A new short-term toxicity assay using <Emphasis Type="Italic">Aspergillus awamori</Emphasis> with recombinant aequorin gene

Background  

Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca²⁺ is a universal intracellular signalling molecule found in all organisms from prokaryotes to highly specialized animal cells. In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca²⁺-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca²⁺]_c changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca²⁺ in the cytosol ([Ca²⁺]_c) and the pattern of these changes (Ca²⁺ signature) is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca²⁺]_c changes in a reproducible and dose dependant manner.     

Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca²⁺ concentrations in the mutant strain Aspergillus awamori 66A containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity of sporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 μM) stimulated spore germination: the number of germinating spores was 2–3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 μM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca²⁺ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 μM) were shown to cause an instantaneous increase in Ca²⁺ concentrations compared to the control and lower amphotericin concentration (2.5 μM). Ca²⁺ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.     

Effect of a chemical analogue of autoinducers of microbial anabiosis on the Ca<Superscript>2+</Superscript> response of mycelial fungi

The microbial alkylhydroxybenzenes (AHB), which are anabiosis autoinducers also termed d₁ factors, participate in the stress response of mycelial fungi, as determined from changes in intracellular Ca²⁺ concentration. By using the genetically modified strain Aspergillus awamori 66A, which produces the recombinant Ca²⁺-dependent protein aequorin, the dynamics of Ca²⁺ was studied in the cytosol of cells exposed to mechanical shock in the presence of protective doses (0.001–0.01% w/vol) of a chemical AHB analogue, 4-n-hexylresorcinol. As under stressful conditions, Ca²⁺ concentration increases in the cell cytosol in response to an enhanced AHB level in a growing fungal culture; thus, AHB is perceived by cells as a stress signal. The level of cell response, which was determined from the amplitude of luminescence dependent on the Ca²⁺ concentration in the cytosol, was related to the physiological age of the cells and the AHB concentration. Micromycete preincubation with AHB was found to protect cells from subsequent stress; this was reflected in the Ca²⁺ response. The protective AHB effect was manifested as (1) a significant decrease in the amplitude of luminescence and, thus, in Ca²⁺ accumulation in the cytosol during subsequent mechanical stress (as compared to the control—mechanical stress only); (2) development of a secondary Ca²⁺ response, which was not observed in the control; and (3) a high level of Ca²⁺ retained in the cytosol for a long time in the presence of AHB (as compared to the control without preincubation with AHB). The mechanisms underlying the AHB effect on Ca²⁺ transport systems are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 741–750.Original Russian Text Copyright © 2004 by Kozlova, Kupriyanova-Ashina, Egorov, El-Registan.     

Analysis of the Ca<Superscript>2+</Superscript> response of mycelial fungi to external effects by the recombinant aequorin method

Using the mutant strain Aspergillus awamori 66A, producing the recombinant Ca²⁺-dependent photosensitive protein aequorin, the dynamics of Ca²⁺ was studied for the first time in the cytosol of micromycetes exposed to stressful factors, such as an increase in extracellular Ca²⁺ to 50 mM, hypoosmotic shock, and mechanical shock. The cell response to stress proved to involve an increase in the Ca²⁺ concentration in the cytosol, which was determined from the amplitude of aequorin luminescence and the time of the amplitude enhancement and relaxation. The level of the Ca²⁺ response depended on the physiological stimulus. Inhibitory analysis with various agents that block Ca²⁺ channels and with agonists that specifically enhance the activity of the channels suggested that (1) the level of Ca²⁺ in the cytosol of micromycetes increases in response to stress because of the ion influx from both the growth medium and intracellular reservoirs and (2) potential-dependent transport systems play the major role in the Ca²⁺ influx into the cytosol of the micromycete cells.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 734–740.Original Russian Text Copyright © 2004 by Kozlova, Egorov, Kupriyanova-Ashina, Rid, El-Registan.     

Effect of calcium on growth of submerged <Emphasis Type="Italic">Terfezia boudieri</Emphasis> mycelium

The effect of Ca²⁺ on mycelial growth in Terfezia boudieri was studied. Terfezia boudieri Chatin (Ascomycotinae) occurs in mycorrhizal association with Helianthemum shrubs in deserts with calcareous soils. External Ca²⁺ stimulated mycelial growth in both liquid media and solidified substrates. The response to Ca²⁺ was very faint in well-aerated culture but pronounced in mycelia immersed in the medium, indicating dependence on mycelial aeration. 2,4-Dinitrophenol (DNP), an inhibitor of oxidative phosphorylation, and succinate, a potential cytoplasm acidifier, inhibited mycelial growth but enhanced the stimulatory effect of Ca²⁺. This effect was reduced by the Ca²⁺ channel blocker verapamil.     

Simultaneous imaging of ER and cytosolic Ca2+ dynamics reveals long-distance ER Ca2+ waves in plants

Calcium ions (Ca²⁺) play a key role in cell signaling across organisms. In plants, a plethora of environmental and developmental stimuli induce specific Ca²⁺ increases in the cytosol as well as in different cellular compartments including the endoplasmic reticulum (ER). The ER represents an intracellular Ca²⁺ store that actively accumulates Ca²⁺ taken up from the cytosol. By exploiting state-of-the-art genetically encoded Ca²⁺ indicators, specifically the ER-GCaMP6-210 and R-GECO1, we report the generation and characterization of an Arabidopsis (Arabidopsis thaliana) line that allows for simultaneous imaging of Ca²⁺ dynamics in both the ER and cytosol at different spatial scales. By performing analyses in single cells, we precisely quantified (1) the time required by the ER to import Ca²⁺ from the cytosol into the lumen and (2) the time required to observe a cytosolic Ca²⁺ increase upon the pharmacological inhibition of the ER-localized P-Type IIA Ca²⁺-ATPases. Furthermore, live imaging of mature, soil-grown plants revealed the existence of a wounding-induced, long-distance ER Ca²⁺ wave propagating in injured and systemic rosette leaves. This technology enhances high-resolution analyses of intracellular Ca²⁺ dynamics at the cellular level and in adult organisms and paves the way to develop new methodologies aimed at defining the contribution of subcellular compartments in Ca²⁺ homeostasis and signaling.

Dual color imaging allows the simultaneous analysis of calcium dynamics in the endoplasmic reticulum and cytosol from single cells to adult entire plants.     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Influence of Ca<Superscript>2+</Superscript> ions on metabolism of active oxygen species in wheat calli cocultured with the bunt pathogen <Emphasis Type="Italic">Tilletia caries</Emphasis>

The effect of Ca²⁺ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O²⁻, H₂O₂, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca²⁺ in the culture medium of calli. The increase of the concentration of Ca²⁺ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.     

Studies on the Crayfish Plague Fungus Aphanomyces astaci

A mycelial suspension of the crayfish plague fungus, Aphanomyces astaci, was able to produce large numbers of zoospores, when transferred to redistilled water, at 20°C, even after storage for months at 2°C. Spore production was greater in redistilled water than in tap water and heavier under shake conditions than under stationary ones. In buffered redistilled water sporulation occurred between pH 5 and 8 and the optimal range was about pH 5 to 7. Of the tested aliphatic alcohols, aldehydes, and carboxylic acids, the long analogues were more toxic to spore formation than the shorter ones. Ethylenediamine-tetraacetic acid (EDTA) prevented sporulation probably by removing some essential metal (s) with an affinity for EDTA near that of calcium. Calcium protected against the toxic effect of lithium, sodium, and potassium. Magnesium, only tested against lithium, had no such protecting effect. Cu²⁺, Ni²⁺, Zn²⁺, Co²⁺, K⁺ Mn²⁺, NH₄⁺, Li⁺, Na⁺, Ca²⁺, Mg²⁺ was the approximate order among tested cations in their ability to stop the swimming stage of the zoospores, the first mentioned being the most effective ones. Nitrate and acetate were more active in the same respect than sulphate, chloride, phosphate, or bicarbonate. The optimal pH range for swimming seemed to be pH 6–7.5, and the maximal range 4.5–9.0. The zoospores showed no chemotactic response to tested substances. The germination ability was as high in horse blood as in crayfish blood. A spore suspension stored for 2 months at 2°C still contained viable spores.     

Colony formation of highly dispersed <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> by controlling extracellular polysaccharides and calcium ion concentrations in aquatic solution

This study isolated extracellular polysaccharides (EPS) as a powder material from cyanobacterial blooms and the powdered EPS was used to trigger colony formation of dispersed unicellular M. aeruginosa by controlling EPS concentration in culture medium. The effect of Ca²⁺ ions on the colony formation of M. aeruginosa was also investigated, then the interaction between EPS and Ca²⁺ ions on colony formation was discussed. The results showed that the addition of the powdered EPS into the medium did not cause morphological changes of M. aeruginosa, suggesting that EPS alone would not induce the colony formation of M. aeruginosa. On the other hand, a high concentration of calcium ions (1000 mg/l) caused colony formation. When EPS and Ca²⁺ ions in the culture medium were adjusted to 200 and 1000 mg/l, respectively, the colony density, the average cell number per colony and the particle size of M. aeruginosa showed ca. 1.7–2.0 times greater values than those in the Ca²⁺ added medium. Calcium ion contributed to the aggregation of M. aeruginosa via crosslinked reaction with negatively charged M. aeruginosa cells, and the addition of EPS possessing negatively charged functional groups such as carboxy groups could enhance the reaction, promoting the crosslinked reaction between EPS and Ca²⁺ ions.     

The developmental patterns of lysosomal enzyme activities during Ca2+-induced sporangium formation in Achlya bisexualis

The present paper describes intracellular changes in ribonuclease specific activity during Ca²⁺-induced sporangium formation in the water mold Achlya bisexualis. The enzymes undergo a decrease in activity prior to crosswall formation followed by an increase in activity during spore cleavage. As spore discharge occurs the RNase activity again decreases. A large percentage of the nuclease activity is associated with a lysosomal-like fraction of the cell, but there is also considerably activity associated with nuclear and microsomal fractions. Addition of cycloheximide or actinomycin D at various times during development prevents further decrease or increase in the enzyme activity. Mixing of cell extracts from different developmental stages provides evidence that inhibitors or activators of the enzyme activity are not responsible for the activity levels evident at the different stages. There is a change in the total levels of presumptive mRNA during Ca²⁺-induced sporangial formation which appears to be associated with the patterns of RNase activity. Utilizing total cellular RNA and Poly(A)⁺ RNA with the crude ribonuclease preparations, no substrate specificity could be ascertained.     

Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus

Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca²⁺-mediated signalling. Ca²⁺ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca²⁺ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuB ^KU80, were engineered to express the bioluminescent Ca²⁺-reporter aequorin. An aequorin-mediated method for routine Ca²⁺ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca²⁺]_c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca²⁺, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The ‘signatures’ of the transient [Ca²⁺]_c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca²⁺-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca²⁺-signalling components in each stress response. Concordant with the involvement of Ca²⁺-calmodulin complexes in these Ca²⁺-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca²⁺-signatures to all the challenges. The Ca²⁺-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca²⁺ in initiating the stress responses.     

Calcium-activated factors in pancreatic islets that inhibit actin polymerization

Pancreatic islet cytosol in combination with 50 μm or lower Ca²⁺ markedly inhibits action polymerization when added to actin under conditions in which actin usually polymerizes. Inhibition by either cytosol or Ca²⁺ alone is minimal and heated cytosol is ineffective in inhibiting actin polymerization. Two inhibitory factors with relative molecular weights of about 200,000 and less than 40,000 can be separated by gel permeation chromatography. The larger factor can inhibit actin polymerization almost 100% in the presence of Ca²⁺. Pancreatic beta cells contain a well-described microfilamentous cell web beneath the plasma membrane hypothesized to be a barrier to insulin granule movement. An hypothesis on the function of the factors is that when beta cell calcium increases, as it does in response to stimuli for insulin secretion, calcium interacts with the factors to locally disrupt a microfilamentous barrier to insulin granule extrusion.     

Analysis of EF-hand-containing proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

In plants, calcium (Ca²⁺) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca²⁺]_cyt), which in turn is thought to regulate cellular and developmental processes via Ca²⁺-binding proteins. Three out of the four classes of Ca²⁺-binding proteins in plants contain Ca²⁺-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca²⁺ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.     

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Summary Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the⁴⁵Ca-exchange method to determine free cellular Ca⁺⁺, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca⁺⁺). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca⁺⁺] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca⁺⁺ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca⁺⁺] results in a release of the fusion-block and an increase in cellular Ca⁺⁺ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca⁺⁺] must exist. The latter suggests further that cell surface Ca⁺⁺ was also critical.     

Calcium is essential for fructan synthesis induction mediated by sucrose in wheat

The role of Ca²⁺ in the induction of enzymes involved in fructan synthesis (FSS) mediated by sucrose was studied in wheat (Triticum aestivum). Increase of FSS enzyme activity and induction of the expression of their coding genes by sucrose were inhibited in leaf blades treated with chelating agents (EDTA, EGTA and BAPTA). Ca²⁺ channel blockers (lanthanum chloride and ruthenium red) also inhibited the FSS response to sucrose, suggesting the participation of Ca²⁺ from both extra- and intra- cellular stores. Sucrose induced a rapid Ca²⁺ influx into the cytosol in wheat leaf and root tissues, shown with the Ca²⁺ sensitive fluorescent probe Fluo-3/AM ester. Our results support the hypothesis that calcium is a component of the sucrose signaling pathway that leads to the induction of fructan synthesis.     

Relationship between vitamin D status and deposition of bound calcium in skeletal muscle of the rat

Summary— The effects of vitamin D on the intramuscular distribution of total and bound calcium, phosphate and on available cytosolic calcium, were investigated in skeletal muscle. Total calcium and phosphorus were measured on ashed subcellular fractions of muscles from vitamin D-repleted and vitamin D-deprived rats. The variations in available calcium were followed by determining the activities of calcium-sensitive enzymes in isolated cytosol. Bound-calcium was revealed ultra-microscopically by pyroantimonate. In vitamin D-repleted muscles, the pyroantimonate method revealed specific areas of intense bound-calcium deposition: the myofibrils, where they formed pronounced lines parallel to the Z-bands. In vitamin D-deficient muscles, the calcium-pyroantimonate deposits appeared clearly reduced. This loss was accompanied by a marked reduction in total calcium and phosphorus in all the subcellular fractions, as compared to vitamin D-repleted muscles. Unexpectedly, the activity of the Ca²⁺-activated isocitrate-dehydrogenase was increased in the cytosol, while that of the Ca²⁺-inhibited pyruvate-kinase decreased. Prolonged vitamin D-administration to vitamin D-repleted rats led to an intensification of calcium-pyroantimonate deposits and a general increase in total calcium and phosphorus, but no change in the cytosolic Ca²⁺-sensitive enzyme activities. Cessation of vitamin D-administration to vitamin D-repleted rats produced a regression of calcium-pyroantimonate deposits, a general decrease of total calcium and phosphate levels, and stimulation of the Ca²⁺-activated isocitrate-dehydrogenase accompanied by lowering of the Ca²⁺-inhibited pyruvate-kinase. The results clearly indicate a correlation between vitamin D-repletion and the total and bound calcium content of skeletal muscle. In addition, they demonstrate an apparent contradiction between the decrease of total and bound calcium, and the activities of cytosolic Ca²⁺ sensitive enzymes during vitamin D-deprivation, which can only be explained by an increase in available calcium. It is suggested that vitamin D stimulates intramuscular mechanisms tending to lower available calcium by inactivating the cation via the formation of calcium chelates.     

Calcium-Based Interactions of Symbiotic Partners in Legumes: Role of Peribacteroid Membrane

Based on experimental evidence, a concept is formulated that mutualistic relationships between pro- and eukaryotic cells during nitrogen-fixing legume–rhizobia symbiosis rely both on selective transfer of metabolites and ion transport, Ca²⁺ in particular, across the peribacteroid membrane (PBM). PBM in the nitrogen-fixing cells of yellow lupine (Lupinus luteus L.) and broad bean (Vicia faba L.) is endowed with a calcium-translocating ATPase that pumps Ca²⁺ into the symbiosome. This pumping ensures, on the one hand, calcium homeostasis in the cytosol of infected plant cells and, on the other hand, it optimizes Ca²⁺ level in symbiosomes, first of all in the bacteroids, because Ca²⁺ is one of the main factors controlling their nitrogenase activity. The balance between the symbiotic partners and the maintenance of optimal Ca²⁺ level in the bacteroids also depends on passive Ca²⁺ efflux from symbiosomes to the plant cell cytosol via calcium channels. The Ca²⁺-transporting mechanisms residing at PBM are characterized.     

Relationship between the Molecular Structure of the Nitrogen Source and the Activity of the Extracellular Lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] upon Submerged Cultivation

The activity of the extracellular lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] and the formation of a pigmented mycelial film by this fungus upon submerged cultivation in a synthetic medium were found to depend on the presence of some amino acids (particularly, asparagine) and Ca²⁺ and Mn²⁺ ions in the medium. Quantum-chemical calculations suggest that the different character of the interaction of amino acids with the aforementioned ions is due to differences in the hydrophobicity of the amino acids rather than to differences in the electron structure of the amino acid zwitterions.