Calcium bioaccessibility increased during gastrointestinal digestion of α-lactalbumin and ß-lactoglobulin.

Calcium bioaccessibility depends on the amount of soluble calcium under intestinal digestion. The changes in calcium during in vitro static digestion of α-lactalbumin and ß-lactoglobulin in presence of calcium chloride (0 mM, 20 mM and 50 mM) were followed by combining electrochemical determination of free calcium with the determination of soluble calcium by inductively coupled plasma optical emission spectroscopy. α-Lactalbumin and, more evident, ß-lactoglobulin were found to increase calcium bioaccessibility with increasing intestinal digestion time by around 5% and 10%, respectively, due to the complex binding of calcium to peptides formed from protein hydrolysis by gastrointestinal enzymes. In vitro digested samples of ß-lactoglobulin in presence of CaCl2 had nearly twice as much complex bound calcium as α-lactalbumin samples. The calcium bioaccessibility decreased significantly with the increasing concentration of added calcium chloride, although the amount of calcium chloride had little effect on the extension of digestion of α-lactalbumin and ß-lactoglobulin. Simulated digestion fluids were found to have a negative effect on calcium bioaccessibility, especially the presence of hydrogen phosphate, and the amount of precipitated calcium increased significantly with increasing amount of added calcium chloride. Based on analysis and visualization by sequences of the peptides formed during digestion of α-lactalbumin and ß-lactoglobulin, it was observed that peptides containing aspartic acid and glutamic acid acting as calcium chelators, may prevent precipitation of calcium in the intestines and increase calcium bioaccessibility. These results provide knowledge for the design of new dairy based functional foods to prevent calcium deficiency.

Theoretical and Experimental Models to Evaluate the Possibility of Corrosion Resistant Concrete for Coastal Offshore Structures.

This study built theoretical and practical models to evaluate the corrosion resistance of concrete for coastal offshore structures in Vietnam. A mathematical model was developed in the form of a system of nonlinear partial differential equations characterizing the diffusion "free calcium hydroxide" in a solid of a concrete structure. The model describes the process of non-stationary mass conductivity observed in the "concrete structure-marine environment" system under non-uniform arbitrary initial conditions, as well as combined boundary conditions of the second and third kind, taking into account the nonlinear nature of the coefficients of mass conductivity k and mass transfer ß. It was shown that the solution of the boundary value problem of non-stationary mass conductivity allows us to conclude about the duration of the service life of a concrete structure, which will be determined by the processes occurring at the interface: in concrete-mass conductivity, depending on the structural and mechanical characteristics of hydraulic structures, and in the liquid phase-mass transfer, determined by the conditions of interaction at the interface of the indicated phases.

Methodology for Cross-Talk Elimination in Simultaneous Voltage and Calcium Optical Mapping Measurements With Semasbestic Wavelengths.

Most cardiac arrhythmias at the whole heart level result from alteration of cell membrane ionic channels and intracellular calcium concentration ([Ca2+] i ) cycling with emerging spatiotemporal behavior through tissue-level coupling. For example, dynamically induced spatial dispersion of action potential duration, QT prolongation, and alternans are clinical markers for arrhythmia susceptibility in regular and heart-failure patients that originate due to changes of the transmembrane voltage (V m) and [Ca2+] i . We present an optical-mapping methodology that permits simultaneous measurements of the V m - [Ca2+] i signals using a single-camera without cross-talk, allowing quantitative characterization of favorable/adverse cell and tissue dynamical effects occurring from remodeling and/or drugs in heart failure. We demonstrate theoretically and experimentally in six different species the existence of a family of excitation wavelengths, we termed semasbestic, that give no change in signal for one dye, and thus can be used to record signals from another dye, guaranteeing zero cross-talk.

Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights.

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos.

Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus-oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

Online cement clinker quality monitoring: A soft sensor model based on multivariate time series analysis and CNN.

The content of free calcium oxide (f-CaO) in cement clinker is an important index for cement quality. Aiming at the characteristics of strong coupling, time-varying delay and highly non-linearity in cement clinker production, a soft sensor model based on multivariate time series analysis and convolutional neural network (MVTS-CNN) is proposed for the online f-CaO content monitoring. Based on the process industry characteristics, the MVTS-CNN modeling involves the detailed analysis of coupling relationship and time-varying delay in cement production and the application of neural network in multivariate time-series feature extraction. The main researches and contributions are fourfold: First, the strong coupling in the production system is further analyzed, and the proposed model is focused on the data coupling between specific processes, not the control coupling. Second, a multivariate time series analysis method is designed to select the time series that may have direct impacts on the f-CaO content in different production conditions, which is founded on the information on time delay range and longest active duration. Third, a multivariate time series feature extraction method is designed and adopted in the MVTS-CNN model to extract the multivariate time series features, such as active duration difference features, coupling features, nonlinear features and key time series features. Fourth, a new timing matching method, which is combined the rough timing matching of multivariate time series and the detailed timing matching of key features, is proposed to deal with the time-varying delay in various production conditions. Compared with traditional CNN, support vector machines (SVM) and long-short term memory networks (LSTM), the results demonstrate that the MVTS-CNN model has higher accuracy, better generalization ability and superior robustness.

Lime Juice Enhances Calcium Bioaccessibility from Yogurt Snacks Formulated with Whey Minerals and Proteins.

Yogurt-based snacks originally with a calcium content between 0.10 and 0.17 mmol/g dry matter were enriched with a whey mineral concentrate and whey protein isolate or hydrolysate. Whey mineral concentrate was added to increase the total amount of calcium by 0.030 mmol/g dry matter. Calcium bioaccessibility was determined following an in vitro protocol including oral, gastric, and intestinal digestion, with special focus on the effect of lime juice quantifying calcium concentration and activity. Calcium bioaccessibility, defined as soluble calcium divided by total calcium after intestinal digestion amounted to between 17 and 25% for snacks without lime juice. For snacks with lime juice, the bioaccessibility increased to between 24 and 40%, an effect attributed to the presence of citric acid. Citric acid increased the calcium solubility both from whey mineral concentrate and yogurt, and the citrate anion kept supersaturated calcium soluble in the chyme. The binding of calcium in the chyme from snacks with or without lime juice was compared electrochemically, showing that citrate increased the amount of bound calcium but with lower affinity. The results indicated that whey minerals, a waste from cheese production, may be utilized in snacks enhancing calcium bioaccessibility when combined with lime juice.

Understanding Calcium-Dependent Conformational Changes in S100A1 Protein: A Combination of Molecular Dynamics and Gene Expression Study in Skeletal Muscle.

The S100A1 protein, involved in various physiological activities through the binding of calcium ions (Ca2+), participates in several protein-protein interaction (PPI) events after Ca2+-dependent activation. The present work investigates Ca2+-dependent conformational changes in the helix-EF hand-helix using the molecular dynamics (MD) simulation approach that facilitates the understanding of Ca2+-dependent structural and dynamic distinctions between the apo and holo forms of the protein. Furthermore, the process of ion binding by inserting Ca2+ into the bulk of the apo structure was simulated by molecular dynamics. Expectations of the simulation were demonstrated using cluster analysis and a variety of structural metrics, such as interhelical angle estimation, solvent accessible surface area, hydrogen bond analysis, and contact analysis. Ca2+ triggered a rise in the interhelical angles of S100A1 on the binding site and solvent accessible surface area. Significant configurational regulations were observed in the holo protein. The findings would contribute to understanding the molecular basis of the association of Ca2+ with the S100A1 protein, which may be an appropriate study to understand the Ca2+-mediated conformational changes in the protein target. In addition, we investigated the expression profile of S100A1 in myoblast differentiation and muscle regeneration. These data showed that S100A1 is expressed in skeletal muscles. However, the expression decreases with time during the process of myoblast differentiation.

Inorganic polyphosphate is required for sustained free mitochondrial calcium elevation, following calcium uptake.

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake.

Hesperidin Improves Colonic Motility in Loeramide-Induced Constipation Rat Model via 5-Hydroxytryptamine 4R/cAMP Signaling Pathway.

Fructus has motivation effect on gastrointestinal tract. Hesperidin is extracts of Fructus, and we attempted to prove its effects on improving the gastrointestinal transmission function and determine the possible mechanisms by a loperamide-induced slow transit constipation (STC) model. Constipation phenotypes were measured in rats with Lop-induced constipation after treatment with hesperidin. The amounts and water content of stool were significantly higher in the hesperidin-treated group than the loperamide-induced model group, whereas food intake was maintained at constant levels. Moreover, intestinal transit rate was increased in the treatment group of hesperidin. Histological alteration was detected by H&E staining, we found that the colon smooth muscle cells and neuron cells of the rats were increased, and the infiltration of inflammatory cells was decreased in the hesperidin-treated group compared with the loperamide-induced model group. 5-Hydroxytryptamine (5-HT) receptor4 fluorescence intensity and intracellular-free calcium ions in colon tissue were increased, and relative protein of cAMP/PKA pathway and p-cAMP response component-binding protein (CREB) pathway were upregulated in the hesperidin-treated group compared with the loperamide-induced model group. Further, SMCs from colon tissue of rats were cultured and identified. We found hesperidin could significantly promote tegaserod-induced increase of 5-HTR4 fluorescence intensity, intracellular calcium ions, relative protein of cAMP/PKA pathway and p-CREB pathway, and cell proliferation and inhibit GR113808-induced decrease of 5-HTR4 fluorescence intensity, 5-HTR4 pathway-related proteins (ADCY3, cAMP, PKA, and p-CREB), intracellular calcium ions, and cell proliferation. The analysis of our data suggested that hesperidin could obviously improve the gastrointestinal transmission function in loperamide-induced STC rat model via increasing the 5-HTR4 and intracellular-free calcium ions to enhance the expression of relative protein of cAMP/PKA pathway and p-CREB pathway. Hesperidin could be used in the treatment of STC, and our data not only provide experimental basis for the treatment of STC in hesperidin but also provides a theoretical reference for clinical treatment.

Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.

Simulated microgravity reduces intracellular-free calcium concentration by inhibiting calcium channels in primary mouse osteoblasts.

Calcium homeostasis in osteoblasts plays fundamental roles in the physiology and pathology of bone tissue. Various types of mechanical stimuli promote osteogenesis and increase bone formation elicit increases in intracellular-free calcium concentration in osteoblasts. However, whether microgravity, a condition of mechanical unloading, exerts an influence on intracellular-free calcium concentration in osteoblasts or what mechanisms may underlie such an effect are unclear. Herein, we show that simulated microgravity reduces intracellular-free calcium concentration in primary mouse osteoblasts. In addition, simulated microgravity substantially suppresses the activities of L-type voltage-sensitive calcium channels, which selectively allow calcium to cross the plasma membrane from the extracellular space. Moreover, the functional expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors, which mediate the release of calcium from intracellular storage, decreased under simulated microgravity conditions. These results suggest that simulated microgravity substantially reduces intracellular-free calcium concentration through inhibition of calcium channels in primary mouse osteoblasts. Our study may provide a novel mechanism for microgravity-induced detrimental effects in osteoblasts, offering a new avenue to further investigate bone loss induced by mechanical unloading.

Calcium and Bone Metabolism Indices.

Calcium and inorganic phosphate are of critical importance for many body functions, thus the regulations of their plasma concentrations are tightly controlled by the concerted actions of reabsorption/excretion in the kidney, absorption in the intestines, and exchange from bone, the major reservoir for calcium and phosphate in the body. Parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)2D) control calcium homeostasis, whereas PTH, 1,25(OH)2D, and bone-derived fibroblast growth factor 23 (FGF 23) control phosphate homeostasis. Hypoparathyroidism can cause hypocalcemia and hyperphosphatemia, whereas deficient vitamin D actions can cause osteomalacia in adults and rickets in children. Hyperparathyroidism, alternatively, can cause hypercalcemia and hypophosphatemia. Laboratory tests of calcium, phosphate, PTH, and 25-hydroxyvitamin D are very useful in the diagnosis of abnormalities associated with calcium and/or phosphate metabolisms. Bone is constantly remodeled throughout life in response to mechanical stress and a need for calcium in extracellular fluids. Metabolic bone diseases such as osteoporosis, osteomalacia in adults or rickets in children, and renal osteodystrophy develop when bone resorption exceeds bone formation. Bone turnover markers (BTM) such as serum N-terminal propeptide of type I procollagen (P1NP) and C-terminal collagen cross-link (CTX) may be useful in predicting future fracture risk or monitoring the response to anti-resorptive therapy. There is a need to standardize sample collection protocols because certain BTMs exhibit large circadian variations and tend to be influenced by food intakes. In the United States, a project to standardize BTM sample collection protocols and to establish the reference intervals for serum P1NP and serum CTX is ongoing. We anticipate the outcome of this project to shine lights on the standardization of BTM assays, sample collection protocols, reference intervals in relation to age, sex, and ethnic origins, and clinical utilities of BTMs. This review will briefly discuss the regulations of calcium and phosphate homeostasis, laboratory's role in the diagnosis, and monitoring of bone and calcium metabolism, as well as the usefulness and controversies of the utilities of BTMs in the diagnosis and monitoring of metabolic bone diseases.

Calcium mediates the cellular response of Chlamydomonas reinhardtii to the emerging aquatic pollutant Triclosan.

The present study was aimed at investigating the role of intracellular free calcium, [Ca2+]c, in the early cellular response of the green alga Chlamydomonas reinhardtii to the emergent pollutant Triclosan (13.8µM; 24h of exposure). There is a growing concern about the persistence and toxicity of this antimicrobial in aquatic environments, where non-target organisms such as C. reinhardtii, a primary producer of ecological relevance, might be severely impacted. A mechanistic study was undertaken which combined flow cytometry protocols, physiological as well as gene expression analysis. As an early response, Triclosan strongly altered [Ca2+]c homeostasis which could be prevented by prechelation with the intracellular calcium chelator BAPTA-AM. Triclosan induced ROS overproduction which ultimately leads to oxidative stress with loss of membrane integrity, membrane depolarization, photosynthesis inhibition and mitochondrial membrane depolarization; within this context, Triclosan also induced an increase in caspase 3/7 activity and altered the expression of metacaspase genes which are indicative of apoptosis. All these adverse outcomes were dependent on [Ca2+]c. Interestingly, an interconnection between [Ca2+]c alterations and increased ROS formation by Triclosan was found. Taken altogether these results shed light on the mechanisms behind Triclosan toxicity in the green alga Chlamydomonas reinhardtii and demonstrate the role of [Ca2+]c in mediating the observed toxicity.

Exploring poisonous mechanism of honeybee, Apis mellifera ligustica Spinola, caused by pyrethroids.

As the important intracellular secondary messengers, calcium channel is the target of many neurotoxic pesticides as calcium homeostasis in the neuroplasm play important role in neuronal functions and behavior in insects. This study investigated the effect of deltamethrin (DM) on calcium channel in the brain nerve cells of adult workers of Apis mellifera ligustica Spinola that were cultured in vitro. The results showed that the intracellular calcium concentration was significantly elevated even with a very low concentration of the DM (3.125×10-2mg/L). Further testing revealed that T-type voltage-gated calcium channels (VGCCs), except for sodium channels, was one of the target of DM on toxicity of Apis mellifera, while DM has no significant effect on the L-type VGCCs, N-methyl-d-aspartate receptor-gated calcium channels and calcium store. These results suggesting that the DM may act on T-type VGCCs in brain cells of honeybees and result in behavioral abnormalities including swarming, feeding, learning, and acquisition.

Hydraulic retention time on vinasse stabilisation with limestone in the acidogenic phase of anaerobic digestion / Tempo de retenção hidráulica em vinhaça estabilizada com calcário na fase acidogênica da digestão anaeróbia

ABSTRACT: The main problem in anaerobic digestion of low-protein residues is the instability caused acidity. The use of limestone at the same time as a neutralizing agent and support material is innovative because stones wear allows the slow release of the calcium carbonate thereby eliminating dispersers. Free calcium content in the system was measured in two plug flow reactors filled with vinasse at initial pH of 4.50. The proportion of 1.8 tonnes of limestone per m³ of vinasse was evaluated at the Hydraulic Retention Times (HRT) of 24, 48, 72, 96 and 120 hours, allowing stabilisation at 96 hours. The ratio of Volatile Acids/Total Alkalinity (VA/TA) ranged from 0.2 to 0.4 and the pH reached 7.0, at the HRT of 120 hours. Increasing the HRT also increased the volatile total solids (VTS) and fixed total solids (TFS) in a similar profile to the measured free calcium content, but calcium remained at the appropriate level of 100 to 250mg l-1. The proportion of limestone/vinasse was adequate to ensure stabilisation, but it is not recommended to reduce the HRT below 96 hours due to the risk of compromising the stability of the anaerobic system.

RESUMO: O maior problema na digestão anaeróbia de resíduos de baixo teor de proteína é a instabilidade por acidez. O uso de calcário ao mesmo tempo como agente neutralizador e material suporte é inovador porque permite eliminar o dosador pois o desgaste das pedras permite a liberação lenta do carbonato de cálcio. O teor de cálcio livre no sistema foi medido em dois reatores tipo Plug flow, preenchidos com vinhaça com pH inicial de 4,50. A proporção de 1,8 toneladas de calcário por m³ de vinhaça foi avaliada com os Tempos de Retenção Hidráulica (TRH) de 24, 48, 72, 96 e 120 horas, permitindo a estabilização em 96 horas. Com um TRH de 120 horas, a relação de Ácidos Voláteis/Alcalinidade (AV/AC) variou de 0,2 à 0,4 e o pH atingiu 7,0. Aumentando o TRH também aumentaram os sólidos voláteis totais (SVT) e sólidos totais fixos (STF) em um perfil semelhante ao teor de cálcio livre medido, mas mantendo níveis adequados de 100 a 250mg L-1. A proporção de calcário/vinhaça mostrou-se adequada para garantir a estabilização, mas não se recomenda a redução do TRH abaixo de 96 horas devido ao risco de comprometer a estabilidade do sistema anaeróbio.

N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils.

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na(+)-free HEPES, both NAC and cysteine induced a marked increase in [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

Comparison Between Measured and Calculated Free Calcium Values at Different Serum Albumin Concentrations.

INTRODUCTION: Free ionic calcium is the metabolically active component of total calcium (TCa) in blood. However, most laboratories report TCa levels that are dependent on serum albumin concentration. Hence, several formulae have evolved to calculate free calcium levels from TCa after adjustment for albumin. However, free calcium can directly be measured using direction selective electrodes rather than spectrophotometric methods used in autoanalyzers. OBJECTIVES: This study compares the levels of free calcium obtained by measurement by direct ion selective electrode (ISE) and the one calculated as a function of TCa by formulae. MATERIALS AND METHODS: A total of 254 serum samples submitted to clinical biochemistry laboratory of a tertiary care hospital were analyzed for total protein, albumin, and TCa by standard spectrophotometric methods and for free calcium by direct ISE. Three commonly used formulae viz. Orrell, Berry et al. and Payne et al. were used to calculate adjusted TCa. Calculated free calcium was obtained by taking 50% of these values. RESULTS: A significant difference (P < 0.05) was observed between calculated free calcium by all the three formulae and measured free calcium estimated by direct ISE using paired t-test and Bland-Altman plots. CONCLUSION: Formulae for predicting free calcium by estimating TCa and albumin lacks consistency in prediction and free calcium should be evaluated by direct measurement.

Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein.

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca(2+)]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca(2+) infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 µM) and SKF-96365 (20 µM) signifi cantly inhibited K49-PLA2-induced [Ca(2+)]i increase. These results suggest that K49-PLA2 modulates [Ca(2+)]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca²⁺ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca²⁺]ᵢ) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca²⁺]ᵢ in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca²⁺]ᵢ in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca²⁺]ᵢ in human neutrophils was observed. In Ca²⁺-free buffer, NAC- and cysteine-induced [Ca²⁺]ᵢ increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca²⁺]ᵢ in human neutrophils occur through Ca²⁺ influx. NAC- and cysteine-induced [Ca²⁺]ᵢ increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na⁺-free HEPES, both NAC and cysteine induced a marked increase in [Ca²⁺]ᵢ in human neutrophils, arguing against the possibility that Na⁺-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca²⁺]ᵢ increasing activity. Our results show that NAC and cysteine induce [Ca²⁺]ᵢ increase through Ca²⁺ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.