs. However, there is a substantial uncertainty concerning why other known routes of significant calcium entry, in particular, VGCCs, are not similarly toxic. Here, we report that in the majority of neurons in rat hippocampal and cortical cultures, maximal L-type VGCC activation induces much lower calcium loading than toxic NMDAR activation. Consequently, few depolarization-activated neurons exhibit calcium deregulation and cell death. Activation of alternative routes of calcium entry induced neuronal death in proportion to the degree of calcium loading. In a small subset of neurons, depolarization evoked stronger calcium elevations, approaching those induced by toxic NMDA. These neurons were characterized by elevated expression of VGCCs and enhanced voltage-gated calcium currents, mitochondrial dysfunction and cell death. Preventing VGCC-dependent mitochondrial calcium loading resulted in stronger cytoplasmic calcium elevations, whereas inhibiting mitochondrial calcium clearance accelerated mitochondrial depolarization. Both observations further implicate mitochondrial dysfunction in VGCC-mediated cell death. Results indicate that neuronal vulnerability tracks the extent of calcium loading but does not appear to depend explicitly on the route of calcium entry.

  17. Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention. (United States)

    Kristian, Tibor; Pivovarova, Natalia B; Fiskum, Gary; Andrews, S Brian


    Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.

  18. Fibroblast-like synoviocytes induce calcium mineral formation and deposition. (United States)

    Sun, Yubo; Mauerhan, David R; Franklin, Atiya M; Zinchenko, Natalia; Norton, Harry James; Hanley, Edward N; Gruber, Helen E


    Calcium crystals are present in the synovial fluid of 65%-100% patients with osteoarthritis (OA) and 20%-39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  19. Fibroblast-Like Synoviocytes Induce Calcium Mineral Formation and Deposition

    Directory of Open Access Journals (Sweden)

    Yubo Sun


    Full Text Available Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA and 20%–39% patients with rheumatoid arthritis (RA. This study sought to investigate the role of fibroblast-like synoviocytes (FLSs in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s. The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  20. Simultaneous electrophysiological recording and calcium imaging of suprachiasmatic nucleus neurons. (United States)

    Irwin, Robert P; Allen, Charles N


    Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca(2+) concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

  1. 21 CFR 184.1187 - Calcium alginate. (United States)


    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium alginate. 184.1187 Section 184.1187 Food... Specific Substances Affirmed as GRAS § 184.1187 Calcium alginate. (a) Calcium alginate (CAS Reg. No. 9005.... Calcium alginate is prepared by the neutralization of purified alginic acid with appropriate pH...

  2. Adsorption of Potassium and Calcium Ions by Variable Charge Soils

    Institute of Scientific and Technical Information of China (English)



    Interactions of potassium and calcium ions with four typical variable charge soils in South China were examined by measuring pK-0.5pCa value with a potassium ion-selective electrode and a calcium ion-selective electrode,and pK value with a potassium ion-selective electrode.The results showed that adsorption of potassium and calcium ions increased with soil suspension pH,and the tendency of the pK-0.5pCa value changing with pH differed with respect to pH range and potassium to calcium ratio.Adsorption of equal amount of calcium and potassium ions led to release of an identical number of protons,suggesting similar adsorption characteristics of these two ions when adsorbed by variable charge soils.Compared with red soil,latosol and lateritic red soil had higher adsorption selectivities for calcium ion.The red soil had a greater affinity for potassium ion than that for calcium ion at low concentration,which seems to result from its possession of 2:1 type minerals,such as vermiculite and mica with a high affinity for potassium ion.The results indicated that adsorption of potassium and calcium ions by the variable charge soils was chiefly caused by the electrostatic attraction between the cations and the soil surfaces.Moreover,it was found that sulfate could affect the adsorption by changing soil surface properties and by forming ion-pair.

  3. Scientific Opinion on the Tolerable Upper Intake Level of calcium

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Dietetic Products, Nutrition and Allergies


    Full Text Available

    Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies was asked to re-evaluate the safety in use of calcium. The Panel was requested to consider if the Tolerable Upper Intake Level (UL for calcium established by the SCF in 2003 (2,500 mg/day for adults, including pregnant and lactating women, which was based on different intervention studies of long duration in which total daily calcium intakes of 2,500 mg from both diet and supplements were tolerated without adverse effects, needed to be changed on the basis of new available evidence. A number of placebo controlled human intervention studies in adults published since then also showed that total daily calcium intakes of 2,500 mg from both diet and supplements are tolerated without adverse effects. The Panel considers that no relationship has been established between long-term calcium intakes from diet and supplements and increased risk of nephrolithiasis, cardiovascular disease or prostate cancer. No new data have become available which would require a revision of the UL for calcium for adults, including pregnant and lactating women, of 2,500 mg. No new data have become available which would allow the setting of a UL for infants, children or adolescents. Data from European populations indicate that intakes of calcium in high consumers among adult males can be close to the UL. Although available data do not allow the setting of a UL for infants, children or adolescents, no risk has been identified with highest current levels of calcium intake in these age groups.

  4. Calcium phosphates for biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Canillas, M.; Pena, P.; Aza, A.H. de; Rodriguez, M.A.


    The history of calcium phosphates in the medicine field starts in 1769 when the first evidence of its existence in the bone tissue is discovered. Since then, the interest for calcium phosphates has increased among the scientific community. Their study has been developed in parallel with new advances in materials sciences, medicine or tissue engineering areas. Bone tissue engineering is the field where calcium phosphates have had a great importance. While the first bioceramics are selected according to bioinert, biocompatibility and mechanical properties with the aim to replace bone tissue damaged, calcium phosphates open the way to the bone tissue regeneration challenge. Nowadays, they are present in the majority of commercial products directed to repair or regenerate damaged bone tissue. Finally, in the last few decades, they have been suggested and studied as drug delivering devices and as vehicles of DNA and RNA for the future generation therapies. (Author)

  5. Calcium-sensing beyond neurotransmitters

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Han, Weiping


    synaptotagmins are located in brain and endocrine cells, and some of these synaptotagmins bind to phospholipids and calcium at levels that trigger regulated exocytosis of SVs and LDCVs. This led to the proposed synaptotagmin-calcium-sensor paradigm, that is, members of the synaptotagmin family function...... as calcium sensors for the regulated exocytosis of neurotransmitters, neuropeptides and hormones. Here, we provide an overview of the synaptotagmin family, and review the recent mouse genetic studies aimed at understanding the functions of synaptotagmins in neurotransmission and endocrine-hormone secretion......Neurotransmitters, neuropeptides and hormones are released through the regulated exocytosis of SVs (synaptic vesicles) and LDCVs (large dense-core vesicles), a process that is controlled by calcium. Synaptotagmins are a family of type 1 membrane proteins that share a common domain structure. Most...

  6. Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons. (United States)

    Luo, Fen-Lan; Yang, Nian; He, Chao; Li, Hong-Li; Li, Chao; Chen, Fang; Xiong, Jia-Xiang; Hu, Zhi-An; Zhang, Jun


    Previous studies have revealed that extremely low frequency electromagnetic field (ELF-EMF) exposure affects neuronal dendritic spine density and NMDAR and AMPAR subunit expressions in the entorhinal cortex (EC). Although calcium signaling has a critical role in control of EC neuronal functions, however, it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell recording and calcium imaging, we record the whole-cell inward currents that contain the voltage-gated calcium currents and show that ELF-EMF (50Hz, 1mT or 3mT, lasting 24h) exposure does not influence these currents. Next, we specifically isolate the high-voltage activated (HVA) and low-voltage activated (LVA) calcium channels-induced currents. Similarly, the activation and inactivation characteristics of these membrane calcium channels are also not influenced by ELF-EMF. Importantly, ELF-EMF exposure reduces the maximum amplitude of the high-K(+)-evoked calcium elevation in EC neurons, which is abolished by thapsigargin, a Ca(2+) ATPase inhibitor, to empty the intracellular calcium stores of EC neurons. Together, these findings indicate that ELF-EMF exposure specifically influences the intracellular calcium dynamics of cultural EC neurons via a calcium channel-independent mechanism.

  7. A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis (United States)

    Guatimosim, C; Romano-Silva, M A; Cruz, J S; Beirão, P S L; Kalapothakis, E; Moraes-Santos, T; Cordeiro, M N; Diniz, C R; Gomez, M V; Prado, M A M


    The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. Using ω-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by ω-agatoxin IVA, an antagonist of P/Q calcium channels. ω-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. It is concluded that Tx3-3 potently inhibits ω-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes. PMID:9351520

  8. Ultrastructure of the calcium-sequestering gastrodermal cell in the hydroid Hydractinia symbiolongicarpus (Cnidaria, Hydrozoa). (United States)

    Dandar-Roh, Alicia M; Rogers-Lowery, Constance L; Zellmann, Erhard; Thomas, Mary Beth


    Large, free-floating crystals of calcium carbonate occur in vacuoles of gastrodermal cells of the hydroid Hydractinia symbiolongicarpus. Here, morphological details about the process by which these cells accumulate and sequester calcium are provided by a cytochemical method designed to demonstrate calcium at the ultrastructural level. Electron-dense material presumably indicative of the presence of calcium was EGTA-sensitive and was shown by parallel electron energy loss spectroscopy (EELS) and energy spectroscopic imaging (ESI) to contain calcium. Calcium occurred in only one cell type, the endodermally derived gastrodermal cell. In these cells, the electron-dense material appeared first as a fine precipitate in the cytosol and nucleus and later as larger deposits and aggregates in the vacuole. During the life cycle, gastrodermal cells of the uninduced planula and the planula during metamorphic induction sequestered calcium. In primary polyps and polyps from established colonies, gastrodermal cells sequestered calcium, but the endodermal secretory cells did not. Our observations support the hypothesis that gastrodermal cells function as a physiological sink for calcium that enters the organism in conjunction with calcium-requiring processes such as motility, secretion, and metamorphosis. Copyright 2004 Wiley-Liss, Inc.

  9. Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue. (United States)

    Cameron, Morven; Kékesi, Orsolya; Morley, John W; Tapson, Jonathan; Breen, Paul P; van Schaik, André; Buskila, Yossi


    Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for 24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

  10. Calcium Orthophosphate Cements and Concretes

    Directory of Open Access Journals (Sweden)

    Sergey V. Dorozhkin


    Full Text Available In early 1980s, researchers discovered self-setting calcium orthophosphate cements, which are a bioactive and biodegradable grafting material in the form of a powder and a liquid. Both phases form after mixing a viscous paste that after being implanted, sets and hardens within the body as either a non-stoichiometric calcium deficient hydroxyapatite (CDHA or brushite, sometimes blended with unreacted particles and other phases. As both CDHA and brushite are remarkably biocompartible and bioresorbable (therefore, in vivo they can be replaced with newly forming bone, calcium orthophosphate cements represent a good correction technique for non-weight-bearing bone fractures or defects and appear to be very promising materials for bone grafting applications. Besides, these cements possess an excellent osteoconductivity, molding capabilities and easy manipulation. Furthermore, reinforced cement formulations are available, which in a certain sense might be described as calcium orthophosphate concretes. The concepts established by calcium orthophosphate cement pioneers in the early 1980s were used as a platform to initiate a new generation of bone substitute materials for commercialization. Since then, advances have been made in the composition, performance and manufacturing; several beneficial formulations have already been introduced as a result. Many other compositions are in experimental stages. In this review, an insight into calcium orthophosphate cements and concretes, as excellent biomaterials suitable for both dental and bone grafting application, has been provided.

  11. Calcium affects on vascular endpoints

    Directory of Open Access Journals (Sweden)

    Patel Vaishali B


    Full Text Available Abstract Calcium is one of the most abundant minerals in the body and its metabolism is one of the basic biologic processes in humans. Although historically linked primarily to bone structural development and maintenance, calcium is now recognized as a key component of many physiologic pathways necessary for optimum health including cardiovascular, neurological, endocrine, renal, and gastrointestinal systems. A recent meta-analysis published in August 2011 showed a potential increase in cardiovascular events related to calcium supplementation. The possible mechanism of action of this correlation has not been well elucidated. This topic has generated intense interest due to the widespread use of calcium supplements, particularly among the middle aged and elderly who are at the most risk from cardiac events. Prior studies did not control for potential confounding factors such as the use of statins, aspirin or other medications. These controversial results warrant additional well-designed studies to investigate the relationship between calcium supplementation and cardiovascular outcomes. The purpose of this review is to highlight the current literature in regards to calcium supplementation and cardiovascular health; and to identify areas of future research.

  12. Zinc modulation of calcium activity at the photoreceptor terminal: a calcium imaging study. (United States)

    Anastassov, Ivan; Shen, Wen; Ripps, Harris; Chappell, Richard L


    There is abundant experimental evidence that zinc ions (Zn(2+)) are present in the synaptic vesicles of vertebrate photoreceptors, and that they are co-released with glutamate. Here we show that increasing the concentration of extracellular zinc (2 μM-2 mM) suppresses the entry of calcium into the synaptic terminals of isolated salamander double cones. The resultant dose-dependent curve was fit by an inverse Hill equation having an IC50 of 38 μM, and Hill coefficient of 1.1. Because there is currently no reliable way to measure the concentration of extracellular zinc, it is not known whether the zinc released under normal circumstances is of physiological significance. In an attempt to circumvent this problem we used zinc chelators to reduce the available pool of endogenous zinc. This enabled us to determine how the absence of zinc affected calcium entry. We found that when intra- or extra-cellular zinc was chelated by 250 μM of membrane-permeable TPEN or 500 μM of membrane-impermeable histidine, there was a significant rise in the depolarization-induced intracellular calcium level within photoreceptor terminals. This increase in internal [Ca(2+)] will undoubtedly lead to a concomitant increase in glutamate release. In addition, we found that blocking the L-type calcium channels that are expressed on the synaptic terminals of photoreceptors with 50 μM nicardipine or 100 μM verapamil abolished the effects of zinc chelation. These findings are a good indication that, when released in vivo, the zinc concentration is sufficient to suppress voltage-gated calcium channels, and reduce the rate of glutamate release from photoreceptor terminals.

  13. Calcium and magnesium concentrations in mature human milk: influence of calcium intake, age and socioeconomic level. (United States)

    Vítolo, M R; Valente Soares, L M; Carvalho, E B; Cardoso, C B


    Concentrations of calcium and magnesium were measured in mature milk, collected between 30 and 90 days after childbirth, from a group of 90 mothers between 14 and 39 years of age, exclusively breastfeeding. The group was divided into three sub-groups: low socioeconomic-level adolescents (LSAd), low socioeconomic-level adults (LSA), and high socioeconomic-level adults (HSA). Each mother's nutritional status was determined using the body-mass index (BMI) and her eating habits, obtained on the basis of a 24-h dietary recall. Adolescent and adult mothers in the low socioeconomic-level group had lower average calcium intake (LSAd = 618.4 +/- 555.2 mg and LSA = 679.4 +/- 411.4 mg) than adult mothers in the higher socioeconomic-level group (853.6 +/- 415.5 mg). The average concentration of calcium in the adolescent mothers' milk (LSAd) was significantly lower (5.30 +/- 1.42 mmol Ca/L, P = 0.01) than that of the two adult groups (LSA = 5.82 +/- 1.55 mmol Ca/L and HSA = 6.40 mmol Ca/L). The average magnesium concentrations for all groups did not show significant differences (LSAd = 1.06 +/- 0.18, LSA = 1.16 +/- 0.23 and HSA = 1.11 +/- 0.23 mmol Mg/L, for P= 0.16). These results indicate that magnesium concentrations in mature human milk do not seem to depend on maternal nutritional status. The condition of adolescence, however, associated with lower calcium intake by the mother, resulted in lower calcium concentrations in the milk secreted when compared to that of adult mothers.

  14. Factors affecting calcium balance in Chinese adolescents. (United States)

    Yin, Jing; Zhang, Qian; Liu, Ailing; Du, Weijing; Wang, Xiaoyan; Hu, Xiaoqi; Ma, Guansheng


    Chinese dietary reference intakes (DRIs) for calcium were developed mainly from studies conducted amongst Caucasians, yet a recent review showed that reference calcium intakes for Asians are likely to be different from those of Caucasians (Lee and Jiang, 2008). In order to develop calcium DRIs for Chinese adolescents, it is necessary to explore the characteristics and potential influencing factors of calcium metabolic balance in Chinese adolescents. A total of 80 students (15.1+/-0.8 years) were recruited stratified by gender from a 1-year calcium supplementation study. Subjects were randomly designed to four groups and supplemented with calcium carbonate tablets providing elemental calcium at 63, 354, 660, and 966 mg/day, respectively. Subjects consumed food from a 3-day cycle menu prepared by staff for 10 days. Elemental calcium in samples of foods, feces, and urine was determined in duplicates by inductively coupled plasma atomic emission spectrometry. The total calcium intake ranged from 352 to 1323 mg/day. The calcium apparent absorption efficiency and retention in boys were significantly higher than that in girls (68.7% vs. 46.4%, 480 mg/day vs. 204 mg/day, PCalcium retention increased with calcium intakes, but did not reach a plateau. Calcium absorption efficiency in boys increased with calcium intake up to 665 mg/day, and decreased after that. In girls, calcium absorption efficiency decreased with calcium intake. Calcium absorption efficiency increased within 1 year after first spermatorrhea in boys, but decreased with pubertal development in girls. Sex, calcium intake, age, and pubertal development were the most important determinants of calcium absorption (R(2)=0.508, Pcalcium intake, age, and pubertal development are important factors for calcium retention and absorption during growth, which should be considered for the development of calcium DRIs for Chinese adolescents.

  15. Protein-specific localization of a rhodamine-based calcium-sensor in living cells. (United States)

    Best, Marcel; Porth, Isabel; Hauke, Sebastian; Braun, Felix; Herten, Dirk-Peter; Wombacher, Richard


    A small synthetic calcium sensor that can be site-specifically coupled to proteins in living cells by utilizing the bio-orthogonal HaloTag labeling strategy is presented. We synthesized an iodo-derivatized BAPTA chelator with a tetramethyl rhodamine fluorophore that allows further modification by Sonogashira cross-coupling. The presented calcium sensitive dye shows a 200-fold increase in fluorescence upon calcium binding. The derivatization with an aliphatic linker bearing a terminal haloalkane-function by Sonogashira cross-coupling allows the localization of the calcium sensor to Halo fusion proteins which we successfully demonstrate in in vitro and in vivo experiments. The herein reported highly sensitive tetramethyl rhodamine based calcium indicator, which can be selectively localized to proteins, is a powerful tool to determine changes in calcium levels inside living cells with spatiotemporal resolution.

  16. High concentration of calcium ions in Golgi apparatus

    Institute of Scientific and Technical Information of China (English)



    The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6-NBD-ceramide,and then the single cells were examined by laser scanning confocal microscopy(LSCFM) for subcellular distributions of Ca2+ and the location of Golgi apparatus.In these cells,the intracellular Ca2+ were found to be highly concentrated in the Golgi apparatus.The changes of distribution of cytosolic high Ca2+ region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium,when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin,the fluorescence of the Golgi region decreased far less than that of the cytosol.Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.

  17. Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

    DEFF Research Database (Denmark)

    Petersen, R H; Knudsen, T; Johansen, Torben


    The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat...... and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation...

  18. 43. Calmodulin regulating calcium sensitivity of Na channels

    Directory of Open Access Journals (Sweden)

    R. Vegiraju


    M complexes across the varying calcium concentrations, the overall pattern indicated that there was a one to one stoichiometry between calmodulin and Nav 1.5. More importantly, it indicated calcium sensitivity of the Na channel. With this research, a definitive answer has been drawn regarding the importance of calmodulin in calcium modulation in Na channels. Not only does this have the effect of creating a foundation for further research into the structure and function of Na channels, but it also gives deep insight into fundamental functions of the channel that can play a major role into the creation of drugs to treat the many cardiac diseases associated with dysfunction of the channel.

  19. Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus. (United States)

    Arrizubieta, María Jesús; Toledo-Arana, Alejandro; Amorena, Beatriz; Penadés, José R; Lasa, Iñigo


    Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.

  20. Spatial wavelet analysis of calcium oscillations in developing neurons.

    Directory of Open Access Journals (Sweden)

    Federico Alessandro Ruffinatti

    Full Text Available Calcium signals play a major role in the control of all key stages of neuronal development, and in particular in the growth and orientation of neuritic processes. These signals are characterized by high spatial compartmentalization, a property which has a strong relevance in the different roles of specific neuronal regions in information coding. In this context it is therefore important to understand the structural and functional basis of this spatial compartmentalization, and in particular whether the behavior at each compartment is merely a consequence of its specific geometry or the result of the spatial segregation of specific calcium influx/efflux mechanisms. Here we have developed a novel approach to separate geometrical from functional differences, regardless on the assumptions on the actual mechanisms involved in the generation of calcium signals. First, spatial indices are derived with a wavelet-theoretic approach which define a measure of the oscillations of cytosolic calcium concentration in specific regions of interests (ROIs along a cell, in our case developing chick ciliary ganglion neurons. The resulting spatial profile demonstrates clearly that different ROIs along the neuron are characterized by specific patterns of calcium oscillations. Next we have investigated whether this inhomogeneity is due just to geometrical factors, namely the surface to volume ratio in the different subcompartments (e.g. soma vs. growth cone or it depends on their specific biophysical properties. To this aim correlation functions are computed between the activity indices and the surface/volume ratio along the cell: the data thus obtained are validated by a statistical analysis on a dataset of [Formula: see text] different cells. This analysis shows that whereas in the soma calcium dynamics is highly correlated to the surface/volume ratio, correlations drop in the growth cone-neurite region, suggesting that in this latter case the key factor is the

  1. The potential for calcium depletion in forest ecosystems of southeastern United States: Review and analysis (United States)

    Huntington, Thomas G.


    Biogeochemical mass balance assessments of calcium status in southeastern forests indicate that losses through harvesting and soil leaching often exceed inputs from atmospheric deposition and weathering. Many forest soils of the southeastern United States are particularly sensitive because these soils and the underlying saprolite from which these soils are derived are largely depleted of weatherable calcium. At most of the intensively studied sites in the southeastern United States, it is estimated that calcium depletion has already reduced or will likely reduce exchangeable soil calcium reserves to less than the estimated requirement for a merchantable forest stand in 150 years or less. At most sites, calcium uptake into merchantable wood equals or exceeds soil leaching losses. Chronic atmospheric deposition of sulfate and nitrate and declining atmospheric deposition of calcium are likely to accelerate calcium depletion. The southeastern U.S. regional distribution of soil calcium pools and calcium fluxes (deposition and uptake in merchantable wood) indicates that the depletion status of the intensively studied sites is representative of a substantially larger area. Where weathering inputs are insufficient to replace leaching and uptake losses, there is a potential for a regional problem in forest nutrition over the long term.

  2. The antiatherogenic potential of calcium antagonists. (United States)

    Weinstein, D B


    Atherosclerosis is an arterial disease characterized by focal accumulation of collagen, elastin, lipids, and calcium at sites associated with macrophage infiltration and altered smooth muscle metabolic function. Studies in several types of animal models, especially cholesterol-fed rabbits, have shown that calcium competitors, calcium chelators, anticalcifying agents, and calcium channel blockers can reduce the accumulation of atherogenic lesion components and thus apparently decrease the progression of lesions. Although there are some conflicting data in the animal model studies using calcium channel antagonists, as a result of differences in experimental designs, it is now apparent that several classes of calcium channel blockers inhibit the progression of early arterial lesions induced by cholesterol feeding. The dihydropyridine calcium channel blockers appear to be more potent antiatherosclerotic agents than other classes of calcium channel antagonists. Several mechanisms involving regulation of endothelial cell, smooth muscle cell, and macrophage metabolic functions may be responsible for the calcium channel blocker effects on early lesion progression. For example, recent studies in cell culture model systems suggest that calcium channel blockers may significantly alter activities that regulate lipoprotein-derived cholesterol accumulation by cells. Some of these activities are independent of calcium flux across voltage-operated calcium channels. Thus, calcium channel blockers may reduce the progression of atherogenic lesions by a combination of decreasing calcium accumulation within arterial wall cells and by altering calcium-independent metabolic activities.

  3. Calcium balance in young adults on a vegan and lactovegetarian diet. (United States)

    Kohlenberg-Mueller, Kathrin; Raschka, Ladislav


    For people in Western countries, the vegan diet has the advantage of low energy intake, but the calcium status of this strictly plant-based diet is still unclear. The aim of this study was to determine the calcium balance of individuals on a vegan diet in comparison with a lactovegetarian diet in a short-term investigation. Seven women and one man, ranging in age from 19 to 24 years, received during the first 10 days a vegan diet based on plant foods and calcium-rich mineral water and a lactovegetarian diet during the following 10 days. Portion size was adapted to the subjects' individual energy requirements. Calcium status was assessed by means of calcium intake in food and calcium output in feces and urine as measured by flame atomic absorption spectrophotometry. In addition, deoxypyridinoline was measured in urine as a marker of bone resorption. The results show a significantly smaller daily calcium intake with an average of 843 +/- 140 mg in the vegan versus 1322 +/- 303 mg in the lactovegetarian diet. Apparent calcium absorption rates were calculated as 26% +/- 15% in the vegan and 24% +/- 8% in the lactovegetarian group (NS). The calcium balance was positive both in the vegan diet (119 +/- 113 mg/day) and in the lactovegetarian diet (211 +/- 136 mg/day) (NS). Deoxypyridinoline excretion showed no significant difference between the two diets (105 +/- 31 and 98 +/- 23 nmol/day). The present results indicate that calcium balance and a marker of bone turnover are not affected significantly when calcium is provided either solely by plant foods or by a diet including dairy products, despite the significantly different calcium intake levels in the diets. We conclude that a well-selected vegan diet maintains calcium status, at least for a short-term period.

  4. Mitochondrial calcium uptake. (United States)

    Williams, George S B; Boyman, Liron; Chikando, Aristide C; Khairallah, Ramzi J; Lederer, W J


    Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (P(O)); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.

  5. Dopaminergic regulation of dendritic calcium: fast multisite calcium imaging. (United States)

    Zhou, Wen-Liang; Oikonomou, Katerina D; Short, Shaina M; Antic, Srdjan D


    Optimal dopamine tone is required for the normal cortical function; however it is still unclear how cortical-dopamine-release affects information processing in individual cortical neurons. Thousands of glutamatergic inputs impinge onto elaborate dendritic trees of neocortical pyramidal neurons. In the process of ensuing synaptic integration (information processing), a variety of calcium transients are generated in remote dendritic compartments. In order to understand the cellular mechanisms of dopaminergic modulation it is important to know whether and how dopaminergic signals affect dendritic calcium transients. In this chapter, we describe a relatively inexpensive method for monitoring dendritic calcium fluctuations at multiple loci across the pyramidal dendritic tree, at the same moment of time (simultaneously). The experiments have been designed to measure the amplitude, time course and spatial extent of action potential-associated dendritic calcium transients before and after application of dopaminergic drugs. In the examples provided here the dendritic calcium transients were evoked by triggering the somatic action potentials (backpropagation-evoked), and puffs of exogenous dopamine were applied locally onto selected dendritic branches.

  6. Microscopic imaging of intracellular calcium in live cells using lifetime-based ratiometric measurements of Oregon Green BAPTA-1. (United States)

    Lattarulo, Carli; Thyssen, Diana; Kuchibholta, Kishore V; Hyman, Bradley T; Bacskaiq, Brian J


    Calcium is a ubiquitous intracellular messenger that has important functions in normal neuronal function. The pathology of Alzheimer's disease has been shown to alter calcium homeostasis in neurons and astrocytes. Several calcium dye indicators are available to measure intracellular calcium within cells, including Oregon Green BAPTA-1 (OGB-1). Using fluorescence lifetime imaging microscopy, we adapted this single wavelength calcium dye into a ratiometric dye to allow quantitative imaging of cellular calcium. We used this approach for in vitro calibrations, single-cell microscopy, high-throughput imaging in automated plate readers, and in single cells in the intact living brain. While OGB is a commonly used fluorescent dye for imaging calcium qualitatively, there are distinct advantages to using a ratiometric approach, which allows quantitative determinations of calcium that are independent of dye concentration. Taking advantage of the distinct lifetime contrast of the calcium-free and calcium-bound forms of OGB, we used time-domain lifetime measurements to generate calibration curves for OGB lifetime ratios as a function of calcium concentration. In summary, we demonstrate approaches using commercially available tools to measure calcium concentrations in live cells at multiple scales using lifetime contrast. These approaches are broadly applicable to other fluorescent readouts that exhibit lifetime contrast and serve as powerful alternatives to spectral or intensity readouts in multiplexing experiments.

  7. Formation of calcium complexes by borogluconate in vitro and during calcium borogluconate infusion in sheep. (United States)

    Farningham, D A


    The effect of borogluconate on plasma calcium fractions was studied in vitro and in vivo in sheep. In vitro calcium chloride was more effective in raising ionised plasma calcium than calcium borogluconate. Sodium borate or gluconate added to blood caused only small decreases in blood ionised calcium. However, together, a synergistic reduction in ionised calcium was observed. Following calcium borogluconate infusions into sheep, total plasma calcium rose primarily because of an increase in the unionised ultrafiltrable fraction. Other changes observed following the infusion were hypercalciuria, decreased glomerular filtration rate and acidosis. Sodium borogluconate administered subcutaneously lowered total plasma calcium. This probably resulted from enhanced calcium excretion. It is suggested that since the anionic component of calcium solutions alters the availability and retention of calcium, it is likely to affect clinical efficacy significantly.

  8. Calcium wave of tubuloglomerular feedback. (United States)

    Peti-Peterdi, János


    ATP release from macula densa (MD) cells into the interstitium of the juxtaglomerular (JG) apparatus (JGA) is an integral component of the tubuloglomerular feedback (TGF) mechanism that controls the glomerular filtration rate. Because the cells of the JGA express a number of calcium-coupled purinergic receptors, these studies tested the hypothesis that TGF activation triggers a calcium wave that spreads from the MD toward distant cells of the JGA and glomerulus. Ratiometric calcium imaging of in vitro microperfused isolated JGA-glomerulus complex dissected from rabbits was performed with fluo-4/fura red and confocal fluorescence microscopy. Activation of TGF by increasing tubular flow rate at the MD rapidly produced a significant elevation in intracellular Ca(2+) concentration ([Ca(2+)](i)) in extraglomerular mesangial cells (by 187.6 +/- 45.1 nM) and JG renin granular cells (by 281.4 +/- 66.6 nM). Subsequently, cell-to-cell propagation of the calcium signal at a rate of 12.6 +/- 1.1 microm/s was observed upstream toward proximal segments of the afferent arteriole and adjacent glomeruli, as well as toward intraglomerular elements including the most distant podocytes (5.9 +/- 0.4 microm/s). The same calcium wave was observed in nonperfusing glomeruli, causing vasoconstriction and contractions of the glomerular tuft. Gap junction uncoupling, an ATP scavenger enzyme cocktail, and pharmacological inhibition of P(2) purinergic receptors, but not adenosine A(1) receptor blockade, abolished the changes in [Ca(2+)](i) and propagation of the calcium wave. These studies provided evidence that both gap junctional communication and extracellular ATP are integral components of the TGF calcium wave.

  9. [Calcium metabolism characteristics in microgravity]. (United States)

    Grigor'ev, A I; Larina, I M; Morukov, B V


    The results of research of calcium exchange parameters at cosmonauts taken part in long space flights (SF) onboard of orbital stations "SALUT" and "MIR" within 1978-1998 were generalized. The analysis of data received during observation of 44 cosmonauts (18 of them have taken part in long SF twice) was done. The observation was carried out before and after SF by duration 30-438 days. The content of a total calcium in blood serum was increased basically by the increase of its ionized fraction after flights of moderate (3-6 months) and large duration (6-14 months) along with the significant increase of PTH and decrease of calcitonin levels. The content of osteocalcin after SF was increased. Three cosmonauts participated in research of calcium kinetics using stable isotopes before, in time and after a 115-day SF. Reduction of intestinal absorption, excretion through a gastrointestinal tract, and increase of calcium excretion with urine were marked in time of SF. In early postflight period a level of intestinal absorption, on the average, was much lower than in SF, and the calcium removal through intestine was increased. Both renal and intestinal excretion of calcium were not normalized in 3.5-4.5 months after end of SF. Increase of resorbtive processes in bone tissues which induced negative bone balance during flight was observed in all test subjects, proceeding from estimations of speed of the basic calcium flows made on the basis of mathematical modeling. The conclusion about decrease in speed of bone tissue remodeling and strengthening of its resorption proves to be true by data of research of biochemical and endocrine markers.

  10. A three-photon microscope with adaptive optics for deep-tissue in vivo structural and functional brain imaging (United States)

    Tao, Xiaodong; Lu, Ju; Lam, Tuwin; Rodriguez, Ramiro; Zuo, Yi; Kubby, Joel


    We developed a three-photon adaptive optics add-on to a commercial two-photon laser scanning microscope. We demonstrated its capability for structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins or calcium indicators deep in the living mouse brain with cellular and subcellular resolution.

  11. Correlating Whole Brain Neural Activity with Behavior in Head-Fixed Larval Zebrafish. (United States)

    Orger, Michael B; Portugues, Ruben


    We present a protocol to combine behavioral recording and imaging using 2-photon laser-scanning microscopy in head-fixed larval zebrafish that express a genetically encoded calcium indicator. The steps involve restraining the larva in agarose, setting up optics that allow projection of a visual stimulus and infrared illumination to monitor behavior, and analysis of the neuronal and behavioral data.

  12. Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes. (United States)

    Kim, H Y; Hanley, M R


    Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.

  13. Calcium supplement: humanity's double-edged sword. (United States)

    Bunyaratavej, Narong; Buranasinsup, Shutipen


    The principle aim of the present study is to investigate the dark side of calcium, pollutions in calcium preparation especially lead (Pb), mercury (Hg) and cadmium (Cd). The collected samples were the different calcium salts in the market and 18 preparations which were classified into 3 groups: Calcium carbonate salts, Chelated calcium and natural-raw calcium. All samples were analyzed for lead, cadmium and mercury by inductively Coupled Plasma Mass Spectrometry (ICP-MS) technique, in house method based on AOAC (2005) 999.10 by ICP-MS. The calcium carbonate and the natural-raw calcium in every sample contained lead at 0.023-0.407 mg/kg of calcium powder. Meanwhile, the natural-raw calcium such as oyster, coral and animal bone showed amount of lead at 0.106-0.384 mg/kg with small amounts of mercury and cadmium. The chelated calcium such as calcium gluconate, calcium lactate and calcium citrate are free of lead.

  14. Mitochondrial response and calcium ion change in apoptotic insect cells induced by SfaMNPV

    Institute of Scientific and Technical Information of China (English)

    XIU Meihong; PENG Jianxin; HONG Huazhu


    Mitochondrial responses and changes of calcium ions in apoptotic insect SL-1 cells induced by Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV) are reported in this paper. By using Rhodamine 123 as a fluorescent labeling probe, flow cytometry analysis and confocal laser scanning microscope observation we observed that the mitochondrial transmembrane potential (△Ψm) began to decrease in SL-1 cells at 4 h post infection and △Ψm reduced continuously with the extension of virus infection. Western blotting indicated that the Bcl-2 level in the mitochondria gradually declined and was down- regulated. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria, which indicated that cytochrome c was released from mitochondria into cytosol. These results suggest that mitochondrion-mediated apoptotic signal transduction pathway exists in apoptotic insect cell induced by SfaMNPV. Cytosolic free calcium ([Ca2+]i) concentration rapidly increased after SfaMNPV infection and the elevated calcium was tested to come partly from extracelllular calcium ion influx. Flow cytometry analysis indicated that the apoptosis in SL-1 cells was not influenced by established cytosolic calcium clamped conditions and the EGTA inhibiting calcium influx. Therefore, neither the elevation of cytosolic calcium ion nor extracellular calcium entry was the inducing factor of apoptosis, which hinted that the depletion of ER Ca2+ store contributed to SL-1 cell apoptosis induced by SfaMNPV.

  15. Effect of calcium and cholecalciferol supplementation on several parameters of calcium status in plasma and urine of captive Asian (Elephas maximus) and African elephants (Loxodonta africana). (United States)

    van Sonsbeek, Gerda R; van der Kolk, Johannes H; van Leeuwen, Johannes P T M; Everts, Hendrik; Marais, Johan; Schaftenaar, Willem


    The aim of the current study was to assess the effect of oral calcium and cholecalciferol supplementation on several parameters of calcium status in plasma and urine of captive Asian (Elephas maximus; n=10) and African elephants (Loxodonta africana; n=6) and to detect potential species differences. Calcium and cholecalciferol supplementation were investigated in a feeding trial using a crossover design consisting of five periods of 28 days each in summer. From days 28-56 (period 2), elephants were fed the Ca-supplemented diet and from days 84-112, elephants were fed the cholecalciferol-supplemented diet (period 4). The control diet was fed during the other periods and was based on their regular ration, and the study was repeated similarly during winter. Periods 1, 3, and 5 were regarded as washout periods. This study revealed species-specific differences with reference to calcium and cholecalciferol supplementation. Asian elephants showed a significant increase in mean plasma total calcium concentration following calcium supplementation during summer, suggesting summer-associated subclinical hypocalcemia in Western Europe. During winter, no effect was seen after oral calcium supplementation, but a significant increase was seen both in mean plasma, total, and ionized calcium concentrations after cholecalciferol supplementation in Asian elephants. In contrast, evidence of subclinical hypocalcemia could be demonstrated neither in summer nor in winter in African elephants, although 28 days of cholecalciferol supplementation during winter reversed the decrease in plasma 1,25(OH)2-cholecalciferol and was followed by a significant increase in mean plasma total calcium concentration. Preliminary findings indicate that the advisable permanent daily intake for calcium in Asian elephants and cholecalciferol in both elephant species at least during winter might be higher than current guidelines. It is strongly recommended to monitor blood calcium concentrations and, if

  16. Fibroblast growth factor-23 negates 1,25(OH)2D3-induced intestinal calcium transport by reducing the transcellular and paracellular calcium fluxes. (United States)

    Khuituan, Pissared; Wongdee, Kannikar; Jantarajit, Walailuk; Suntornsaratoon, Panan; Krishnamra, Nateetip; Charoenphandhu, Narattaphol


    The calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been known to stimulate intestinal calcium transport via both transcellular and paracellular pathways. Recently, we reported that the 1,25(OH)2D3-enhanced calcium transport in the mouse duodenum could be abolished by fibroblast growth factor (FGF)-23, but the targeted calcium transport pathway has been elusive. Herein, the 1,25(OH)2D3-enhanced calcium transport was markedly inhibited by FGF-23 and inhibitors of the basolateral calcium transporters, NCX1 and PMCA1b, suggesting the negative effect of FGF-23 on the transcellular calcium transport. Similar results could be observed in the intestinal epithelium-like Caco-2 monolayer. Although the Arrhenius plot indicated that FGF-23 decreased the potential barrier (e.g., activation energy) of the paracellular calcium movement, FGF-23 was found to modestly decrease the 1,25(OH)2D3-enhanced paracellular calcium transport and calcium permeability. Moreover, FGF-23 affected the 1,25(OH)2D3-induced change in duodenal water permeability as determined by tritiated water, but both 1,25(OH)2D3 and FGF-23 were without effects on the transepithelial fluxes of paracellular markers, (3)H-mannitol and (14)C-polyethylene glycol. It could be concluded that FGF-23 diminished the 1,25(OH)2D3-enhanced calcium absorption through the transcellular and paracellular pathways. Our findings have thus corroborated the presence of a bone-kidney-intestinal axis of FGF-23/vitamin D system in the regulation of calcium homeostasis.

  17. Calcium imaging of individual erythrocytes: problems and approaches. (United States)

    Kaestner, Lars; Tabellion, Wiebke; Weiss, Erwin; Bernhardt, Ingolf; Lipp, Peter


    Although in erythrocytes calcium is thought to be important in homeostasis, measurements of this ion concentration are generally seen as rather problematic because of the auto-fluorescence or absorption properties of the intracellular milieu. Here, we describe experiments to assess the usability of popular calcium indicators such as Fura-2, Indo-1 and Fluo-4. In our experiments, Fluo-4 turned out to be the preferable indicator because (i) its excitation and emission properties were least influenced by haemoglobin and (ii) it was the only dye for which excitation light did not lead to significant auto-fluorescence of the erythrocytes. From these results, we conclude that the use of indicators such as Fura-2 together with red blood cells has to be revisited critically. We thus utilized Fluo-4 in erythrocytes to demonstrate a robust but heterogeneous calcium increase in these cells upon stimulation by prostaglandin E(2) and lysophosphatidic acid. For the latter stimulus, we recorded emission spectra of individual erythrocytes to confirm largely unaltered Fluo-4 emission. Our results emphasize that in erythrocytes measurements of intracellular calcium are reliably possible with Fluo-4 and that other indicators, especially those requiring UV-excitation, appear less favourable.

  18. Magnesium: origin and role in calcium-treated inclusions

    CSIR Research Space (South Africa)

    Pistorius, PC


    Full Text Available . The predicted fraction liquid in the inclusion was estimated from the ternary alumina-magnesia-lime phase diagram. Inclusions with higher CaO contents generally had lower MgO contents, indicating that the calcium wire is not the origin of the magnesium...

  19. Influence of calcium on ceramide-1-phosphate monolayers

    Directory of Open Access Journals (Sweden)

    Joana S. L. Oliveira


    Full Text Available Ceramide-1-phosphate (C1P plays an important role in several biological processes, being identified as a key regulator of many protein functions. For instance, it acts as a mediator of inflammatory responses. The mediation of the inflammation process happens due to the interaction of C1P with the C2 domain of cPLA2α, an effector protein that needs the presence of submicromolar concentrations of calcium ions. The aim of this study was to determine the phase behaviour and structural properties of C1P in the presence and absence of millimolar quantities of calcium in a well-defined pH environment. For that purpose, we used monomolecular films of C1P at the soft air/liquid interface with calcium ions in the subphase. The pH was varied to change the protonation degree of the C1P head group. We used surface pressure versus molecular area isotherms coupled with other monolayer techniques as Brewster angle microscopy (BAM, infrared reflection–absorption spectroscopy (IRRAS and grazing incidence X-ray diffraction (GIXD. The isotherms indicate that C1P monolayers are in a condensed state in the presence of calcium ions, regardless of the pH. At higher pH without calcium ions, the monolayer is in a liquid-expanded state due to repulsion between the negatively charged phosphate groups of the C1P molecules. When divalent calcium ions are added, they are able to bridge the highly charged phosphate groups, enhancing the regular arrangement of the head groups. Similar solidification of the monolayer structure can be seen in the presence of a 150 times larger concentration of monovalent sodium ions. Therefore, calcium ions have clearly a strong affinity for the phosphomonoester of C1P.

  20. High-calcium diet modulates effects of long-term prolactin exposure on the cortical bone calcium content in ovariectomized rats. (United States)

    Charoenphandhu, Narattaphol; Tudpor, Kukiat; Thongchote, Kanogwun; Saengamnart, Wasana; Puntheeranurak, Supaporn; Krishnamra, Nateetip


    diet. The present results thus indicated that the adult cortical bones were potentially direct targets of prolactin. Moreover, the effects of high physiological prolactin on cortical bones were age dependent and were observed only under the modulation of high-calcium diet condition.

  1. Regulation of Microstructure of Calcium Carbonate Crystals by Egg White Protein

    Institute of Scientific and Technical Information of China (English)

    ZHU Wen-kun; LUO Xue-gang; ZHANG Chi; DUAN Tao; ZHOU Jian


    Crystal growth of calcium carbonate in biological simulation was investigated via egg white protein with different volume fractions,during which calcium carbonate was synthesized by calcium chloride and sodium carbonate.The morphology,thermal properties and microstructure of the calcium carbonate micro-to-nanoscale crystals were characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM),Fourier transform infrared spectroscopy(FTIR),thermogravimetric analysis(TG)and X-ray diffraction(XRD)analysis.The results show that the volume fraction of egg white protein has great influence on the shape,size and morphology of calcium carbonate crystals.The calcium carbonate crystals were the mixtures of calcite-vaterite-like crystals including spherical and rough surface,which are different from that formed in pure water.With the increase of egg white protein concentration,the diameter of calcium carbonate crystals changed,the amount of formed spherical calcium carbonate particles decreased and that of vaterite increased.These results indicate that the coordination and electrostatic interaction between egg white protein and Ca2+ significantly affect the calcium carbonate crystalization.

  2. Effect of calcium and phosphorus ion implantation on the corrosion resistance and biocompatibility of titanium. (United States)

    Krupa, D; Baszkiewicz, J; Kozubowski, J A; Lewandowska-Szumieł, M; Barcz, A; Sobczak, J W; Biliński, A; Rajchel, A


    This paper is concerned with the corrosion resistance and biocompatibility of titanium after surface modification by the ion implantation of calcium or phosphorus or calcium + phosphorus. Calcium and phosphorus ions were implanted in a dose of 10(17) ions/cm(2). The ion beam energy was 25 keV. The microstructure of the implanted layers was examined by TEM. The chemical composition of the surface layers was determined by XPS and SIMS. The corrosion resistance was examined by electrochemical methods in a simulated body fluid (SBF) at a temperature of 37 degrees C. The biocompatibility was evaluated in vitro. As shown by TEM results, the surface layers formed during calcium, phosphorus and calcium + phosphorus implantation were amorphous. The results of the electrochemical examinations (Stern's method) indicate that the calcium, phosphorus and calcium + phosphorus implantation into the surface of titanium increases its corrosion resistance in stationary conditions after short- and long-term exposures in SBF. Potentiodynamic tests show that the calcium-implanted samples undergo pitting corrosion during anodic polarisation. The breakdown potentials measured are high (2.5 to 3 V). The good biocompatibility of all the investigated materials was confirmed under the specific conditions of the applied examination, although, in the case of calcium implanted titanium it was not as good as that of non-implanted titanium.

  3. Incorporation of calcium buffers into salamander retinal rods: a rejection of the calcium hypothesis of phototransduction. (United States)

    Lamb, T D; Matthews, H R; Torre, V


    The suction-electrode technique was used to monitor the photocurrent of isolated retinal rods from the tiger salamander, by drawing in the light-sensitive outer segment, or sometimes the inner segment. Calcium buffers or other agents were then introduced into the rod cytoplasm by the 'whole-cell patch-clamp' technique. A patch pipette was sealed against the region of the rod protruding from the suction pipette (usually the inner segment), and the membrane patch was ruptured to obtain a whole-cell recording. Several lines of evidence indicated that the pipette contents diffused into the outer segment, and showed that the cell could be adequately voltage clamped. With only trace quantities of chelator in the patch pipette (to bind stray calcium), a gradual decline of the dark current and slowing of responses was usually observed over a period of 10-20 min after rupture of the patch. When the patch pipette contained no added calcium and 10 mM of the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (free Ca2+ ca. 10(-9) M) rupture of the patch led, over a period of a few minutes, to an increase in mean dark current, an increased duration of responses, a substantial increase in flash sensitivity, and a pronounced overshoot in the recovery phase, but with virtually no change in the rising phase of the response to bright flashes. Similar results were obtained when EGTA was used in place of BAPTA, and also in the few cases when successful rupture of the outer segment membrane was obtained. With the free calcium concentration in the patch pipette buffered to the higher level of 1 microM (with 10 mM-Ca2+/11 mM-BAPTA) the results were qualitatively similar to those obtained with BAPTA alone, except that the mean dark current did not increase. This is consistent with a resting free calcium concentration in darkness in the region of 1 microM. In the presence of bright steady illumination with BAPTA in the cell the suppression of outer segment

  4. Vitamin D and intestinal calcium absorption. (United States)

    Christakos, Sylvia; Dhawan, Puneet; Porta, Angela; Mady, Leila J; Seth, Tanya


    The principal function of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. Calcium is absorbed by both an active transcellular pathway, which is energy dependent, and by a passive paracellular pathway through tight junctions. 1,25Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) the hormonally active form of vitamin D, through its genomic actions, is the major stimulator of active intestinal calcium absorption which involves calcium influx, translocation of calcium through the interior of the enterocyte and basolateral extrusion of calcium by the intestinal plasma membrane pump. This article reviews recent studies that have challenged the traditional model of vitamin D mediated transcellular calcium absorption and the crucial role of specific calcium transport proteins in intestinal calcium absorption. There is also increasing evidence that 1,25(OH)(2)D(3) can enhance paracellular calcium diffusion. The influence of estrogen, prolactin, glucocorticoids and aging on intestinal calcium absorption and the role of the distal intestine in vitamin D mediated intestinal calcium absorption are also discussed.

  5. Methylene blue adsorption on graphene oxide/calcium alginate composites. (United States)

    Li, Yanhui; Du, Qiuju; Liu, Tonghao; Sun, Jiankun; Wang, Yonghao; Wu, Shaoling; Wang, Zonghua; Xia, Yanzhi; Xia, Linhua


    Graphene oxide has been used as an adsorbent in wastewater treatment. However, the dispersibility in aqueous solution and the biotoxicity to human cells of graphene oxide limits its practical application in environmental protection. In this research, a novel environmental friendly adsorbent, calcium alginate immobilized graphene oxide composites was prepared. The effects of pH, contact time, temperature and dosage on the adsorption properties of methylene blue onto calcium alginate immobilized graphene oxide composites were investigated. The equilibrium adsorption data were described by the Langmuir and Freundlich isotherms. The maximum adsorption capacity obtained from Langmuir isotherm equation was 181.81 mg/g. The pseudo-first order, pseudo-second order, and intraparticle diffusion equation were used to evaluate the kinetic data. Thermodynamic analysis of equilibriums indicated that the adsorption reaction of methylene blue onto calcium alginate immobilized graphene oxide composites was exothermic and spontaneous in nature.

  6. Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data. (United States)

    Pnevmatikakis, Eftychios A; Soudry, Daniel; Gao, Yuanjun; Machado, Timothy A; Merel, Josh; Pfau, David; Reardon, Thomas; Mu, Yu; Lacefield, Clay; Yang, Weijian; Ahrens, Misha; Bruno, Randy; Jessell, Thomas M; Peterka, Darcy S; Yuste, Rafael; Paninski, Liam


    We present a modular approach for analyzing calcium imaging recordings of large neuronal ensembles. Our goal is to simultaneously identify the locations of the neurons, demix spatially overlapping components, and denoise and deconvolve the spiking activity from the slow dynamics of the calcium indicator. Our approach relies on a constrained nonnegative matrix factorization that expresses the spatiotemporal fluorescence activity as the product of a spatial matrix that encodes the spatial footprint of each neuron in the optical field and a temporal matrix that characterizes the calcium concentration of each neuron over time. This framework is combined with a novel constrained deconvolution approach that extracts estimates of neural activity from fluorescence traces, to create a spatiotemporal processing algorithm that requires minimal parameter tuning. We demonstrate the general applicability of our method by applying it to in vitro and in vivo multi-neuronal imaging data, whole-brain light-sheet imaging data, and dendritic imaging data.

  7. The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Charles Godbout

    Full Text Available Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.

  8. The effect of calcium gluconate and other calcium supplements as a dietary calcium source on magnesium absorption in rats. (United States)

    Chonan, O; Takahashi, R; Yasui, H; Watanuki, M


    The effects of commercially available calcium supplements (calcium carbonate, calcium gluconate, oyster shell preparation and bovine bone preparation) and gluconic acid on the absorption of calcium and magnesium were evaluated for 30 days in male Wistar rats. There were no differences in the apparent absorption ratio of calcium among rats fed each calcium supplement; however, the rats fed the calcium gluconate diet had a higher apparent absorption ratio of magnesium than the rats fed the other calcium supplements. Dietary gluconic acid also more markedly stimulated magnesium absorption than the calcium carbonate diet, and the bone (femur and tibia) magnesium contents of rats fed the gluconic acid diet were significantly higher than those of the rats fed the calcium carbonate diet. Furthermore, the weight of cecal tissue and the concentrations of acetic acid and butyric acid in cecal digesta of rats fed the calcium gluconate diet or the gluconic acid diet were significantly increased. We speculate that the stimulation of magnesium absorption in rats fed the calcium gluconate diet is a result of the gluconic acid component and the effect of gluconic acid on magnesium absorption probably results from cecal hypertrophy, magnesium solubility in the large intestine and the effects of volatile fatty acids on magnesium absorption.

  9. Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes

    Directory of Open Access Journals (Sweden)

    Dmitry eSamigullin


    Full Text Available At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 рА and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 µM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

  10. Calcium fertilization increases the concentration of calcium in sapwood and calcium oxalate in foliage of red spruce (United States)

    Kevin T. Smith; Walter C. Shortle; Jon H. Connolly; Rakesh Minocha; Jody Jellison


    Calcium cycling plays a key role in the health and productivity of red spruce forests in the northeastern US. A portion of the flowpath of calcium within forests includes translocation as Ca2+ in sapwood and accumulation as crystals of calcium oxalate in foliage. Concentrations of Ca in these tree tissues have been used as markers of...

  11. Topotactic exchange and intercalation of calcium phosphate (United States)

    Lima, Cicero B. A.; Airoldi, Claudio


    The precursor (NH 4) 2Ca(H 2PO 4) 2ṡH 2O (CaAP) compound was obtained by combining a calcium chloride solution with dibasic ammonium phosphate. After submitting it to a thermal treatment, crystalline calcium phosphate, Ca(H 2PO 4) 2ṡH 2O (CaP) was isolated. X-ray diffraction patterns for this compound indicated good crystallinity, with a peak at 2θ=12.8°, to give an interlamellar distance of 697 pm, which changed to 1550 pm, when the reaction employed phenylphosphonic acid, and to 1514 pm when intercalated with methylamine. Phosphorus and calcium analysis from colorimetric and gravimetric methods gave for CaP 24.2 and 15.8%, respectively, to yield a P:Ca molar ratio equal to two. The phosphorus nuclear magnetic resonance presented a peak centered at -1.23 ppm, in agreement with the existence of phosphate groups in protonated form. CaAP showed a mass loss of 21.2% in the 466 to 541 K interval due to ammonia and water elimination to yield Ca(PO 3) 3, and CaP can be dehydrated at 440 K for 6 h. A topotactical exchange occurred when CaP is intercalated with methylamine or reacted with phenylphosphonic acid to yield the phosphonate compound and the infrared spectrum of the resulting compound clearly showed the presence of PO 4 and PO 3 groups. The topotactic exchange was also demonstrated by X-ray diffractometry in following the stages of decomposition from 527 to 973 K.

  12. Fabrications of zinc-releasing biocement combining zinc calcium phosphate to calcium phosphate cement. (United States)

    Horiuchi, Shinya; Hiasa, Masahiro; Yasue, Akihiro; Sekine, Kazumitsu; Hamada, Kenichi; Asaoka, Kenzo; Tanaka, Eiji


    Recently, zinc-releasing bioceramics have been the focus of much attention owing to their bone-forming ability. Thus, some types of zinc-containing calcium phosphate (e.g., zinc-doped tricalcium phosphate and zinc-substituted hydroxyapatite) are examined and their osteoblastic cell responses determined. In this investigation, we studied the effects of zinc calcium phosphate (ZCP) derived from zinc phosphate incorporated into calcium phosphate cement (CPC) in terms of its setting reaction and MC3T3-E1 osteoblast-like cell responses. Compositional analysis by powder X-ray diffraction analysis revealed that HAP crystals were precipitated in the CPC containing 10 or 30wt% ZCP after successfully hardening. However, the crystal growth observed by scanning electron microscopy was delayed in the presence of additional ZCP. These findings indicate that the additional zinc inhibits crystal growth and the conversion of CPC to the HAP crystals. The proliferation of the cells and alkaline phosphatase (ALP) activity were enhanced when 10wt% ZCP was added to CPC. Taken together, ZCP added CPC at an appropriate fraction has a potent promotional effect on bone substitute biomaterials.

  13. Simultaneous Sodium and Calcium Imaging from Dendrites and Axons. (United States)

    Miyazaki, Kenichi; Ross, William N


    Dynamic calcium imaging is a major technique of neuroscientists. It can reveal information about the location of various calcium channels and calcium permeable receptors, the time course, magnitude, and location of intracellular calcium concentration ([Ca(2+)]i) changes, and indirectly, the occurrence of action potentials. Dynamic sodium imaging, a less exploited technique, can reveal analogous information related to sodium signaling. In some cases, like the examination of AMPA and NMDA receptor signaling, measurements of both [Ca(2+)]i and [Na(+)]i changes in the same preparation may provide more information than separate measurements. To this end, we developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiologic events. This approach has advantages over sequential imaging because the preparation may not respond identically in different trials. We designed custom dichroic and emission filters to allow the separate detection of the fluorescence of sodium and calcium indicators loaded together into a single neuron in a brain slice from the hippocampus of Sprague-Dawley rats. We then used high-intensity light emitting diodes (LEDs) to alternately excite the two indicators at the appropriate wavelengths. These pulses were synchronized with the frames of a CCD camera running at 500 Hz. Software then separated the data streams to provide independent sodium and calcium signals. With this system we could detect [Ca(2+)]i and [Na(+)]i changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal-to-noise ratio (S/N).

  14. Calcium release from experimental dental materials. (United States)

    Okulus, Zuzanna; Buchwald, Tomasz; Voelkel, Adam


    The calcium release from calcium phosphate-containing experimental dental restorative materials was examined. The possible correlation of ion release with initial calcium content, solubility and degree of curing (degree of conversion) of examined materials was also investigated. Calcium release was measured with the use of an ion-selective electrode in an aqueous solution. Solubility was established by the weighing method. Raman spectroscopy was applied for the determination of the degree of conversion, while initial calcium content was examined with the use of energy-dispersive spectroscopy. For examined materials, the amount of calcium released was found to be positively correlated with solubility and initial calcium content. It was also found that the degree of conversion does not affect the ability of these experimental composites to release calcium ions. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Mechanism of store-operated calcium entry

    Indian Academy of Sciences (India)

    Devkanya Dutta


    Activation of receptors coupled to the phospholipase C/IP3 signalling pathway results in a rapid release of calcium from its intracellular stores, eventually leading to depletion of these stores. Calcium store depletion triggers an influx of extracellular calcium across the plasma membrane, a mechanism known as the store-operated calcium entry or capacitative calcium entry. Capacitative calcium current plays a key role in replenishing calcium stores and activating various physiological processes. Despite considerable efforts, very little is known about the molecular nature of the capacitative channel and the signalling pathway that activates it. This review summarizes our current knowledge about store operated calcium entry and suggests possible hypotheses for its mode of activation.

  16. The ins and outs of mitochondrial calcium. (United States)

    Finkel, Toren; Menazza, Sara; Holmström, Kira M; Parks, Randi J; Liu, Julia; Sun, Junhui; Liu, Jie; Pan, Xin; Murphy, Elizabeth


    Calcium is thought to play an important role in regulating mitochondrial function. Evidence suggests that an increase in mitochondrial calcium can augment ATP production by altering the activity of calcium-sensitive mitochondrial matrix enzymes. In contrast, the entry of large amounts of mitochondrial calcium in the setting of ischemia-reperfusion injury is thought to be a critical event in triggering cellular necrosis. For many decades, the details of how calcium entered the mitochondria remained a biological mystery. In the past few years, significant progress has been made in identifying the molecular components of the mitochondrial calcium uniporter complex. Here, we review how calcium enters and leaves the mitochondria, the growing insight into the topology, stoichiometry and function of the uniporter complex, and the early lessons learned from some initial mouse models that genetically perturb mitochondrial calcium homeostasis.

  17. Familial hypocalciuric hypercalcemia and calcium sensing receptor

    DEFF Research Database (Denmark)

    Mrgan, Monija; Nielsen, Sanne; Brixen, Kim


    Familial hypocalciuric hypercalcemia (FHH) is a lifelong, benign autosomal dominant disease characterized by hypercalcemia, normal to increased parathyroid hormone level, and a relatively low renal calcium excretion. Inactivation of the calcium-sensing receptor in heterozygous patients results in...

  18. Vitamin D, Calcium, and Bone Health (United States)

    ... Bone Health Featured Resource Find an Endocrinologist Search Vitamin D, Calcium, and Bone Health March 2012 Download ... also helps keep your bones strong. Why are vitamin D and calcium important to bone health? Vitamin ...

  19. Using elemental ratios of calcium and strontium to track calcium availability in the freshwater zooplankton Daphnia pulicaria (United States)

    Peters, Stephen C.; Lockwood, Ryan; Williamson, Craig E.; Saros, Jasmine E.


    The assimilation of elements by an organism is dependent on both the environmental availability of the element and the processes of the organism. For some elements, organisms have a challenging time discriminating between nearly identical chemical analogs, for example, calcium and strontium. We tested the hypothesis that in environments where a desired element is scarce, the organism will assimilate a chemically similar analog at an increased rate. Populations of Daphnia pulicaria were manipulated using a microcosm in situ experiment and the results of that experiment tested with a field survey. Experimental results indicated a correlation between higher environmental calcium concentration and lower [Sr]/[Ca] ratios (R2 = 0.91, p < 0.05), suggesting that Daphnia in high calcium environments will assimilate more calcium relative to strontium. Field survey results across eight lakes confirmed that as lake calcium concentration increased, the value of [Sr]/[Ca] between the organism and the lake water decreased (R2 = 0.60, p < 0.05). Measurement of the elemental ratio of major and trace element analogs within organisms compared to their environments may be a useful tool for measuring the relative bioavailability of the major element, and provide insight into elemental limitation in other calcifying aquatic invertebrates.

  20. Calcium regulation in endosymbiotic organelles of plants


    Bussemer, Johanna; Vothknecht, Ute C.; Chigri, Fatima


    In plant cells calcium-dependent signaling pathways are involved in a large array of biological processes in response to hormones, biotic/abiotic stress signals and a variety of developmental cues. This is generally achieved through binding of calcium to diverse calcium-sensing proteins, which subsequently control downstream events by activating or inhibiting biochemical reactions. Regulation by calcium is considered as a eukaryotic trait and has not been described for prokaryotes. Neverthele...

  1. Variable efficacy of calcium carbonate tablets. (United States)

    Kobrin, S M; Goldstein, S J; Shangraw, R F; Raja, R M


    Orally administered calcium carbonate tablets are commonly prescribed as a calcium supplement and for their phosphate-binding effects in renal failure patients. Two cases are reported in which a commercially available brand of calcium carbonate tablets appeared to be ineffective. Formal investigation of the bioavailability of this product revealed it to have impaired disintegration and dissolution and a lack of clinical efficacy. Recommendations that will enable physicians to avoid prescribing and pharmacists to avoid dispensing ineffective calcium carbonate tablets are proposed.

  2. Calcium channel blockers and Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Yi Tan; Yulin Deng; Hong Qing


    Alzheimer's disease is characterized by two pathological hallmarks: amyloid plaques and neurofi-brillary tangles. In addition, calcium homeostasis is disrupted in the course of human aging. Recent research shows that dense plaques can cause functional alteration of calcium signals in mice with Alzheimer's disease. Calcium channel blockers are effective therapeutics for treating Alzheimer's disease. This review provides an overview of the current research of calcium channel blockers in-volved in Alzheimer's disease therapy.

  3. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. (United States)

    Shen, Dongbiao; Wang, Xiang; Li, Xinran; Zhang, Xiaoli; Yao, Zepeng; Dibble, Shannon; Dong, Xian-ping; Yu, Ting; Lieberman, Andrew P; Showalter, Hollis D; Xu, Haoxing


    Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

  4. Synchronous circadian voltage rhythms with asynchronous calcium rhythms in the suprachiasmatic nucleus. (United States)

    Enoki, Ryosuke; Oda, Yoshiaki; Mieda, Michihiro; Ono, Daisuke; Honma, Sato; Honma, Ken-Ichi


    The suprachiasmatic nucleus (SCN), the master circadian clock, contains a network composed of multiple types of neurons which are thought to form a hierarchical and multioscillator system. The molecular clock machinery in SCN neurons drives membrane excitability and sends time cue signals to various brain regions and peripheral organs. However, how and at what time of the day these neurons transmit output signals remain largely unknown. Here, we successfully visualized circadian voltage rhythms optically for many days using a genetically encoded voltage sensor, ArcLightD. Unexpectedly, the voltage rhythms are synchronized across the entire SCN network of cultured slices, whereas simultaneously recorded Ca(2+) rhythms are topologically specific to the dorsal and ventral regions. We further found that the temporal order of these two rhythms is cell-type specific: The Ca(2+) rhythms phase-lead the voltage rhythms in AVP neurons but Ca(2+) and voltage rhythms are nearly in phase in VIP neurons. We confirmed that circadian firing rhythms are also synchronous and are coupled with the voltage rhythms. These results indicate that SCN networks with asynchronous Ca(2+) rhythms produce coherent voltage rhythms.

  5. Conditions and constraints for astrocyte calcium signaling in the hippocampal mossy fiber pathway. (United States)

    Haustein, Martin D; Kracun, Sebastian; Lu, Xiao-Hong; Shih, Tiffany; Jackson-Weaver, Olan; Tong, Xiaoping; Xu, Ji; Yang, X William; O'Dell, Thomas J; Marvin, Jonathan S; Ellisman, Mark H; Bushong, Eric A; Looger, Loren L; Khakh, Baljit S


    The spatiotemporal activities of astrocyte Ca²⁺ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca²⁺ and glutamate indicators as well as pharmacogenetic and electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca²⁺ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca²⁺ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca²⁺ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.

  6. Teaching Calcium-Induced Calcium Release in Cardiomyocytes Using a Classic Paper by Fabiato (United States)

    Liang, Willmann


    This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In…

  7. Calcium mobilisation following shell damage in the Pacific oyster, Crassostrea gigas. (United States)

    Sillanpää, J K; Ramesh, K; Melzner, F; Sundh, H; Sundell, K


    concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage.

  8. 21 CFR 582.3189 - Calcium ascorbate. (United States)


    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium ascorbate. 582.3189 Section 582.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3189 Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance...

  9. 21 CFR 182.3189 - Calcium ascorbate. (United States)


    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium ascorbate. 182.3189 Section 182.3189 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Calcium ascorbate. (a) Product. Calcium ascorbate. (b) Conditions of use. This substance is...

  10. 21 CFR 582.7187 - Calcium alginate. (United States)


    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium alginate. 582.7187 Section 582.7187 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium alginate. (a) Product. Calcium alginate. (b) Conditions of use. This substance is...

  11. 21 CFR 582.5217 - Calcium phosphate. (United States)


    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium phosphate. 582.5217 Section 582.5217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Supplements 1 § 582.5217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  12. 21 CFR 582.1217 - Calcium phosphate. (United States)


    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium phosphate. 582.1217 Section 582.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Additives § 582.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  13. 21 CFR 182.1217 - Calcium phosphate. (United States)


    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Calcium phosphate. 182.1217 Section 182.1217 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Substances § 182.1217 Calcium phosphate. (a) Product. Calcium phosphate (mono-, di-, and tribasic)....

  14. Abnormalities of serum calcium and magnesium (United States)

    Neonatal hypocalcemia is defined as a total serum calcium concentration of <7 mg/dL or an ionized calcium concentration of <4 mg/dL (1mmol/L). In very low birth weight (VLBW) infants, ionized calcium values of 0.8 to 1 mmol/L are common and not usually associated with clinical symptoms. In larger in...

  15. Acute calcium homeostasis in MHS swine. (United States)

    Harrison, G G; Morrell, D F; Brain, V; Jaros, G G


    To elucidate a pathogenesis for the reduction in bone calcium content observed in MHS individuals, we studied the acute calcium homeostasis of MHS swine. This was achieved by the serial measurement, with a calcium selective electrode, of calcium transients in Landrace MHS (five) and control Landrace/large white cross MH negative (five) swine following IV bolus injection of calcium gluconate 0.1 mmol X kg-1--a dose which induced an acute 45 per cent increase in plasma ionised calcium. Experimental animals were anaesthetised with ketamine 10 mg X kg-1 IM, thiopentone (intermittent divided doses) 15-25 mg X kg-1 (total) IV and N2O/O2 (FIO2 0.3) by IPPV to maintain a normal blood gas, acid/base state. The plasma ionised calcium decay curve observed in MHS swine did not differ from that of control normal swine. Further it was noted that the induced acute rise in plasma ionised calcium failed to trigger the MH syndrome in any MHS swine. It is concluded that the mechanisms of acute calcium homeostasis in MHS swine are normal. An explanation for the reduction in bone calcium content observed in MHS individuals must be sought, therefore, through study of the slow long-term component of the calcium regulatory process. In addition, the conventional strictures placed on the use, in MHS patients, of calcium gluconate are called in question.

  16. 21 CFR 582.6219 - Calcium phytate. (United States)


    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Calcium phytate. 582.6219 Section 582.6219 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL... Calcium phytate. (a) Product. Calcium phytate. (b) Conditions of use. This substance is...

  17. Multifaceted Role of Calcium in Cancer. (United States)

    Sarode, Gargi S; Sarode, Sachin C; Patil, Shankargouda


    Role of calcium in bone remodeling and tooth remineral-ization is well known. However, calcium also plays a very imperative role in many biochemical reactions, which are essential for normal functioning of cells. The calcium associated tissue homeostasis encompasses activities like proliferation, cell death, cell motility, oxygen, and nutrient supply.

  18. Predictive value of derived calcium figures based on the measurement of ionised calcium. (United States)

    Gardner, M D; Dryburgh, F J; Fyffe, J A; Jenkins, A S


    The algorithms used in this hospital to assess calcium status are calculated ionised serum calcium and the serum calcium concentration adjusted for albumin. In order to establish their clinical usefulness, they were compared with the ionised calcium concentration measured on the Nova 2 instrument in patients with various calcium and protein abnormalities. Good correlation was found between the measured and calculated values. The predictive values for the calculated results and for total serum calcium concentrations are presented. In this series, the derived values were useful in predicting the serum ionised calcium concentration of the patients studied.

  19. Calcium Orthophosphate-Based Bioceramics

    Directory of Open Access Journals (Sweden)

    Sergey V. Dorozhkin


    Full Text Available Various types of grafts have been traditionally used to restore damaged bones. In the late 1960s, a strong interest was raised in studying ceramics as potential bone grafts due to their biomechanical properties. A bit later, such synthetic biomaterials were called bioceramics. In principle, bioceramics can be prepared from diverse materials but this review is limited to calcium orthophosphate-based formulations only, which possess the specific advantages due to the chemical similarity to mammalian bones and teeth. During the past 40 years, there have been a number of important achievements in this field. Namely, after the initial development of bioceramics that was just tolerated in the physiological environment, an emphasis was shifted towards the formulations able to form direct chemical bonds with the adjacent bones. Afterwards, by the structural and compositional controls, it became possible to choose whether the calcium orthophosphate-based implants remain biologically stable once incorporated into the skeletal structure or whether they were resorbed over time. At the turn of the millennium, a new concept of regenerative bioceramics was developed and such formulations became an integrated part of the tissue engineering approach. Now calcium orthophosphate scaffolds are designed to induce bone formation and vascularization. These scaffolds are often porous and harbor different biomolecules and/or cells. Therefore, current biomedical applications of calcium orthophosphate bioceramics include bone augmentations, artificial bone grafts, maxillofacial reconstruction, spinal fusion, periodontal disease repairs and bone fillers after tumor surgery. Perspective future applications comprise drug delivery and tissue engineering purposes because calcium orthophosphates appear to be promising carriers of growth factors, bioactive peptides and various types of cells.

  20. Engineering crystal growth of calcium hydrogenphosphate dihydrate

    Energy Technology Data Exchange (ETDEWEB)

    Sikiric, M.; Babic-Ivancic, V. [Institut Rudjer Boskovic, Zagreb (Croatia); Milat, O. [Zagreb Univ. (Croatia). Inst. za Fiziku; Sarig, S.; Fueredi-Milhofer, H. [Hebrew Univ., Jerusalem (Israel). Inst. of Applied Chemistry


    The factors underlying calcium hydrogenphosphate dihydrate (CaHPO{sub 4}.2H{sub 2}O, DCPD) interactions with several structurally different additives: glutamic and aspartic acid, sodium citrate, hexaammonium tetrapolyphosphate, calcium phytate and polyaspartic acid were studied. DCPD crystals were prepared under controlled conditions by fast mixing of the anionic and cationic reactant solutions and subsequent growth without further stirring in the course of 24 hours at 37 C. The initial conditions were c(CaCl{sub 2}) = c(Na{sub 2}HPO{sub 4}) = 0.021 mol dm{sup -3}, c(NaCl) = 0.3 mol dm{sup -3}, pH{sub i} 5.5. The respective additive was added to the anionic component prior to pH adjustment. Crystals were characterized by X-ray diffraction, while their morphology was observed by optical and scanning electron microscopy (SEM). The Miller indices of the crystal faces were determined from SEM micrographs, after the orientation of the most prominent face was ascertained by the Weissenberg method. Mechanism of additive-DCPD crystals interaction depends on size and structure of additive molecule, structural fit between organic molecule and the ionic structure of particular crystal face. Small molecules (ions) specifically adsorb on lateral faces by electrostatic interactions, while macromolecules and molecules with hindered structure specifically adsorb on dominant (010) face, for which certain degree of structural fit is necessary. (orig.)

  1. Apical entry channels in calcium-transporting epithelia. (United States)

    Peng, Ji-Bin; Brown, Edward M; Hediger, Matthias A


    The identification of the apical calcium channels CaT1 and ECaC revealed the key molecular mechanisms underlying apical calcium entry in calcium-transporting epithelia. These channels are regulated directly or indirectly by vitamin D and dietary calcium and undergo feedback control by intracellular calcium, suggesting their rate-limiting roles in transcellular calcium transport.

  2. Effect of curd washing on the properties of reduced-calcium and standard-calcium Cheddar cheese. (United States)

    Hou, Jia; McSweeney, Paul L H; Beresford, Thomas P; Guinee, Timothy P


    Washed (W) and nonwashed (NW) variants of standard (SCa) and reduced-calcium (RCa) Cheddar cheeses were made in triplicate, ripened for a 270-d period, and analyzed for composition and changes during maturation. Curd washing was applied to cheeses to give a target level of lactose plus lactic acid in cheese moisture of 3.9 g/100 g in the W cheese, compared with a value of 5.3 g/100 g of lactose plus lactic acid in cheese moisture in the control NW cheeses. The 4 cheese types were denoted standard calcium nonwashed (SCaNW), standard calcium washed (SCaW), reduced-calcium nonwashed (RCaNW), and reduced-calcium washed (RCaW). The mean calcium level was 760 mg/100 g in the SCaNW and SCaW and 660 mg/100 g in the RCaNW and RCaW cheeses. Otherwise the gross composition of all cheeses was similar, each with protein, fat, and moisture levels of ~26, 32, and 36 g/100 g, respectively. Curd washing significantly reduced the mean level of lactic acid in the SCaW cheese and residual lactose in both SCaW and RCaW cheeses. The mean pH of the standard-calcium cheese over the 270-d ripening period increased significantly with curd washing and ripening time, in contrast to the reduced-calcium cheese, which was not affected by the latter parameters. Otherwise curd washing had little effect on changes in populations of starter bacteria or nonstarter lactic acid bacteria, proteolysis, rheology, or color of the cheese during ripening. Descriptive sensory analysis at 270 d indicated that the SCaW cheese had a nuttier, sweeter, less fruity, and less rancid taste than the corresponding SCaNW cheese. In contrast, curd washing was not as effective in discriminating between the RCaW and RCaNW cheeses. The RCaW cheese had a more buttery, caramel odor and flavor, and a more bitter, less sweet, and nutty taste than the SCaW cheese, whereas the RCaNW had a more pungent and less fruity flavor, a less fruity odor, a saltier, more-bitter, and less acidic taste, and a more astringent mouthfeel than

  3. Desalted duck egg white peptides promote calcium uptake by counteracting the adverse effects of phytic acid. (United States)

    Hou, Tao; Liu, Weiwei; Shi, Wen; Ma, Zhili; He, Hui


    The structure of the desalted duck egg white peptides-calcium chelate was characterized by fluorescence spectroscopy, fourier transform infrared spectroscopy, and dynamic light scattering. Characterization results showed structural folding and aggregation of amino acids or oligopeptides during the chelation process. Desalted duck egg white peptides enhanced the calcium uptake in the presence of oxalate, phosphate and zinc ions in Caco-2 monolayers. Animal model indicated that desalted duck egg white peptides effectively enhanced the mineral absorption and counteracted the deleterious effects of phytic acid. These findings suggested that desalted duck egg white peptides might promote calcium uptake in three pathways: 1) desalted duck egg white peptides bind with calcium to form soluble chelate and avoid precipitate; 2) the chelate is absorbed as small peptides by enterocyte; and 3) desalted duck egg white peptides regulate the proliferation and differentiation of enterocytes through the interaction with transient receptor potential vanilloid 6 calcium channel.

  4. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent. (United States)

    Davis, F M; Azimi, I; Faville, R A; Peters, A A; Jalink, K; Putney, J W; Goodhill, G J; Thompson, E W; Roberts-Thomson, S J; Monteith, G R


    Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of

  5. [Calcium distribution in the egg cell, zygote and proembryo of lettuce (Lactuca sativa L.)]. (United States)

    Qiu, Yi Lan; Liu, Ru Shi; Wei, Dong Mei; Tian, Hui Qiao


    Potassium antimonite precipitation was used to located calcium in the egg cells (before and after anthesis), zygotes and proembryos of lettuce (Lactuca sativa L.). A few calcium precipitates (ppts) were located in the small vacuoles of cytoplasm of egg cell at 3 d before anthesis, when egg cells just formed. Then the small vacuoles fused to form some bigger vacuoles in egg cell at 2d before anthesis. Calcium ppts increased evidently in the cytoplasm and nucleus of egg cells at this time. At 1d before anthesis, a biggest vacuole located at the micropyle end of the cell and its nucleus was pushed toward the chalazal end of the cell, which made an evident cellular polarity. The number of calcium ppts in the egg cell markedly decreased, suggesting that change of calcium distribution may be related to the development of egg cell. After anthesis and before fertilization, calcium ppts were still few in the egg cells, and most of them were accumulated in the nucleus, especially in the vacuoles of nucleolus. At 4h after anthesis, egg cell was fertilized and the wall at the chalazal end of egg cell was formed completely. Calcium ppts evidently increased again in egg cell, and some big ppts appeared in the karyoplasm of nucleus and abundant small ppts in the large vacuole. At 9h after anthesis, zygote completed its first division. Calcium ppts in the nucleus and cytoplasm of two-celled proembryo began to decrease, and only some ones accumulated in the vacuoles of nucleolus. At 18h after anthesis, zygote divided several times and became a multi-celled proembryo. Calcium ppts in the cells of proembryo ulteriorly diminished but there were many ppts on the surface of proembryo. The result indicates that calcium in egg cell, zygote and the cells of proembryo orderly changes its temporal and spatial position, which suggests that calcium may play a role during the development of egg cell and zygote.

  6. Differential CaMKII regulation by voltage-gated calcium channels in the striatum. (United States)

    Pasek, Johanna G; Wang, Xiaohan; Colbran, Roger J


    Calcium signaling regulates synaptic plasticity and many other functions in striatal medium spiny neurons to modulate basal ganglia function. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a major calcium-dependent signaling protein that couples calcium entry to diverse cellular changes. CaMKII activation results in autophosphorylation at Thr286 and sustained calcium-independent CaMKII activity after calcium signals dissipate. However, little is known about the mechanisms regulating striatal CaMKII. To address this, mouse brain slices were treated with pharmacological modulators of calcium channels and punches of dorsal striatum were immunoblotted for CaMKII Thr286 autophosphorylation as an index of CaMKII activation. KCl depolarization increased levels of CaMKII autophosphorylation ~2-fold; this increase was blocked by an LTCC antagonist and was mimicked by treatment with pharmacological LTCC activators. The chelation of extracellular calcium robustly decreased basal CaMKII autophosphorylation within 5min and increased levels of total CaMKII in cytosolic fractions, in addition to decreasing the phosphorylation of CaMKII sites in the GluN2B subunit of NMDA receptors and the GluA1 subunit of AMPA receptors. We also found that the maintenance of basal levels of CaMKII autophosphorylation requires low-voltage gated T-type calcium channels, but not LTCCs or R-type calcium channels. Our findings indicate that CaMKII activity is dynamically regulated by multiple calcium channels in the striatum thus coupling calcium entry to key downstream substrates.

  7. Reduction of calcium flux from the extracellular region and endoplasmic reticulum by amorphous nano-silica particles owing to carboxy group addition on their surface

    Directory of Open Access Journals (Sweden)

    Akira Onodera


    Full Text Available Several studies have reported that amorphous nano-silica particles (nano-SPs modulate calcium flux, although the mechanism remains incompletely understood. We thus analyzed the relationship between calcium flux and particle surface properties and determined the calcium flux route. Treatment of Balb/c 3T3 fibroblasts with nano-SPs with a diameter of 70 nm (nSP70 increased cytosolic calcium concentration, but that with SPs with a diameter of 300 or 1000 nm did not. Surface modification of nSP70 with a carboxy group also did not modulate calcium flux. Pretreatment with a general calcium entry blocker almost completely suppressed calcium flux by nSP70. Preconditioning by emptying the endoplasmic reticulum (ER calcium stores slightly suppressed calcium flux by nSP70. These results indicate that nSP70 mainly modulates calcium flux across plasma membrane calcium channels, with subsequent activation of the ER calcium pump, and that the potential of calcium flux by nano-SPs is determined by the particle surface charge.

  8. The relationship between habitual dietary phosphorus and calcium intake, and bone mineral density in young Japanese women: a cross-sectional study. (United States)

    Ito, Sanae; Ishida, Hiromi; Uenishi, Kazuhiro; Murakami, Kentaro; Sasaki, Satoshi


    Phosphorus and calcium are essential for bone health. There is a concern that a low calcium/phosphorus intake ratio resulting from low calcium intake coupled with high phosphorus intake may have a negative effect on bone mineral status, especially in Western countries. The objective of this study was to examine cross-sectionally the influence of habitual phosphorus and calcium intake and the calcium/phosphorus intake ratio on the bone mineral density (BMD) in 441 young Japanese women (aged 18-22) whose calcium/phosphorus intake ratio was assumed to be lower than young Western women. We also ascertained the relationship between dietary intake and serum or urinary measurements of phosphorus and calcium. Parathyroid hormone (PTH) and 25-hydroxy vitamin D (25(OH)D) were also examined for 214 of the 441 subjects. Phosphorus and calcium intake and the calcium/phosphorus intake ratio had significant positive correlations with urinary phosphorus. Calcium intake and the calcium/phosphorus intake ratio independently had positive and significant associations with BMD in the distal radius adjusted for postmenarcheal age, body mass index, and physical activity. There were no significant associations with BMD in the lumbar spine and femoral neck. These results indicate that in young Japanese women, phosphorus intake did not have a significantly negative effect on bone mineral density, and calcium intake and calcium/phosphorus intake ratio had a small but significant association only in a site-specific manner with BMD.

  9. Mercury Control with Calcium-Based Sorbents and Oxidizing Agents

    Energy Technology Data Exchange (ETDEWEB)

    Thomas K. Gale


    This Final Report contains the test descriptions, results, analysis, correlations, theoretical descriptions, and model derivations produced from many different investigations performed on a project funded by the U.S. Department of Energy, to investigate calcium-based sorbents and injection of oxidizing agents for the removal of mercury. Among the technologies were (a) calcium-based sorbents in general, (b) oxidant-additive sorbents developed originally at the EPA, and (c) optimized calcium/carbon synergism for mercury-removal enhancement. In addition, (d) sodium-tetrasulfide injection was found to effectively capture both forms of mercury across baghouses and ESPs, and has since been demonstrated at a slipstream treating PRB coal. It has been shown that sodium-tetrasulfide had little impact on the foam index of PRB flyash, which may indicate that sodium-tetrasulfide injection could be used at power plants without affecting flyash sales. Another technology, (e) coal blending, was shown to be an effective means of increasing mercury removal, by optimizing the concentration of calcium and carbon in the flyash. In addition to the investigation and validation of multiple mercury-control technologies (a through e above), important fundamental mechanism governing mercury kinetics in flue gas were elucidated. For example, it was shown, for the range of chlorine and unburned-carbon (UBC) concentrations in coal-fired utilities, that chlorine has much less effect on mercury oxidation and removal than UBC in the flyash. Unburned carbon enhances mercury oxidation in the flue gas by reacting with HCl to form chlorinated-carbon sites, which then react with elemental mercury to form mercuric chloride, which subsequently desorbs back into the flue gas. Calcium was found to enhance mercury removal by stabilizing the oxidized mercury formed on carbon surfaces. Finally, a model was developed to describe these mercury adsorption, desorption, oxidation, and removal mechanisms, including

  10. Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation.

    Directory of Open Access Journals (Sweden)

    Pei-Rung Wu

    Full Text Available BACKGROUND: Human endothelial nitric oxide synthase (eNOS requires calcium-bound calmodulin (CaM for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50 of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q or at C-terminal (B34Q lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding.

  11. Presynaptic calcium signalling in cerebellar mossy fibres

    DEFF Research Database (Denmark)

    Thomsen, Louiza Bohn; Jörntell, Henrik; Midtgaard, Jens


    Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX....... Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon......)-sensitive fast Na(+) spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers...

  12. Store-operated calcium signaling in neutrophils. (United States)

    Clemens, Regina A; Lowell, Clifford A


    Calcium signals in neutrophils are initiated by a variety of cell-surface receptors, including formyl peptide and other GPCRs, FcRs, and integrins. The predominant pathway by which calcium enters immune cells is termed SOCE, whereby plasma membrane CRAC channels allow influx of extracellular calcium into the cytoplasm when intracellular ER stores are depleted. The identification of 2 key families of SOCE regulators, STIM calcium "sensors" and ORAI calcium channels, has allowed for genetic manipulation of SOCE pathways and provided valuable insight into the molecular mechanism of calcium signaling in immune cells, including neutrophils. This review focuses on our current knowledge of the molecules involved in neutrophil SOCE and how study of these molecules has further informed our understanding of the role of calcium signaling in neutrophil activation.

  13. Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels. (United States)

    Jiang, Shaojuan Amy; Campusano, Jorge M; Su, Hailing; O'Dowd, Diane K


    Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.

  14. Role of Calcium Signaling in the Transcriptional Regulation of the Apicoplast Genome of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Sabna Cheemadan


    Full Text Available Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1 in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1.

  15. Synthesis and reaction behavior of calcium silicate hydrate in basic system

    Institute of Scientific and Technical Information of China (English)

    刘桂华; 贺强; 李小斌; 彭志宏; 周秋生


    At the molar ratio of CaO to SiO2 of 1, with calcium hydroxide and sodium silicate, calcium silicate hydrate was synthesized at 50, 100, 170 ℃, respectively. The results show that temperature favors the formation of calcium silicate hydrate with perfect structure. When calcium silicate hydrate reacts with caustic solution, the decomposition rate of calcium silicate hydrate increases with the increasing caustic concentration and decreases with the raising synthesis temperature and the prolongation of reaction time. The decomposition rate is all less than 1.2 % in caustic solution, and XRD pattern of the residue after reaction with caustic solution is found as the same as that of original calcium silicate hydrate, which indicates the stable existence of calcium silicate hydrate in caustic solution.When reacted with soda solution, the decomposition rate increases with the increasing soda concentration and reaction time, while decreases with the synthesis temperature. The decomposition rate is more than 2% because CaO · SiO2 · H2O(CSH( Ⅰ )), except Ca5 (OH)2Si6O16 · 4H2O and Ca6Si6O17 (OH)2, is decomposed. So the synthesis temperature and soda concentration should be controlled in the process of transformation of sodium aluminosilicate hydrate into calcium silicate hydrate.

  16. The interactive roles of zinc and calcium in mitochondrial dysfunction and neurodegeneration. (United States)

    Pivovarova, Natalia B; Stanika, Ruslan I; Kazanina, Galina; Villanueva, Idalis; Andrews, S Brian


    Zinc has been implicated in neurodegeneration following ischemia. In analogy with calcium, zinc has been proposed to induce toxicity via mitochondrial dysfunction, but the relative role of each cation in mitochondrial damage remains unclear. Here, we report that under conditions mimicking ischemia in hippocampal neurons - normal (2 mM) calcium plus elevated (> 100 μM) exogenous zinc - mitochondrial dysfunction evoked by glutamate, kainate or direct depolarization is, despite significant zinc uptake, primarily governed by calcium. Thus, robust mitochondrial ion accumulation, swelling, depolarization, and reactive oxygen species generation were only observed after toxic stimulation in calcium-containing media. This contrasts with the lack of any mitochondrial response in zinc-containing but calcium-free medium, even though zinc uptake and toxicity were strong under these conditions. Indeed, abnormally high, ionophore-induced zinc uptake was necessary to elicit any mitochondrial depolarization. In calcium- and zinc-containing media, depolarization-induced zinc uptake facilitated cell death and enhanced accumulation of mitochondrial calcium, which localized to characteristic matrix precipitates. Some of these contained detectable amounts of zinc. Together these data indicate that zinc uptake is generally insufficient to trigger mitochondrial dysfunction, so that mechanism(s) of zinc toxicity must be different from that of calcium. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  17. Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.

    Directory of Open Access Journals (Sweden)

    Zhong Yao

    Full Text Available The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.

  18. Voltage gated calcium channels negatively regulate protective immunity to Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Shashank Gupta

    Full Text Available Mycobacterium tuberculosis modulates levels and activity of key intracellular second messengers to evade protective immune responses. Calcium release from voltage gated calcium channels (VGCC regulates immune responses to pathogens. In this study, we investigated the roles of VGCC in regulating protective immunity to mycobacteria in vitro and in vivo. Inhibiting L-type or R-type VGCC in dendritic cells (DCs either using antibodies or by siRNA increased calcium influx in an inositol 1,4,5-phosphate and calcium release calcium activated channel dependent mechanism that resulted in increased expression of genes favoring pro-inflammatory responses. Further, VGCC-blocked DCs activated T cells that in turn mediated killing of M. tuberculosis inside macrophages. Likewise, inhibiting VGCC in infected macrophages and PBMCs induced calcium influx, upregulated the expression of pro-inflammatory genes and resulted in enhanced killing of intracellular M. tuberculosis. Importantly, compared to healthy controls, PBMCs of tuberculosis patients expressed higher levels of both VGCC, which were significantly reduced following chemotherapy. Finally, blocking VGCC in vivo in M. tuberculosis infected mice using specific antibodies increased intracellular calcium and significantly reduced bacterial loads. These results indicate that L-type and R-type VGCC play a negative role in M. tuberculosis infection by regulating calcium mobilization in cells that determine protective immunity.

  19. Evidence for glutamate-mediated activation of hippocampal neurons by glial calcium waves. (United States)

    Hassinger, T D; Atkinson, P B; Strecker, G J; Whalen, L R; Dudek, F E; Kossel, A H; Kater, S B


    Communication from astrocytes to neurons has recently been reported by two laboratories, but different mechanisms were though to underlie glial calcium wave activation of associated neurons. Neuronal calcium elevation by glia observed in the present report is similar to that reported previously, where an increase in neuronal calcium was demonstrated in response to glial stimulation. In the present study hippocampal neurons plated on a confluent glial monolayer displayed a transient increase in intracellular calcium following a short delay after the passage of a wave of increased calcium in underlying glia. Activated cells displayed action potentials in response to glial waves and showed antineurofilament immunoreactivity. Finally, the N-methyl-D-aspartate glutamate receptor antagonist DL-2-amino-5-phosphonovaleric acid and the non-NMDA glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione significantly reduced the responsiveness of neurons to glial calcium waves. Our results indicate that hippocampal neurons growing on hippocampal or cortical astrocytes respond to glial calcium waves with elevations in calcium and increased electrical activity. Furthermore, we show that in most cases this communication appears to be mediated by ionotropic glutamate receptor channels.

  20. Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels. (United States)

    Zemkova, Hana; Tomić, Melanija; Kucka, Marek; Aguilera, Greti; Stojilkovic, Stanko S


    Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

  1. Calcium channel antagonists in hypertension. (United States)

    Ambrosioni, E; Borghi, C


    The clinical usefulness of calcium entry-blockers for the treatment of high blood pressure is related to their capacity to act upon the primary hemodynamic derangement in hypertension: the increased peripheral vascular resistance. They can be used alone or in combination with other antihypertensive agents for the treatment of various forms of hypertensive disease. The calcium entry-blockers appear to be the most useful agents for the treatment of hypertension in the elderly and for the treatment of hypertension associated with ischemic heart disease, pulmonary obstructive disease, peripheral vascular disease, and supraventricular arrhythmias. They are effective in reducing blood pressure in pregnancy-associated hypertension and must be considered as first-line therapy for the treatment of hypertensive crisis.

  2. The calcium-alkali syndrome


    Arroyo, Mariangeli; Fenves, Andrew Z.; Emmett, Michael


    The milk-alkali syndrome was a common cause of hypercalcemia, metabolic alkalosis, and renal failure in the early 20th century. It was caused by the ingestion of large quantities of milk and absorbable alkali to treat peptic ulcer disease. The syndrome virtually vanished after introduction of histamine-2 blockers and proton pump inhibitors. More recently, a similar condition called the calcium-alkali syndrome has emerged as a common cause of hypercalcemia and alkalosis. It is usually caused b...

  3. Calcium phosphate polymer hybrid materials



    Calcium phosphate (CaP) is of strong interest to the medical field because of its potential for bone repair, gene transfection, etc.1-3 Nowadays, the majority of the commercially available materials are fabricated via “classical” materials science approaches, i.e. via high temperature or high pressure approaches, from rather poorly defined slurries, or from organic solvents.3,4 Precipitation of inorganics with (polymeric) additives from aqueous solution on the other hand enables the synthesis...

  4. The effects of thermal stimuli on intracellular calcium change and histamine releases in rat basophilic leukemia mast cells (United States)

    Wu, Zu-Hui; Zhu, Dan; Chen, Ji-Yao; Zhou, Lu-Wei


    The effects of thermal stimuli on rat basophilic leukemia mast cells were studied. The cells in calcium-contained or calcium-free buffers were thermally stimulated in the temperature range of 25-60 °C. The corresponding calcium ion concentration in cells [Ca2+]i as well as the released histamine from cells was measured with fluorescence staining methods. The ruthenium red (RR), a block of membrane calcium channels (transient receptor potential family V (TRPV)), was used in experiments. Under the stimulus of 25-50 °C, no significant difference on [Ca2+]i was found between these three groups of the cells in calcium-contained buffer without or with RR and cells in calcium-free saline, indicating that the increased calcium in cytosol did not result from the extracellular buffer but came from the intracellular calcium stores. The [Ca2+]i continuously increased under the temperature of 50-60 °C, but the RR and calcium-free saline can obviously diminish the [Ca2+]i increase at these high temperatures, reflecting that the opening of the TRPV2 channels leads to a calcium influx resulting in the [Ca2+]i increment. The histamine release also became significant in these cases. Since the released histamine is a well-known mediator for the microcirculation promotion, the histamine release from mast cells could be one of the mechanisms of thermal therapy.

  5. Store-Operated Calcium Channels. (United States)

    Prakriya, Murali; Lewis, Richard S


    Store-operated calcium channels (SOCs) are a major pathway for calcium signaling in virtually all metozoan cells and serve a wide variety of functions ranging from gene expression, motility, and secretion to tissue and organ development and the immune response. SOCs are activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the first characterization of SOCs through electrophysiology, the identification of the STIM proteins as ER Ca(2+) sensors and the Orai proteins as store-operated channels has enabled rapid progress in understanding the unique mechanism of store-operate calcium entry (SOCE). Depletion of Ca(2+) from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca(2+) depletion is coupled to the activation of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation, Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE, and the functions of STIM and Orai in physiology and disease.

  6. Kinetics of calcium sulfoaluminate formation from tricalcium aluminate, calcium sulfate and calcium oxide

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xuerun, E-mail:; Zhang, Yu; Shen, Xiaodong, E-mail:; Wang, Qianqian; Pan, Zhigang


    The formation kinetics of tricalcium aluminate (C{sub 3}A) and calcium sulfate yielding calcium sulfoaluminate (C{sub 4}A{sub 3}$) and the decomposition kinetics of calcium sulfoaluminate were investigated by sintering a mixture of synthetic C{sub 3}A and gypsum. The quantitative analysis of the phase composition was performed by X-ray powder diffraction analysis using the Rietveld method. The results showed that the formation reaction 3Ca{sub 3}Al{sub 2}O{sub 6} + CaSO{sub 4} → Ca{sub 4}Al{sub 6}O{sub 12}(SO{sub 4}) + 6CaO was the primary reaction < 1350 °C with and activation energy of 231 ± 42 kJ/mol; while the decomposition reaction 2Ca{sub 4}Al{sub 6}O{sub 12}(SO{sub 4}) + 10CaO → 6Ca{sub 3}Al{sub 2}O{sub 6} + 2SO{sub 2} ↑ + O{sub 2} ↑ primarily occurred beyond 1350 °C with an activation energy of 792 ± 64 kJ/mol. The optimal formation region for C{sub 4}A{sub 3}$ was from 1150 °C to 1350 °C and from 6 h to 1 h, which could provide useful information on the formation of C{sub 4}A{sub 3}$ containing clinkers. The Jander diffusion model was feasible for the formation and decomposition of calcium sulfoaluminate. Ca{sup 2+} and SO{sub 4}{sup 2−} were the diffusive species in both the formation and decomposition reactions. -- Highlights: •Formation and decomposition of calcium sulphoaluminate were studied. •Decomposition of calcium sulphoaluminate combined CaO and yielded C{sub 3}A. •Activation energy for formation was 231 ± 42 kJ/mol. •Activation energy for decomposition was 792 ± 64 kJ/mol. •Both the formation and decomposition were controlled by diffusion.

  7. Postprandial Energy Metabolism in the Regulation of Body Weight: Is there a Mechanistic Role for Dietary Calcium?

    Directory of Open Access Journals (Sweden)

    Mario J. Soares


    Full Text Available There has been much interest in the mechanisms by which calcium may attenuate weight gain or accelerate body fat loss. This review focuses on postprandial energy metabolism and indicates that dietary calcium increases whole body fat oxidation after single and multiple meals. There is, as yet, no conclusive evidence for a greater diet induced thermogenesis, an increased lipolysis or suppression of key lipogenic enzyme systems. There is however convincing evidence that higher calcium intakes promote a modest energy loss through increased fecal fat excretion. Overall, there is a role for dietary calcium in human energy metabolism. Future studies need to define threshold intakes for metabolic and gastrointestinal outcomes.

  8. Effect of dietary calcium and 1,25-(OH)2D3 on the expression of calcium transport genes in calbindin-D9k and -D28k double knockout mice. (United States)

    Ko, Sang-Hwan; Choi, Kyung-Chul; Oh, Goo Taeg; Jeung, Eui-Bae


    The phenotypes of calbindin-D9k (CaBP-9k) and -28k (CaBP-28k) single knockout (KO) mice are similar to wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we generated CaBP-9k/CaBP-28k double knockout (DKO) mice in order to investigate the importance of CaBP-9k and CaBP-28k in active calcium processing. Under normal dietary conditions, DKO mice did not exhibit any changes in phenotype or the expression of active calcium transport genes as compared to WT or CaBP-28k KO mice. Under calcium-deficient dietary conditions, the phenotype and expression of calcium transport genes in CaBP-28k KO mice were similar to WT, whereas in DKO mice, serum calcium levels and bone length were decreased. The intestinal and renal expression of transient receptor potential vanilloid member 6 (TRPV6) mRNA was significantly decreased in DKO mice fed a calcium-deficient diet as compared to CaBP-28k KO or WT mice, and DKO mice died after 4 weeks on a calcium-deficient diet. Body weight, bone mineral density (BMD) and bone length were significantly reduced in all mice fed a calcium and 1,25-(OH)(2)D(3)-deficient diet, as compared to a normal diet, and none of the mice survived more than 4 weeks. These results indicate that deletion of CaBP-28k alone does not affect body calcium homeostasis, but that deletion of CaBP-9k and CaBP-28k has a significant effect on calcium processing under calcium-deficient conditions, confirming the importance of dietary calcium and 1,25-(OH)(2)D(3) during growth and development.

  9. Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin (United States)

    Yang, T.; Poovaiah, B. W.


    Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H(2)O(2)) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H(2)O(2) into water and oxygen, is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H(2)O(2) and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H(2)O(2). Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H(2)O(2) levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H(2)O(2) homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

  10. Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin (United States)

    Yang, T.; Poovaiah, B. W.


    Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H(2)O(2)) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H(2)O(2) into water and oxygen, is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H(2)O(2) and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H(2)O(2). Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium/CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium/CaM can down-regulate H(2)O(2) levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H(2)O(2) homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

  11. Measuring intracellular calcium dynamics of HeLa cells exposed to nitric oxide by microplate fluorescence reader (United States)

    Huang, Yimei; Chen, Jiangxu; Yang, Hongqin; Zheng, Liqin; Wang, Yuhua; Li, Hui; Xie, Shusen


    Nitric oxide (NO) has been reported to have the ability to promote or inhibit the proliferation and metastasis of cancer cells. It appears to have an effect on inducing calcium transient, which participates in essential cellular signaling in the physiological and pathological processes. Our work was intended to study the effects of exogenous NO on intracellular calcium dynamics of HeLa cells with Fluo-3, a calcium fluorescent indicator by microplate fluorescence reader. The results showed that after NO donor was injected into the wells, intracellular Ca2+ fluorescence intensity increased significantly compared with that of control group. Furthermore, the calcium transient activated by NO was mainly due to the calcium release from intracellular calcium stores. These would be helpful to further recognize the role of NO involved in cancer cell proliferation and metastasis.

  12. Tau causes synapse loss without disrupting calcium homeostasis in the rTg4510 model of tauopathy.

    Directory of Open Access Journals (Sweden)

    Katherine J Kopeikina

    Full Text Available Neurofibrillary tangles (NFTs of tau are one of the defining hallmarks of Alzheimer's disease (AD, and are closely associated with neuronal degeneration. Although it has been suggested that calcium dysregulation is important to AD pathogenesis, few studies have probed the link between calcium homeostasis, synapse loss and pathological changes in tau. Here we test the hypothesis that pathological changes in tau are associated with changes in calcium by utilizing in vivo calcium imaging in adult rTg4510 mice that exhibit severe tau pathology due to over-expression of human mutant P301L tau. We observe prominent dendritic spine loss without disruptions in calcium homeostasis, indicating that tangles do not disrupt this fundamental feature of neuronal health, and that tau likely induces spine loss in a calcium-independent manner.

  13. Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein. (United States)

    An, Bang; Li, Boqiang; Qin, Guozheng; Tian, Shiping


    In this article, we investigated the effect of exogenous calcium on improving viability of Debaryomyces hansenii and Pichia membranaefaciens under heat stress, and evaluated the role of calcium in reducing oxidant damage of proteins in the yeast cells. The results indicated that high concentration of exogenous calcium in culture medium was beneficial for enhancing the tolerance of the biocontrol yeasts to heat stress. The possible mechanism of calcium improving the viability of yeasts was attributed to enhancement of antioxidant enzyme activities, decrease in ROS accumulation and reduction of oxidative damage of intracellular protein in yeast cells under heat stress. D. hansenii is more sensitive to calcium as compared to P. membranaefaciens. Our results suggest that application of exogenous calcium combined with biocontrol yeasts is a practical approach for the control of postharvest disease in fruit.

  14. The role of calcium in endotoxin-induced release of calcitonin gene-related peptide (CGRP) from rat spinal cord

    Institute of Scientific and Technical Information of China (English)

    唐跃明; 韩启德; 王宪


    In the present study, the role of calcium in endotoxin-induced CGRP release was studied. 2 .5-50 μg/mL endotoxin and 1 -10 mmol/L caffeine caused concentration-dependent increase of CGRP release from rat spinal cord in vitro. However, no additive effect could he found when caffeine and endotoxin were concomitantly incubated. By using capsaicin, Ca2+-free medium, Omega-Conotoxin, nifedipine, W-7, ryanodine, MgCl2, Tris-ATP, rutheni-um red, the results indicate that the release of CGRP evoked by endotoxin from the sensory fibers of rat spinal cord is dependent on extracellular calcium. After entering into the cell through the N-type calcium channel, calcium binds to calmodulin, and triggers calcium release from intracellular calcium store by activating the caffeine-sensitive but ryan-odine-insensitive mechanism.

  15. —Part I. Interaction of Calcium and Copper-Calcium Alloy with Electrolyte (United States)

    Zaikov, Yurii P.; Batukhtin, Victor P.; Shurov, Nikolay I.; Ivanovskii, Leonid E.; Suzdaltsev, Andrey V.


    This paper describes the interaction between calcium and molten CaCl2 and the solubility of calcium in this melt, depending on the calcium content in the copper-calcium alloy that comes in contact with the molten CaCl2. The negative influence of the dissolved calcium on the current efficiency was verified. The negative effects of moisture and CaO impurities on the calcium current efficiency were demonstrated. The dependence of the current efficiency and the purity of the metal obtained by the electrolysis conditions were studied in a laboratory electrolyzer (20 to 80 A).

  16. Autogenous vein graft thrombosis following exposure to calcium-free solutions (calcium paradox). (United States)

    Nozick, J H; Farnsworth, P; Montefusco, C M; Parsonnet, V; Ruigrok, T J; Zimmerman, A N


    The morphological and functional effects of calcium-free and calcium-containing solutions on canine jugular vein intima were examined under conditions which closely resemble those techniques currently employed in peripheral vascular and aortocoronary bypass surgery. Veins that had been exposed only to calcium-containing solutions remained patent for the duration of the experimental period. Vein perfusion with a calcium-free solution, however, resulted in disruption of the jugular vein intima once calcium ions were reintroduced. Autogenous as a femoral arterial graft became thrombosed within 60 minutes. It is therefore suggested that vein grafts of autogenous origin be irrigated with calcium-containing solutions to prevent intimal damage and thrombosis.

  17. Calcium channel as a potential anticancer agent. (United States)

    Kriazhev, L


    Anticancer treatment in modern clinical practices includes chemotherapy and radiation therapy with or without surgical interventions. Efficiency of both methods varies greatly depending on cancer types and stages. Besides, chemo- and radiotherapy are toxic and damaging that causes serious side effects. This fact prompts the search for alternative methods of antitumor therapy. It is well known that prolonged or high increase of intracellular calcium concentration inevitably leads to the cell death via apoptosis or necrosis. However, stimulation of cell calcium level by chemical agents is hardly achievable because cells have very sophisticated machinery for maintaining intracellular calcium in physiological ranges. This obstacle can be overridden, nevertheless. It was found that calcium channels in so called calcium cells in land snails are directly regulated by extracellular calcium concentration. The higher the concentration the higher the calcium intake is through the channels. Bearing in mind that extracellular/intracellular calcium concentration ratio in human beings is 10,000-12,000 fold the insertion of the channel into cancer cells would lead to fast and uncontrollable by the cells calcium intake and cell death. Proteins composing the channel may be extracted from plasma membrane of calcium cells and sequenced by mass-spectrometry or N-terminal sequencing. Either proteins or corresponding genes could be used for targeted delivery into cancer cells.

  18. The Role of Calcium in Osteoporosis (United States)

    Arnaud, C. D.; Sanchez, S. D.


    Calcium requirements may vary throughout the lifespan. During the growth years and up to age 25 to 30, it is important to maximize dietary intake of calcium to maintain positive calcium balance and achieve peak bone mass, thereby possibly decreasing the risk of fracture when bone is subsequently lost. Calcium intake need not be greater than 800 mg/day during the relatively short period of time between the end of bone building and the onset of bone loss (30 to 40 years). Starting at age 40 to 50, both men and women lose bone slowly, but women lose bone more rapidly around the menopause and for about 10 years after. Intestinal calcium absorption and the ability to adapt to low calcium diets are impaired in many postmenopausal women and elderly persons owing to a suspected functional or absolute decrease in the ability of the kidney to produce 1,25(OH)2D2. The bones then become more and more a source of calcium to maintain critical extracellular fluid calcium levels. Excessive dietary intake of protein and fiber may induce significant negative calcium balance and thus increase dietary calcium requirements. Generally, the strongest risk factors for osteoporosis are uncontrollable (e.g., sex, age, and race) or less controllable (e.g., disease and medications). However, several factors such as diet, physical activity, cigarette smoking, and alcohol use are lifestyle related and can be modified to help reduce the risk of osteoporosis.

  19. Vitamin D-enhanced duodenal calcium transport. (United States)

    Wongdee, Kannikar; Charoenphandhu, Narattaphol


    For humans and rodents, duodenum is a very important site of calcium absorption since it is exposed to ionized calcium released from dietary complexes by gastric acid. Calcium traverses the duodenal epithelium via both transcellular and paracellular pathways in a vitamin D-dependent manner. After binding to the nuclear vitamin D receptor, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] upregulates the expression of several calcium transporter genes, e.g., TRPV5/6, calbindin-D9k, plasma membrane Ca(2+)-ATPase1b, and NCX1, thereby enhancing the transcellular calcium transport. This action has been reported to be under the regulation of parathyroid-kidney-intestinal and bone-kidney-intestinal axes, in which the plasma calcium and fibroblast growth factor-23 act as negative feedback regulators, respectively. 1,25(OH)2D3 also modulates the expression of tight junction-related genes and convective water flow, presumably to increase the paracellular calcium permeability and solvent drag-induced calcium transport. However, vitamin D-independent calcium absorption does exist and plays an important role in calcium homeostasis under certain conditions, particularly in neonatal period, pregnancy, and lactation as well as in naturally vitamin D-impoverished subterranean mammals.

  20. Calcium-enhanced aggregation of serum amyloid P component and its inhibition by the ligands heparin and heparan sulphate. An electron microscopic and immunoelectrophoretic study

    DEFF Research Database (Denmark)

    Nielsen, EH; Sørensen, Inge Juul; Vilsgaard, K;


    -like structures were formed already at 2 mM calcium. At 25 mM calcium, large aggregates with a crystalline array occasionally exhibiting cylinders predominated. Binding of the ligands heparin and heparan sulphate to SAP completely abolished the calcium-enhanced aggregation, but the distribution of the SAP...... in the absence of calcium ions. However, aggregation is greatly enhanced even at low concentrations (2 mM) of calcium. SAP's tendency to self-aggregation is abolished after its binding to heparin or heparin sulphate. Furthermore, our TEM studies indicate that purified human SAP freed of its natural ligands has...

  1. Calcium isotopes in wine (United States)

    Holmden, C. E.


    The δ 44/40Ca values of bottled wine vary between -0.76% to -1.55% on the seawater scale and correlate weakly with inverse Ca concentration and Mg/Ca ratio, such that the lowest δ 44/40Ca values have the highest Ca concentrations and lowest Mg/Ca ratios. The correlation is notable in the sense that the measured wines include both whites and reds sampled from different wine growing regions of the world, and cover a wide range of quality. Trends among the data yield clues regarding the cause of the observed isotopic fractionation. White wines, and wines generally perceived to be of lower quality, have lower δ 44/40Ca values compared to red wines and wines of generally perceived higher quality. Quality was assessed qualitatively through sensory evaluation, price, and scores assigned by critics. The relationship between δ 44/40Ca and wine quality was most apparent when comparing wines of one varietal from one producer from the same growing region. In the vineyard, wine quality is related to factors such as the tonnage of the crop and the ripeness of the grapes at the time of harvesting, the thickness of the skins for reds, the age of the vines, as well as the place where the grapes were grown (terroir). Quality is also influenced by winemaking practices such as fermentation temperature, duration of skin contact, and barrel ageing. Accordingly, the relationship between δ 44/40Ca and wine quality may originate during grape ripening in the vineyard or during winemaking in the cellar. We tested the grape ripening hypothesis using Merlot grapes sampled from a vineyard in the Okanagan, British Columbia, using sugar content (degrees Brix) as an indicator of ripeness. The grapes were separated into pulp, skin, and pip fractions and were analyzed separately. Thus far, there is no clear evidence for a systematic change in δ 44/40Ca values associated with progressive ripening of grapes in the vineyard. On the day of harvesting, the δ 44/40Ca value of juice squeezed from

  2. Calcium intake is weakly but consistently negatively associated with iron status in girls and women in six European countries

    NARCIS (Netherlands)

    Vijver, L.P.L. van de; Kardinaal, A.F.M.; Charzewska, J.; Rotily, M.; Charles, P.; Maggiolini, M.; Ando, S.; Väänänen. K.; Wajszczyk, B.; Heikkinen, J.; Deloraine, A.; Schaafsma, G.


    Several studies indicate that intake of calcium can inhibit iron absorption especially when taken simultaneously. In the CALEUR study, a cross-sectional study among girls (mean 13.5 y) and young women (mean 22.0 y) in six European countries, the association between calcium intake and iron status was


    Directory of Open Access Journals (Sweden)

    O. D. Ostroumova


    Full Text Available Data on the updated approach to the choice of two-component antihypertensive combinations for different clinical situations are presented. Advantages and indications for combination of an angiotensin converting enzyme (ACE inhibitor and dihydropyridine calcium antagonist are considered. Data on the efficacy and safety of the combination of calcium antagonist of the third generation, lercanidipine, and ACE inhibitor, enalapril, are presented.

  4. In situ fiber-optical monitoring of cytosolic calcium in tissue explant cultures

    CERN Document Server

    Ryser, Manuel; Geiser, Marianne; Frenz, Martin; Rička, Jaro


    We present a fluorescence-lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber-optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green-5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s.

  5. Use of ultrafiltration probes in sheep to collect interstitial fluid for measurement of calcium and magnesium. (United States)

    Janle, E M; Sojka, J E


    Studies of calcium and magnesium changes in living animals usually involve blood, urine, and fecal samples. These samples provide only information on whole-body averages and give no indication of differences between tissues. Ultrafiltration probes were developed to sample interstitial fluid from muscle, bone, and subcutaneous tissue of sheep to provide a tool for investigating tissue differences in calcium and magnesium concentrations. The potential of the probes for mineral distribution studies was demonstrated in sheep by using infusion of a calcium gluconate solution.

  6. Calcium-acting drugs modulate expression and development of chronic tolerance to nicotine-induced antinociception in mice. (United States)

    Damaj, M I


    Initial studies in our laboratory suggested that tolerance to nicotine is thought to involve neuronal adaptation not only at the level of the drug-receptor interaction but at postreceptor events such as calcium-dependent second messengers. The present study was undertaken to investigate the hypothesis that L-type calcium channels and calcium-dependent calmodulin protein kinase II are involved in the development and expression of nicotine tolerance. To that end, the effects of modulation of L-type calcium channels (through the use of inhibitors or activators) as well as calcium-dependent calmodulin protein kinase II inactivation were studied in a mouse model of tolerance where mice were infused with nicotine in minipumps (24 mg/kg/day) for 14 days. In addition, the activity of calcium-dependent calmodulin protein kinase II in the lumbar spinal cord region obtained from nicotine-tolerant mice was measured. Our data showed that chronic administration of L-type calcium channel antagonists nimodipine (1 and 5 mg/kg) and verapamil (10 mg/kg) prevented the development of tolerance to nicotine-induced antinociception. In contrast, chronic exposure of BAYK8644 [(+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester], a calcium channel activator, enhanced nicotine's tolerance. Moreover, a significant increase in both dependent and independent calcium-dependent calmodulin protein kinase II activity was seen in the spinal cord in nicotine-tolerant mice. Finally, spinal administration of 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-phenylpiperazine (KN-62), a calcium-dependent calmodulin protein kinase II antagonist, reduced the expression of tolerance to nicotine-induced antinociception in mice. In conclusion, our data indicate that calcium-dependent mechanisms such as L-type calcium channels and calcium-dependent calmodulin protein kinase II activation are involved in the expression and development of nicotine

  7. Impact Vaporization as a Possible Source of Mercury's Calcium Exosphere (United States)

    Killen, Rosemary M.; Hahn, Joseph M.


    Mercury's calcium exosphere varies in a periodic way with that planet's true anomaly. We show that this pattern can be explained by impact vaporization from interplanetary dust with variations being due to Mercury's radial and vertical excursions through an interplanetary dust disk having an inclination within 5 degrees of the plane of Mercury's orbit. Both a highly inclined dust disk and a two-disk model (where the two disks have a mutual inclination) fail to reproduce the observed variation in calcium exospheric abundance with Mercury true anomaly angle. However, an additional source of impacting dust beyond the nominal dust disk is required near Mercury's true anomaly (?) 25deg +/-5deg. This is close to but not coincident with Mercury's true anomaly (?=45deg) when it crosses comet 2P/Encke's present day orbital plane. Interestingly, the Taurid meteor storms at Earth, which are also due to Comet Encke, are observed to occur when Earth's true anomaly is +/-20 or so degrees before and after the position where Earth and Encke orbital planes cross. The lack of exact correspondence with the present day orbit of Encke may indicate the width of the potential stream along Mercury's orbit or a previous cometary orbit. The extreme energy of the escaping calcium, estimated to have a temperature greater than 50000 K if the source is thermal, cannot be due to the impact process itself but must be imparted by an additional mechanism such as dissociation of a calcium-bearing molecule or ionization followed by recombination.

  8. Synthesis and Characterization of Nanoparticles of Calcium Pyrophosphate (United States)

    Vasant, Sonal R.; Joshi, M. J.

    Calcium phosphate based biomaterials play important roles in clinical applications. Calcium pyrophosphate (CPP), a kind of calcium phosphate, can be used as a bone substitution material as well as a bone graft. Because of its similarity to inorganic component of bone and teeth it can be used for surface coating of metallic dental and orthopedic implants. In the present study, calcium pyrophosphate dihydrate (CPPD) nanoparticles were synthesized using surfactant mediated approach. Crystalline nature and average crystallite size was studied using Powder XRD. The CPPD nanocrystallites were found to be triclinic from powder XRD. The TEM study indicated that CPPD nanoparticles were in the range of 13 nm to 20 nm. The presence of various bonds was confirmed by FTIR spectroscopy. The amount of water of hydration and the thermal stability was studied by thermogravimetry. The variations of various dielectric parameters with the frequency of applied field in 3.2 kHz to 32 MHz range and within a temperature range from 60°C to 120°C were studied. The formation of other phases such as β-CPP and α-CPP on heating of CPPD at 900°C and 1250°C, respectively, were studied by the Powder XRD. The results are discussed.

  9. FRET imaging of calcium signaling in live cells in the microenvironment. (United States)

    Qian, Tongcheng; Lu, Shaoying; Ma, Hongwei; Fang, Jing; Zhong, Wenxuan; Wang, Yingxiao


    The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on a micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and a reduced peak of a transient intracellular calcium rise compared to control cells without constraints. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that a micro-patterned surface can have a profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new light on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.

  10. Calcium signals can freely cross the nuclear envelope in hippocampal neurons: somatic calcium increases generate nuclear calcium transients

    Directory of Open Access Journals (Sweden)

    Bading Hilmar


    Full Text Available Abstract Background In hippocampal neurons, nuclear calcium signaling is important for learning- and neuronal survival-associated gene expression. However, it is unknown whether calcium signals generated by neuronal activity at the cell membrane and propagated to the soma can unrestrictedly cross the nuclear envelope to invade the nucleus. The nuclear envelope, which allows ion transit via the nuclear pore complex, may represent a barrier for calcium and has been suggested to insulate the nucleus from activity-induced cytoplasmic calcium transients in some cell types. Results Using laser-assisted uncaging of caged calcium compounds in defined sub-cellular domains, we show here that the nuclear compartment border does not represent a barrier for calcium signals in hippocampal neurons. Although passive diffusion of molecules between the cytosol and the nucleoplasm may be modulated through changes in conformational state of the nuclear pore complex, we found no evidence for a gating mechanism for calcium movement across the nuclear border. Conclusion Thus, the nuclear envelope does not spatially restrict calcium transients to the somatic cytosol but allows calcium signals to freely enter the cell nucleus to trigger genomic events.

  11. Fortification of all-purpose wheat-flour tortillas with calcium lactate, calcium carbonate, or calcium citrate is acceptable. (United States)

    Romanchik-Cerpovicz, Joelle E; McKemie, Rebecca J


    Fortification helps provide adequate nutrients for individuals not meeting daily needs. Foods may be fortified with calcium to assist individuals with lactose intolerance and others preferring not to consume traditional forms of dairy. This study examined the quality of all-purpose wheat-flour tortillas fortified with calcium lactate, calcium carbonate, or calcium citrate. These tortillas were compared to similarly prepared nonfortified flour tortillas (control) and commercial nonfortified flour tortillas. Calcium-fortified tortillas contained 114 mg elemental calcium per standard serving (48 g tortilla), an 8.6-fold increase compared to nonfortified tortillas. Moisture contents and rollabilities of all tortillas were similar. Consumers (N=87) evaluated each tortilla in duplicate using a hedonic scale and reported liking the appearance, texture, flavor, aftertaste, and overall acceptability of all tortillas. However, the appearance of control tortillas was preferred over commercial tortillas (P<0.01), whereas the aftertaste of commercial tortillas or those fortified with calcium carbonate was preferred over the control (P<0.05). Despite these differences, consumers were equally willing to purchase both fortified and nonfortified tortillas, suggesting that appearance and aftertaste may not influence willingness to purchase. Overall, this study shows that fortification of flour tortillas with various forms of calcium is a feasible alternative calcium source.

  12. Dentin-cement Interfacial Interaction: Calcium Silicates and Polyalkenoates


    Atmeh, A.R.; Chong, E.Z.; Richard, G; Festy, F.; Watson, T.F.


    The interfacial properties of a new calcium-silicate-based coronal restorative material (Biodentine™) and a glass-ionomer cement (GIC) with dentin have been studied by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), micro-Raman spectroscopy, and two-photon auto-fluorescence and second-harmonic-generation (SHG) imaging. Results indicate the formation of tag-like structures alongside an interfacial layer called the “mineral infiltration zone”, where the alkaline c...

  13. Parathormone, Calcium And Phosphorus In Autotransplanted Parathyroid, Total Thyriodectomized Patients


    Abdel Salam, I. M. [ابراهيم عبد السلام; M. Murad; Gamil, M. M.


    The success of parathyroid autotransplantation was indicated by the postoperative assay of serum parathormone in thirteen out of fourteen patients to whom total thyroidectomy was carried out because of thyroid cancer during the past three years. Four glands were autotransplanted in four patients and froam two to three glamds were trasplanted in the remaineng nime patients. All patients were followed up with daily calcium and phosphate determinations. Patients with low ca"1"1" level and with h...

  14. Octupole strength in the neutron-rich calcium isotopes

    CERN Document Server

    Riley, L A; Agiorgousis, M L; Baugher, T R; Bazin, D; Bowry, M; Cottle, P D; DeVone, F G; Gade, A; Glowacki, M T; Gregory, S D; Haldeman, E B; Kemper, K W; Lunderberg, E; Noji, S; Recchia, F; Sadler, B V; Scott, M; Weisshaar, D; Zegers, R G T


    Low-lying excited states of the neutron-rich calcium isotopes $^{48-52}$Ca have been studied via $\\gamma$-ray spectroscopy following inverse-kinematics proton scattering on a liquid hydrogen target using the GRETINA $\\gamma$-ray tracking array. The energies and strengths of the octupole states in these isotopes are remarkably constant, indicating that these states are dominated by proton excitations.

  15. Altered calcium signaling in cancer cells. (United States)

    Stewart, Teneale A; Yapa, Kunsala T D S; Monteith, Gregory R


    It is the nature of the calcium signal, as determined by the coordinated activity of a suite of calcium channels, pumps, exchangers and binding proteins that ultimately guides a cell's fate. Deregulation of the calcium signal is often deleterious and has been linked to each of the 'cancer hallmarks'. Despite this, we do not yet have a full understanding of the remodeling of the calcium signal associated with cancer. Such an understanding could aid in guiding the development of therapies specifically targeting altered calcium signaling in cancer cells during tumorigenic progression. Findings from some of the studies that have assessed the remodeling of the calcium signal associated with tumorigenesis and/or processes important in invasion and metastasis are presented in this review. The potential of new methodologies is also discussed. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

  16. Calcium levels during the initiation of labor. (United States)

    Papandreou, Lampros; Chasiotis, Georgios; Seferiadis, Konstantinos; Thanasoulias, Nikos C; Dousias, Vasilis; Tsanadis, Georgios; Stefos, Theodor


    To investigate the physiological role of calcium in the labor process. Eighty-eight term healthy pregnant women who gave birth to normal healthy neonates participated in our study. We compared calcium levels between pregnant women who had normal delivery and those who underwent scheduled cesarean section. The control group consisted of pregnant women with gestation > or =37 weeks without contractions. The groups were compared with respect to calcium levels: (a) in maternal blood serum; (b) in blood serum of the neonates and mothers; and (c) in blood serum between neonates. Significantly higher calcium levels were found in the group of pregnant women who delivered vaginally compared to those who delivered by scheduled cesarean section and those of the control group. We assume that the increased calcium levels during the first stage of labor are involved with a possible role of calcium in the mechanism of initiation of labor.

  17. Induced calcium carbonate precipitation using Bacillus species. (United States)

    Seifan, Mostafa; Samani, Ali Khajeh; Berenjian, Aydin


    Microbially induced calcium carbonate precipitation is an emerging process for the production of self-healing concrete. This study was aimed to investigate the effects and optimum conditions on calcium carbonate biosynthesis. Bacillus licheniformis, Bacillus sphaericus, yeast extract, urea, calcium chloride and aeration were found to be the most significant factors affecting the biomineralization of calcium carbonate. It was noticed that the morphology of microbial calcium carbonate was mainly affected by the genera of bacteria (cell surface properties), the viscosity of the media and the type of electron acceptors (Ca(2+)). The maximum calcium carbonate concentration of 33.78 g/L was achieved at the optimum conditions This value is the highest concentration reported in the literature.

  18. Movement of endogenous calcium in the elongating zone of graviresponding roots of Zea mays (United States)

    Moore, R.; Cameron, I. L.; Smith, N. K.


    Endogenous calcium (Ca) accumulates along the lower side of the elongating zone of horizontally oriented roots of Zea mays cv. Yellow Dent. This accumulation of Ca correlates positively with the onset of gravicurvature, and occurs in the cytoplasm, cell walls and mucilage of epidermal cells. Corresponding changes in endogenous Ca do not occur in cortical cells of the elongating zone of intact roots. These results indicate that the calcium asymmetries associated with root gravicurvature occur in the outermost layers of the root.

  19. Colorectal Chemoprevention with Calcium and Vitamin D | Division of Cancer Prevention (United States)

    DESCRIPTION: In this application we propose to complete CA098286, a double-blind, randomized, controlled trial of supplementation with vitamin D and/or calcium for the prevention of colorectal adenomas. The study builds on extensive epidemiological and experimental data indicating that both vitamin D and calcium have anti-neoplastic effects in the large bowel and that these agents interact, each requiring the other for full effect. Despite the strong supporting |

  20. Overbased Calcium sulfonate Detergent Technology Overview

    Institute of Scientific and Technical Information of China (English)

    MA Qing-gao; MUIR Ronald J.


    Overbased calcium sulfonate is used widely as detergent in automotive and marine lubricants, as well as various industrial oil applications. In this paper, the process to produce overbased calcium sulfonate is overviewed. The sulfonate structure and molecular weight and its molecular weight distribution, the enclosed calcium carbonate nanoparticle size and crystalline structure, properties of the carrier oil, all influence its properties, such as stability, viscosity, and detergency of the system.

  1. Synthesis of Calcium Silicate (Casio3) Using Calcium Fluoride, Quartz and Microbes

    National Research Council Canada - National Science Library

    B. Gopal Krishna; M. Jagannadha Rao


    .... In this paper, synthesis of calcium silicate (CaSiO3) using calcium fluoride (CaF2) and quartz (SiO2) under microbial environment in a laboratory is being adopted to produce the required material...

  2. [Calcium carbide of different crystal formation synthesized by calcium carbide residue]. (United States)

    Lu, Zhong-yuan; Kang, Ming; Jiang, Cai-rong; Tu, Ming-jing


    To recycle calcium carbide residue effectively, calcium carbide of different crystal form, including global aragonite, calcite and acicular calcium carbide was synthesized. Both the influence of pretreatment in the purity of calcium carbide, and the influence of temperatures of carbonization reaction, release velocity of carbon dioxide in the apparition of calcium carbide of different crystal form were studied with DTA-TG and SEM. The result shows that calcium carbide residue can take place chemistry reaction with ammonia chlorinate straight. Under the condition that pH was above 7, the purity of calcium carbide was above 97%, and the whiteness was above 98. Once provided the different temperatures of carbonization reaction and the proper release velocity of carbon dioxide, global aragonite, calcite and acicular calcium carbide were obtained.

  3. Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement (United States)

    Yoshiki, Keisuke; Azuma, Hiroki; Yoshioka, Kazuhiko; Hashimoto, Mamoru; Araki, Tsutomu

    We have investigated the fluorescence lifetime properties of 8 calcium ion probes, calcium-green-1, calcium green-2, calcium green-5N, calcium orange, oregon green 488 BAPTA-6F, fluo-3, fluo-4, and fluo-5N. We found that the decay time of calcium green-5N varied more sensitively with calcium concentration than calcium green-1 which was known to be a highly sensitive probe. We have also found that the center of observable range of calcium concentration by fluorescence lifetime measurement is lower than that by fluorescence intensity measurement.

  4. Osteoblasts detect pericellular calcium concentration increase via neomycin-sensitive voltage gated calcium channels. (United States)

    Sun, Xuanhao; Kishore, Vipuil; Fites, Kateri; Akkus, Ozan


    The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in

  5. Calcium binding proteins and calcium signaling in prokaryotes. (United States)

    Domínguez, Delfina C; Guragain, Manita; Patrauchan, Marianna


    With the continued increase of genomic information and computational analyses during the recent years, the number of newly discovered calcium binding proteins (CaBPs) in prokaryotic organisms has increased dramatically. These proteins contain sequences that closely resemble a variety of eukaryotic calcium (Ca(2+)) binding motifs including the canonical and pseudo EF-hand motifs, Ca(2+)-binding β-roll, Greek key motif and a novel putative Ca(2+)-binding domain, called the Big domain. Prokaryotic CaBPs have been implicated in diverse cellular activities such as division, development, motility, homeostasis, stress response, secretion, transport, signaling and host-pathogen interactions. However, the majority of these proteins are hypothetical, and only few of them have been studied functionally. The finding of many diverse CaBPs in prokaryotic genomes opens an exciting area of research to explore and define the role of Ca(2+) in organisms other than eukaryotes. This review presents the most recent developments in the field of CaBPs and novel advancements in the role of Ca(2+) in prokaryotes.

  6. Altered calcium signaling following traumatic brain injury

    Directory of Open Access Journals (Sweden)

    John Thomas Weber


    Full Text Available Cell death and dysfunction after traumatic brain injury (TBI is caused by a primary phase, related to direct mechanical disruption of the brain, and a secondary phase which consists of delayed events initiated at the time of the physical insult. Arguably, the calcium ion contributes greatly to the delayed cell damage and death after TBI. A large, sustained influx of calcium into cells can initiate cell death signaling cascades, through activation of several degradative enzymes, such as proteases and endonucleases. However, a sustained level of intracellular free calcium is not necessarily lethal, but the specific route of calcium entry may couple calcium directly to cell death pathways. Other sources of calcium, such as intracellular calcium stores, can also contribute to cell damage. In addition, calcium-mediated signal transduction pathways in neurons may be perturbed following injury. These latter types of alterations may contribute to abnormal physiology in neurons that do not necessarily die after a traumatic episode. This review provides an overview of experimental evidence that has led to our current understanding of the role of calcium signaling in death and dysfunction following TBI.

  7. Regulation of cardiomyocyte autophagy by calcium. (United States)

    Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio


    Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. Copyright © 2016 the American Physiological Society.

  8. The remodeling transient and the calcium economy. (United States)

    Aloia, J F; Arunabh-Talwar, S; Pollack, S; Yeh, J K


    The remodeling transient describes a change in bone mass that lasts one remodeling cycle following an intervention that disturbs the calcium economy. We demonstrated the transient in a study of the response of bone density to calcium/vitamin D3 supplementation and show the hazards of misinterpretation if the transient is not considered. The remodeling transient describes a change in bone mass that lasts for one remodeling cycle following an intervention that disturbs the calcium economy. We report an intervention with calcium and vitamin D supplementation in 208 postmenopausal African-American women where the remodeling transient was considered a priori in the study design. Both groups (calcium alone vs. calcium + 20 microg (800 IU) vitamin D3) were ensured a calcium intake in excess of 1200 mg/day. There were no differences between the two groups in changes in BMD over time. These BMD changes were therefore interpreted to reflect increased calcium intake in both groups but not any influence of vitamin D. A transient increase in bone mineral density was observed during the first year of study, followed by a decline. The remodeling period was estimated at about 9 months, which is similar to histomorphometric estimates. It is problematic to draw conclusions concerning interventions that influence the calcium economy without considering the remodeling transient in study design. Studies of agents that effect bone remodeling must be carried out for at least two remodeling cycles and appropriate techniques must be used in data analysis.

  9. Disease causing mutations of calcium channels. (United States)

    Lorenzon, Nancy M; Beam, Kurt G


    Calcium ions play an important role in the electrical excitability of nerve and muscle, as well as serving as a critical second messenger for diverse cellular functions. As a result, mutations of genes encoding calcium channels may have subtle affects on channel function yet strongly perturb cellular behavior. This review discusses the effects of calcium channel mutations on channel function, the pathological consequences for cellular physiology, and possible links between altered channel function and disease. Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca(2+) ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca(2+) stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores.

  10. Sintering of calcium phosphate bioceramics. (United States)

    Champion, E


    Calcium phosphate ceramics have become of prime importance for biological applications in the field of bone tissue engineering. This paper reviews the sintering behaviour of these bioceramics. Conventional pressureless sintering of hydroxyapatite, Ca10(PO4)6(OH)2, a reference compound, has been extensively studied. Its physico-chemistry is detailed. It can be seen as a competition between two thermally activated phenomena that proceed by solid-state diffusion of matter: densification and grain growth. Usually, the objective is to promote the first and prevent the second. Literature data are analysed from sintering maps (i.e. grain growth vs. densification). Sintering trajectories of hydroxyapatite produced by conventional pressureless sintering and non-conventional techniques, including two-step sintering, liquid phase sintering, hot pressing, hot isostatic pressing, ultrahigh pressure, microwave and spark plasma sintering, are presented. Whatever the sintering technique may be, grain growth occurs mainly during the last step of sintering, when the relative bulk density reaches 95% of the maximum value. Though often considered very advantageous, most assisted sintering techniques do not appear very superior to conventional pressureless sintering. Sintering of tricalcium phosphate or biphasic calcium phosphates is also discussed. The chemical composition of calcium phosphate influences the behaviour. Similarly, ionic substitutions in hydroxyapatite or in tricalcium phosphate create lattice defects that modify the sintering rate. Depending on their nature, they can either accelerate or slow down the sintering rate. The thermal stability of compounds at the sintering temperature must also be taken into account. Controlled atmospheres may be required to prevent thermal decomposition, and flash sintering techniques, which allow consolidation at low temperature, can be helpful.

  11. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels



    Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure plan...

  12. The Role of Calcium in Prevention and Treatment of Osteoporosis. (United States)

    Heaney, Robert P.


    Osteoporosis results from several factors. Calcium deficiency is only one, and high calcium intake will prevent only those cases in which calcium is the limiting factor. Calcium cannot reverse, but only arrest, bone loss. A high calcium intake for every member of the population is advocated. (Author/MT)

  13. Geomagnetic Indices Bulletin (GIB) (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Geomagnetic Indices Bulletin is a one page sheet containing the magnetic indices Kp, Ap, Cp, An, As, Am and the provisional aa indices. The bulletin is published...

  14. Calcium imaging perspectives in plants. (United States)

    Kanchiswamy, Chidananda Nagamangala; Malnoy, Mickael; Occhipinti, Andrea; Maffei, Massimo E


    The calcium ion (Ca2+) is a versatile intracellular messenger. It provides dynamic regulation of a vast array of gene transcriptions, protein kinases, transcription factors and other complex downstream signaling cascades. For the past six decades, intracellular Ca2+ concentration has been significantly studied and still many studies are under way. Our understanding of Ca2+ signaling and the corresponding physiological phenomenon is growing exponentially. Here we focus on the improvements made in the development of probes used for Ca2+ imaging and expanding the application of Ca2+ imaging in plant science research.

  15. The Electronic Structure of Calcium

    DEFF Research Database (Denmark)

    Jan, J.-P.; Skriver, Hans Lomholt


    The electronic structure of calcium under pressure is re-examined by means of self-consistent energy band calculations based on the local density approximation and using the linear muffin-tin orbitals (LMTO) method with corrections to the atomic sphere approximation included. At zero pressure.......149 Ryd, respectively, relative to the s band, give the best possible agreement. Under increasing pressure the s and p electrons are found to transfer into the d band, and Ca undergoes metal-semimetal-metal electronic transitions. Calculations of the bandstructure and the electronic pressure, including...

  16. Preparation and mechanical property of core-shell type chitosan/calcium phosphate composite fiber

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Atsushi [Japan Society for the Promotion of Science, Ikenohata1-1-1, Daitou-ku, Tokyo 110-0008 (Japan) and Creative Research Initiative ' Sousei' , Hokkaido University, Sapporo, Hokkaido 001-0021 (Japan)]. E-mail:; Ikoma, Toshiyuki [Biomaterials Research Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044 (Japan); Kobayashi, Hisatoshi [Biomaterials Research Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044 (Japan)]. E-mail:; Tanaka, Junzo [Creative Research Initiative ' Sousei' , Hokkaido University, Sapporo, Hokkaido 001-0021 (Japan); Biomaterials Research Center, National Institute for Materials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044 (Japan)


    Core-shell type chitosan/calcium phosphate composite fibers were prepared by a facile wet spinning method; the chitosan aqueous solution with PO{sub 4} ions was dropped and coagulated in the ethanol/calcium hydroxide solutions at different mixed ratio. X-ray diffraction (XRD) patterns indicated that the crystal phases of calcium phosphates in the composite fibers were a low-crystalline hydroxyapatite (HAp; Ca{sub 10}(PO{sub 4}){sub 6}(OH){sub 2})or the low-crystalline hydroxyapatite/brushite mixture depended on the ratio of ethanol/calcium hydroxide solutions. The inorganic contents were ca. 60 wt.% by using the TG-DTA analysis. The energy-dispersive X-ray spectroscopy (EDS) analysis indicated that Ca and P atoms were mainly distributed on the outer layer of the composite fiber to grow calcium phosphate crystals; however, a little amount of P atom still remained at the inside of the fiber. This indicated that the composite fibers formed a unique core-shell structure with shell of calcium phosphate and core of chitosan. The mechanical property of the fibers was reinforced by the initial concentration of chitosan solution.

  17. Calcium (United States)

    ... tingling in the fingers, convulsions, and abnormal heart rhythms that can lead to death if not corrected. ... that includes weight-bearing physical activity (such as walking and running). Osteoporosis is a disease of the ...

  18. Calcium (United States)

    ... for dinner. Create mini-pizzas by topping whole-wheat English muffins or bagels with pizza sauce and ... Fitness Center Vitamin D Smart Supermarket Shopping Lactose Intolerance Vitamins and Minerals Vitamin Chart Mineral Chart Food ...

  19. Testosterone increases urinary calcium excretion and inhibits expression of renal calcium transport proteins.

    NARCIS (Netherlands)

    Hsu, Y.J.; Dimke, H.; Schoeber, J.P.H.; Hsu, S.C.; Lin, S.H.; Chu, P.; Hoenderop, J.G.J.; Bindels, R.J.M.


    Although gender differences in the renal handling of calcium have been reported, the overall contribution of androgens to these differences remains uncertain. We determined here whether testosterone affects active renal calcium reabsorption by regulating calcium transport proteins. Male mice had hig

  20. 76 FR 51991 - Determination That PENTETATE CALCIUM TRISODIUM (Trisodium Calcium Diethylenetriaminepentaacetate... (United States)


    ... new drug applications (ANDAs) for PENTETATE CALCIUM TRISODIUM (Ca-DTPA) solution for intravenous or... ANDA that does not refer to a listed drug. PENTETATE CALCIUM TRISODIUM (Ca-DTPA) solution for... HUMAN SERVICES Food and Drug Administration Determination That PENTETATE CALCIUM TRISODIUM...