Changes In Calcium Concentration Research Articles

The change of female progesterone level and blood calcium concentration in perimenopausal women with benign paroxysmal positional vertigo

Objective: The estrogen level and blood calcium concentration changes were studied on menopausal women with benign paroxysmal positional vertigo (BPPV). Methods: Between January 2015 and January 2016, 70 menopause women with BPPV in outpatient clinics of Department of Otorhinolaryngology, Inner Mongolia Medical University Affiliated Hospital were included in this study as research group, while 30 menopause healthy women who came to hospital for check-up were included as control group. Serum levels of estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone (PRO), testosterone (T), serum prolactin (PRL) and the calcium concentration were analysed and comparied between research group and control group. SPSS17.0 statistical software was used to analyze the data. χ(2) test was used to compare the percentage of decreased serum level of sex hormone, and t test was used to compare the serum level of sex hormone and calcium concentration of two groups. Results: In research group, sex hormone decreased proportion of E2 (91%) and PRO (67%) were obviously higher than those in control group (χ(2) value was 8.13, 10.28, respectively, all P<0.05). The E2 and PRO in research group were significantly lower than those in control group ((33.18±31.45) pmol/L vs (64.92 ±31.52) pmol/L, (0.64±0.48) nmol/L vs (1.02±0.60) nmol/L, t value was 6.238, 8.566, respectively, all P<0.05). There was no statistically significant difference of the level of LH, PRL, T, FSH and blood calcium concentration in research group compared with control group ((29.81±13.13) U/L vs (27.21±10.19) U/L, (0.49±0.20) nmol/L vs (0.49±0.15) nmol/L, (0.56±0.42) nmol/L vs (0.73±0.62) nmol/L, (64.25±31.44) U/L vs (60.38±29.97) U/L, (2.28±0.17) mmol/L vs (2.32±0.21) mmol/L, t value was 13.427, 14.876, 7.505, 12.090, 7.532, respectively, all P>0.05). Conclusion: The level of E2 and PRO decrease obviously in postmenopausal women with BPPV, which can cause the inner ear microcirculation disorder , may be one of the risk factors of BPPV.

Investigation of triamterene as an inhibitor of the TGR5 receptor: identification in cells and animals.

BackgroundG-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) has been shown to participate in glucose homeostasis. In animal models, a TGR5 agonist increases incretin secretion to reduce hyperglycemia. Many agonists have been developed for clinical use. However, the effects of TGR5 blockade have not been studied extensively, with the exception of studies using TGR5 knockout mice. Therefore, we investigated the potential effect of triamterene on TGR5.MethodsWe transfected the TGR5 gene into cultured Chinese hamster ovary cells (CHO-K1 cells) to express TGR5. Then, we applied a fluorescent indicator to examine the glucose uptake of these transfected cells. In addition, NCI-H716 cells that secrete incretin were also evaluated. Fura-2, a fluorescence indicator, was applied to determine the changes in calcium concentrations. The levels of cyclic adenosine monophosphate (cAMP) and glucagon-like peptide (GLP-1) were estimated using enzyme-linked immunosorbent assay kits. Moreover, rats with streptozotocin (STZ)-induced type 1-like diabetes were used to investigate the effects in vivo.ResultsTriamterene dose dependently inhibits the increase in glucose uptake induced by TGR5 agonists in CHO-K1 cells expressing the TGR5 gene. In cultured NCI-H716 cells, TGR5 activation also increases GLP-1 secretion by increasing calcium levels. Triamterene inhibits the increased calcium levels by TGR5 activation through competitive antagonism. Moreover, the GLP-1 secretion and increased cAMP levels induced by TGR5 activation are both dose dependently reduced by triamterene. However, treatment with KB-R7943 at a dose sufficient to block the Na+/Ca2+ exchanger (NCX) failed to modify the responses to TGR5 activation in NCI-H716 cells or CHO-K1 cells expressing TGR5. Therefore, the inhibitory effects of triamterene on TGR5 activation do not appear to be related to NCX inhibition. Blockade of TGR5 activation by triamterene was further characterized in vivo using the STZ-induced diabetic rats.ConclusionBased on the obtained data, we identified triamterene as a reliable inhibitor of TGR5. Therefore, triamterene can be developed as a clinical inhibitor of TGR5 activation in future studies.

Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca2+ ] were significantly decreased in the rat sepsis model compared with controls. Decreased intracellular [Ca2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017.

Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes.

Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown. Rapid stimulation frequency-dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency. These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially targeted Förster resonance energy transfer (FRET)-based calcium indicator (4mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+ ]m ) was measured at different stimulation frequencies (0.1-4Hz) and external calcium concentrations (1.8-3.6mm) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura-4AM. The increases in [Ca2+ ]m during electrical stimulation at 0.1Hz were rapid (rise time=49±2ms), while the decreases in [Ca2+ ]m occurred more slowly (decay half time=1.17±0.07s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+ ]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium-calcium exchanger (mNCE) resulted in a rise in [Ca2+ ]m at baseline and, paradoxically, in an acceleration of Ca2+ release. rapid increases in [Ca2+ ]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat-to-beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering.

Src tyrosine kinases contribute to serotonin-mediated contraction by regulating calcium-dependent pathways in rat skeletal muscle arteries.

The Src tyrosine kinase family contributes to the signalling mechanism mediating serotonin (5-hydroxytryptamine (5-HT))-induced vasoconstriction. These kinases were reported to influence the calcium sensitivity of the contractile apparatus. Whether Src kinases affect also the intracellular calcium concentration during constriction of intact arteries is unknown. Thus, we tested the hypothesis that constriction of arteries is associated with a Src kinase-dependent alteration of the intracellular calcium concentration. Contractility of gracilis arteries of Wistar rats was studied using isometric and isobaric myography. The intracellular calcium concentration was measured simultaneously with tension by FURA-2 fluorimetry. Inhibition of Src kinases with 10μM PP2, 30μM dasatinib and 100μM AZM 475271 resulted in a strong attenuation of 5-HT-induced contractions. Vessel incubation with 10μM PP3, an inactive analogue of PP2, had no effect. Removal of the endothelium did not alter vessel contractile responses to 5-HT nor the action of the Src-kinase inhibitor PP2. The PP2-mediated inhibition of 5-HT-induced contraction was associated with a reduced response of [Ca2+]i to 5-HT. In particular, inhibition of Src kinases attenuates 5-HT-induced calcium influx as well as calcium release from intracellular stores. In contrast, the calcium sensitivity of the contractile apparatus and the filling state of the sarcoplasmic reticulum were not influenced by Src kinases during 5-HT-induced contractions. We conclude that Src kinase activation is a powerful mechanism to produce vasoconstriction of small skeletal muscle arteries of rats. This effect is endothelium-independent. The data further suggest that the action of c-Src kinases is associated with a change in the intracellular calcium concentration that involves Ca2+ entry and Ca2+ release pathways.

Probing Deep Brain Circuitry: New Advances in in Vivo Calcium Measurement Strategies.

The study of neuronal ensembles in awake and behaving animals is a critical question in contemporary neuroscience research. Through the examination of calcium fluctuations, which are correlated with neuronal activity, we are able to better understand complex neural circuits. Recently, the development of technologies including two-photon microscopy, miniature microscopes, and fiber photometry has allowed us to examine calcium activity in behaving subjects over time. Visualizing changes in intracellular calcium in vivo has been accomplished utilizing GCaMP, a genetically encoded calcium indicator. GCaMP allows researchers to tag cell-type specific neurons with engineered fluorescent proteins that alter their levels of fluorescence in response to changes in intracellular calcium concentration. Even with the evolution of GCaMP, in vivo calcium imaging had yet to overcome the limitation of light scattering, which occurs when imaging from neural tissue in deep brain regions. Currently, researchers have created in vivo methods to bypass this problem; this Review will delve into three of these state of the art techniques: (1) two-photon calcium imaging, (2) single photon calcium imaging, and (3) fiber photometry. Here we discuss the advantages and disadvantages of the three techniques. Continued advances in these imaging techniques will provide researchers with unparalleled access to the inner workings of the brain.

Modified Calcium Homeostasis in Aged Mouse Skeletal Muscle

In aging decreased physical activity and reduced muscle mass (sarcopenia) leads to impaired muscle force and increased fatigability accompanied by a decline in sarcoplasmic reticulum (SR) calcium release. As an essential trace element selenium plays a significant role in muscle functions, as in selenium deficiency skeletal muscle disorders manifesting in muscle pain, fatigue, proximal weakness, and serum creatine kinase elevation could develop. Here in vivo physical activity of control and myostatin deficient (Cmpt) mice during 22 months were examined. Their performance in grip and voluntary wheel tests reached its maximum 3 month after birth and declined after 10 month. This decrease was faster in Cmpt mice as the average daily running distance decreased to 37% in control and to 25% in Cmpt mice in old age. In vitro force was measured on soleus and extensor digitorum longus (EDL) muscles from 20-month-old control, Cmpt, and selenium supplemented mice. Albeit the absolute force was higher in Cmpt than in control mice, after normalization to cross section EDL was significantly stronger in the latter (4.70±0.88 vs 7.61±0.96 mN/mm2, respectively). Selenium supplementation significantly increased the maximal force of EDL (10.89±0.60 mN/mm2). Changes in intracellular calcium concentration were measured on enzymatically isolated intact flexor digitorum brevis muscle fibers using Fura-2. The rate of KCl depolarization-evoked SR calcium release was greater in selenium supplemented than in control animals (691±100 vs 481±33 µM/s, respectively). Western-blot analysis revealed no change in the expression of the dihydropyridine receptor in the three animal groups while that of the ryanodine receptor declined with aging which was reversed by long-term training. Our results support the positive effects of selenium and training on SR calcium release in old age associated muscle weakness. On the other hand, the increased muscle mass of Cmpt mice during their lifespan doesn’t improve their physical performance in old age. Supported by: NKFIH-K-115461

Calcium Channel Opening Rather than the Release of ATP Causes the Apoptosis of Osteoblasts Induced by Overloaded Mechanical Stimulation

Background: Stress fracture is one of the most common overuse injuries in athletes. Overloaded mechanical stimulation is an important factor affecting stress fractures, but the mechanism is unclear. Methods: MC3T3-E1 cells and a polycaprolactone (PCL) scaffold were co-cultured, and finite element analysis (FEA) was used to analyze the load-carrying capability. Cell proliferation was investigated with CCK-8 assays. An alkaline phosphatase (AKP) activity assay was used to evaluate cell differentiation. Cell apoptosis was analyzed using Hoechst/ PI double-labeling, Caspase-3 activity and lactate dehydrogenase (LDH) activity assays. Realtime PCR and Western blotting were used to examine the gene and protein expression, respectively, of Caspase-3 and Caspase-9. Assays of the intracellular calcium with fluorescent probe technique and extracellular ATP with fluorometric assay kit were used to analyze the changes in the intracellular calcium concentration induced by calcium channel opening and the release of ATP, respectively, at different operation times. Results: When the apparent strain reached 10000 µε, the strain scope of fber at levels greater than 4000 µε was 60%. Overloading for 4 days and operation times of 0.5 h and 2 h increased the cell number and AKP secretion. However, apoptosis genes were activated at the same time, and the operation time of 2 h had a significantly greater effect than 0.5 h. At 8 days, the cell numbers were greater for the operation time of 0.5 h than for 2 h, and the 2-h groups had the fastest apoptosis rate. Overloading for 1 day increased intracellular calcium levels and ATP release. The increase in intracellular calcium could be blocked by the addition of N-ethylmaleimide (NEM) or Hank's medium. Overloading for 8 days increased intracellular calcium levels but decreased extracellular ATP, and verapamil blocked the increase in intracellular calcium. Conclusion: We found that a simultaneous ‘double effect’ on osteoblasts was induced by overloading, which promoted cell proliferation, differentiation and apoptosis. Short-term overloading could open the cell membrane calcium channels and release calcium stores to elevate intracellular calcium levels, thereby promoting the proliferation and differentiation of cells to a greater extent than the effect of apoptosis. For long-term overloading, calcium channel opening in the membrane could lead to overloading of intracellular calcium levels, inducing an apoptosis effect that is greater than the effect on proliferation and differentiation.

Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader.

Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others ( Burgos et al., 2007 ; Martínez et al., 2016 ). Changes in intracellular calcium concentration can be measured using the calcium sensitive fluorescent ratiometric dye fura-2 AM. This method is a high throughput way to measure agonist mediated calcium responses.

Evaluation of a hollow fiber supported liquid membrane device as a chemical surrogate for the measurements of zinc (II) bioavailability using two microalgae strains as biological references

The environmental bioavailability of zinc (II), i.e., the uptake of the element by an organism, was determined using two microalgae species, Scenedesmus acutus and Pseudokirchneriella subcapitata, and estimated using hollow fiber supported liquid membrane (HF-SLM) device as the chemical surrogate. Several experimental conditions were studied including the presence of organic matter, inorganic anions and concomitant cations and pH. The results show strong positive correlation coefficients between the responses given by the HF-SLM and the microalgae species (r = 0.900 for S. acutus and r = 0.876 for P. subcapitata) in multivariate environments (changes in pH, calcium, humic and citrate concentrations). The maximum amount of zinc (II) retained by the HF-SLM (4.7 × 10−8 mol/cm2) was higher than those for P. subcapitata and S. acutus (9.4 × 10−11 mol/cm2 and 6.2 × 10−11 mol/cm2, respectively). The variation in pH (pH 5.5–9) was the variable with the greatest effect on zinc internalization in all systems, increasing approximately 2.5 times for P. subcapitata and 5.5 times for S. acutus respect to pH = 5.5, while the presence of humic acids did not affect the response. The species’ concentration analysis of the experimental design at pH = 5.5 indicated that the amount of internalized zinc (II) by the HF-SLM and both microalgae species is strongly dependent on the free zinc concentration (r = 0.910 for the HF-SLM, r = 0.922 for S. acutus and r = 0.954 for P. subcapitata); however, at pH = 9.0, the amount of internalized zinc (II) is strongly dependent on the sum of free zinc and labile species (r = 0.912 for the HF-SLM, r = 0.947 for S. acutus and r = 0.900 for P. subcapitata). The presence of inorganic ligands (chloride, sulfate, phosphate, carbonate, and nitrate) and metal ions (cobalt (II), copper (II), nickel (II), chromium (VI), lead (II) and cadmium (II)) produced different behaviors both in the chemical surrogate and the biological references. The results showed that the synthetic device can mimic biological uptake in the presence of humic acids, nitrate, sulfate, and phosphate, and pH within the range 5.5–9 when S. acutus was used as the biological reference, considering the simultaneous contribution of the Zn2+ and ZnOH+ labile species depending on the chemical composition of the medium.

Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans

Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.

Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

In this study, we report for the first time that the primary cilium acts as a crucial sensor for electrical field stimulation (EFS)-enhanced osteogenic response in osteoprogenitor cells. In addition, primary cilia seem to functionally modulate effects of EFS-induced cellular calcium oscillations. Primary cilia are organelles that have recently been implicated to play a crucial sensor role for many mechanical and chemical stimuli on stem cells. Here, we investigate the role of primary cilia in EFS-enhanced osteogenic response of human adipose-derived stem cells (hASCs) by knocking down 2 primary cilia structural proteins, polycystin-1 and intraflagellar protein-88. Our results indicate that structurally integrated primary cilia are required for detection of electrical field signals in hASCs. Furthermore, by measuring changes of cytoplasmic calcium concentration in hASCs during EFS, our findings also suggest that primary cilia may potentially function as a crucial calcium-signaling nexus in hASCs during EFS.-Cai, S., Bodle, J. C., Mathieu, P. S., Amos, A., Hamouda, M., Bernacki, S., McCarty, G., Loboa, E. G. Primary cilia are sensors of electrical field stimulation to induce osteogenesis of human adipose-derived stem cells.

Cytosolic Calcium Concentration Changes in Neuronal Cells Under Clinorotation and in Parabolic Flight Missions

All life on earth has been established under conditions of stable gravity of 1g. Nevertheless, in numerous experiments the direct gravity dependence of biological processes has been shown on all levels of organization, from single molecules to humans. To study the effects especially of microgravity on biological systems, a variety of platforms are available, from drop towers to the ISS. Due to the costs of these platforms and their limited availability, as an alternative, numerous simulators have been developed for so called “simulated” microgravity. A classical systems is a clinostat, basically rotating a sample around one axis, and by integration of the gravity vector for 360° arguing that thus the effects of gravity are depleted. Indeed, a variety of studies has shown that taking out the direction of gravity from a biological system often results in consequences similar to the exposure of the system to real microgravity. Nevertheless, the opposite has been shown, too, and as a consequence the relevance of clinostats in microgravity research is still under discussion. To get some more insight into this problem we have constructed a small fluorescence clinostat and have studied the effects of clinorotation on the cytosolic calcium concentration of neuroglioma cells. The results have been compared to experiments with identical cells in real microgravity, utilizing parabolic flight missions. Our results show that in case of a cell suspension used in a small florescence clinostat within a tube diameter of 2mm, the effects of clinorotation are comparable to those under real microgravity, both showing a significant increase in intracellular calcium concentration.

The Role of Ca2+ Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca2+]i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (∆Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca2+]i and ROS production, and reduced GSH content, catalase activity, ∆Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca2+ and drop in ∆Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca2+-independent portion of oxidative stress and cytotoxicity emerges.

Calcium role in human carcinogenesis: a comprehensive analysis and critical review of literature.

The central role played by calcium ion in biological systems has generated an interest for its potential implication in human malignancies. Thus, lines of research, on possible association of calcium metabolism regulation with tumorigenesis, implying disruptions and/or alterations of known molecular pathways, have been extensively researched in the recent decades. This paper is a critical synthesis of these findings, based on a functional approach of the calcium signaling toolkit. It provides strong support that this ubiquitous divalent cation is involved in cancer initiation, promotion, and progression. Different pathways have been outlined, involving equally different molecular and cellular structures. However, if the association between calcium and cancer can be described as constant, it is not always linear. We have identified several influencing factors among which the most relevant are (i) the changes in local or tissular concentrations of free calcium and (ii) the histological and physiological types of tissue involved. Such versatility at the molecular level may probably account for the conflicting findings reported by the epidemiological literature on calcium dietary intake and the risk to develop certain cancers such as the prostatic or mammary neoplasms. However, it also fuels the hypothesis that behind each cancer, a specific calcium pathway can be evidenced. Identifying such molecular interactions is probably a promising approach for further understanding and treatment options for the disease.

Imaging Calcium in Drosophila at Egg Activation.

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in &lt;em&gt;Drosophila&lt;/em&gt; at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton. In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Changes in blood morphology and chosen biochemical parameters in ultra-marathon runners during a 100-km run in relation to the age and speed of runners.

The objective of the study was to reveal morphology, electrolyte and chosen biochemical parameters in terms of health risk in runners in reference to their age and running speed in the case of running a distance of 100 km, which occur after 12 h or 24 h of recovery. Fourteen experienced, male, amateur, ultra-marathon runners, divided into two age and two speed groups took part in the 100-km run. Blood samples for analyses indexes were collected from the ulnar vein just before the run, after 25 km, 50 km, 75 km and 100 km, as well as 12 h and 24 h after termination of the run. The sustained ultramarathon run along with the distance covered (p < 0.05) caused an increase in myoglobin (max 90-fold), bilirubin (max 2.8-fold) and total antioxidant status (max 1.15-fold), which also continued during the recovery. Significant changes in the number of white blood cells were observed with each sequential course and could be associated with muscle damage. The electrolyte showed changes towards slight hyperkalemia, but no changes in natrium and calcium concentrations. There were no significant differences between the age and speed groups for all the parameters after completing the 100-km run as well as after 12 h and 24 h of recovery. Considering changes in blood morphology and chosen biochemical parameters in ultra-marathon runners during a 100-km run it can be stated that such an exhausting effort may be dangerous for human health due to metabolic changes and large damage to the organs. Negative metabolic changes are independent of age of an ultramarathon runner and occur both in younger (32±5.33 years) and older participants (50.56±9.7 years). It can be concluded that organ damage and negative metabolic changes during a 100-km run occur similarly in participants less experienced as well as in well trained runners. Int J Occup Med Environ Health 2016;29(5):801-814.

Sendai Virus Induces Persistent Olfactory Dysfunction in a Murine Model of PVOD via Effects on Apoptosis, Cell Proliferation, and Response to Odorants.

BackgroundViral infection is a common cause of olfactory dysfunction. The complexities of studying post-viral olfactory loss in humans have impaired further progress in understanding the underlying mechanism. Recently, evidence from clinical studies has implicated Parainfluenza virus 3 as a causal agent. An animal model of post viral olfactory disorders (PVOD) would allow better understanding of disease pathogenesis and represent a major advance in the field.ObjectiveTo develop a mouse model of PVOD by evaluating the effects of Sendai virus (SeV), the murine counterpart of Parainfluenza virus, on olfactory function and regenerative ability of the olfactory epithelium.MethodsC57BL/6 mice (6–8 months old) were inoculated intranasally with SeV or ultraviolet (UV)-inactivated virus (UV-SeV). On days 3, 10, 15, 30 and 60 post-infection, olfactory epithelium was harvested and analyzed by histopathology and immunohistochemical detection of S-phase nuclei. We also measured apoptosis by TUNEL assay and viral load by real-time PCR. The buried food test (BFT) was used to measure olfactory function of mice at day 60. In parallel, cultured murine olfactory sensory neurons (OSNs) infected with SeV or UV-SeV were tested for odorant-mixture response by measuring changes in intracellular calcium concentrations indicated by fura-4 AM assay.ResultsMice infected with SeV suffered from olfactory dysfunction, peaking on day 15, with no loss observed with UV-SeV. At 60 days, four out of 12 mice infected with SeV still had not recovered, with continued normal function in controls. Viral copies of SeV persisted in both the olfactory epithelium (OE) and the olfactory bulb (OB) for at least 60 days. At day 10 and after, both unit length labeling index (ULLI) of apoptosis and ULLI of proliferation in the SeV group was markedly less than the UV-SeV group. In primary cultured OSNs infected by SeV, the percentage of cells responding to mixed odors was markedly lower in the SeV group compared to UV-SeV (P = 0.007).ConclusionWe demonstrate that SeV impairs olfaction, persists in OE and OB tissue, reduces their regenerative ability, and impairs the normal physiological function of OSNs without gross cytopathology. This mouse model shares key features of human post-viral olfactory loss, supporting its future use in studies of PVOD. Further testing and development of this model should allow us to clarify the pathophysiology of PVOD.

The calcium-frequency response in the rat ventricular myocyte: an experimental and modelling study.

In the majority of species, including humans, increased heart rate increases cardiac contractility. This change is known as the force-frequency response (FFR). The majority of mammals have a positive force-frequency relationship (FFR). In rat the FFR is controversial. We derive a species- and temperature-specific data-driven model of the rat ventricular myocyte. As a measure of the FFR, we test the effects of changes in frequency and extracellular calcium on the calcium-frequency response (CFR) in our model and three altered models. The results show a biphasic peak calcium-frequency response, due to biphasic behaviour of the ryanodine receptor and the combined effect of the rapid calmodulin buffer and the frequency-dependent increase in diastolic calcium. Alterations to the model reveal that inclusion of Ca(2+) /calmodulin-dependent protein kinase II (CAMKII)-mediated L-type channel and transient outward K(+) current activity enhances the positive magnitude calcium-frequency response, and the absence of CAMKII-mediated increase in activity of the sarco/endoplasmic reticulum Ca(2+) -ATPase induces a negative magnitude calcium-frequency response. An increase in heart rate affects the strength of cardiac contraction by altering the Ca(2+) transient as a response to physiological demands. This is described by the force-frequency response (FFR), a change in developed force with pacing frequency. The majority of mammals, including humans, have a positive FFR, and cardiac contraction strength increases with heart rate. However, the rat and mouse are exceptions, with the majority of studies reporting a negative FFR, while others report either a biphasic or a positive FFR. Understanding the differences in the FFR between humans and rats is fundamental to interpreting rat-based experimental findings in the context of human physiology. We have developed a novel model of rat ventricular electrophysiology and calcium dynamics, derived predominantly from experimental data recorded under physiological conditions. As a measure of FFR, we tested the effects of changes in stimulation frequency and extracellular calcium concentration on the simulated Ca(2+) transient characteristics and showed a biphasic peak calcium-frequency relationship, consistent with recent observations of a shift from negative to positive FFR when approaching the rat physiological frequency range. We tested the hypotheses that (1) inhibition of Ca(2+) /calmodulin-dependent protein kinase II (CAMKII)-mediated increase in sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) activity, (2) CAMKII modulation of SERCA, L-type channel and transient outward K(+) current activity and (3) Na(+) /K(+) pump dynamics play a significant role in the rat FFR. The results reveal a major role for CAMKII modulation of SERCA in the peak Ca(2+) -frequency response, driven most significantly by the cytosolic calcium buffering system and changes in diastolic Ca(2+) .