The relationship between cellular and calcium responses of <Emphasis Type="Italic">Aspergillus awamori</Emphasis> to external influences

The cellular and Ca²⁺ responses to physiological stimuli of different nature were studied in the experiments with the strain Aspergillus awamori 66A containing recombinant aequorin, a Ca²⁺-dependent photosensitive protein. The relationship between the cellular response registered by changes in the development of the mycelial fungus (colony growth, hyphal branching, and the rate of spore formation) and the level and duration of calcium flares in the cytosol was assessed. The physical or chemical stimuli (mechanical effect, osmotic shock) inducing short-time calcium flares in the cytosol did not influence significantly the development of A. awamori grown in liquid or on solid nutrient media. The action on the 24-h A. awamori culture of the Ca²⁺-selective ionophore (A23187) inducing long-term changes in calcium homeostasis caused disorders in the fungus development and morphology (hyperbranching of mycelial hyphae, formation of spherical cells, and inhibition of colony growth and spore formation). Thus, it was established that the development of cellular response in the micromycete correlated with the duration of the calcium flare in the cytosol.     

Intracellular targeting of the photoprotein aequorin: A new approach for measuring, in living cells, Ca2+ concentrations in defined cellular compartments

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca²⁺ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a mitochondrially-targeted aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca²⁺, [Ca²⁺]_m, were obtained, which showed that [Ca²⁺]_m is low at rest (<0.5>M), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria sense very accurately the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and after cell stimulation [Ca²⁺]_m rises to values capable of activating the Ca²⁺-sensitive mitochondrial dehydrogenases.     

Regulation of phosphoglycerate mutase in developing forespores and dormant spores ofBacillus megaterium by thein vivo levels of phosphoglycerate mutase inhibitor

Bacillus megaterium accumulated 3-phosphoglycerate during sporulation which was utilized during spore germination. During sporulation a protein was synthesized before or at the start of 3-phosphoglycerate accumulation inside the developing spores about 1.5 h before dipicolinic acid accumulation. This protein has an affinity for Mn²⁺ and other divalent metal ions and inhibits phosphoglycerate mutase activity which has been shown to require Mn²⁺ However, the levels of the inhibitor decreased considerably (75–85%) during spore germination. No appreciable amount of the inhibitor was detected in the vegetable cell and mother cell compartment; however, the forespore compartment possesses an activity comparable to that of dormant spores. The partially purified inhibitor has a molecular weight of 11,000 and possesses both high and low affinity binding sites for Mn²⁺ and Ca²⁺ as determined by Scatchard plot analysis.     

Antifungal Activity of Thymol against Aspergillus awamori and Botrytis aclada Isolated from Stored Onion Bulbs

The antifungal activity of thymol against Aspergillus awamori F23 and Botrytis aclada F15 in onions was examined through direct treatment with amended media and gaseous treatment with I-plates (plastic plates containing central partitions). The protective and curative control efficacy of thymol was examined 24 h before and after the inoculation of onion bulbs with the fungal isolates. Mycelial growth, sporulation, and spore germination of the isolates were inhibited on potato dextrose agar amended with various concentrations of thymol or acetic acid (positive control). Overall, thymol produced a stronger inhibitory effect on the mycelial growth and development of the isolates than acetic acid. Following gaseous treatment in I-plates, mycelial growth, sporulation, and spore germination of the isolates were inhibited at higher concentrations of thymol or acetic acid; however, acetic acid showed a little effect on the sporulation and spore germination of the isolates. Following the treatment of onion bulbs with 1000 mg L⁻¹ of thymol 24 h before and after fungal inoculation, lesion diameter was greatly reduced compared with that following treatment with 0.5% ethanol (solvent control). Onion bulbs sprayed with thymol 24 h before fungal inoculation generally showed reduced lesion diameters by isolate F23 but not in isolate F15 compared with those sprayed 24 h after fungal inoculation. Collectively, thymol effectively inhibited the growth and development of A. awamori and B. aclada on amended media and in I-plates. In addition, spraying or fumigation of thymol is more desirable for effectively controlling these postharvest fungal pathogens during long-term storage conditions.     

Intracellular targeting of the photoprotein aequorin: A new approach for measuring,in living cells,Ca<Superscript>2+</Superscript> concentrations in defined cellular compartments

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca²⁺ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a “mitochondrially-targeted” aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca²⁺, [Ca²⁺]_m, were obtained, which showed that [Ca²⁺]_m is low at rest (<0.5 μM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria “sense” very accurately the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and after cell stimulation [Ca²⁺]_m rises to values capable of activating the Ca²⁺-sensitive mitochondrial dehydrogenases.     

An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy

Changes in membrane and macromolecular fluidity which may accompany the differentiation processes of sporulation and germination in Bacillus megaterium K.M. are examined by electron spin and nuclear magnetic resonance spectroscopy. No change in membrane lipid fluidity is observed in isolated forespores up to stage VI. Between stage VI and release of mature spores, the ESR spectrum of doxylstearic acid spin labels becomes polycrystalline. This change in spectral fluidity is completely reversed during germination and is paralleled by the rapid release of Ca²⁺ from the spore. NMR studies also show that the mature spore has reduced macromolecular mobility and an increased non-exchangeable water pool compared with vegetative cells.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

Biochemical characterization of the plasma membrane Ca2+ pumping ATPase activity present in the gill cells of Mytilus galloprovincialis LAM

• 1.1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca²⁺-activated ATPase activity is present. The ATPase has high Ca²⁺ affinity (K_ma = 0.3 μM).
• 2.2. The optimum assay conditions to evaluate the enzymatic activity of the Ca²⁺-stimulated ATPase at 19°C are: 120–300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca²⁺ ATPase activity is stimulated by DTT (0.5–1 mM) and it is inhibited by low concentrations of vanadate (10–50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive.
• 3.3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca²⁺ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La³⁺; (c) the mol. wt of Ca²⁺ ATPase is about 140 kDa.
• 4.4. Low Ca²⁺ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca²⁺ uptake by plasma membrane inside-out vesicles.
• 5.5. The results indicate that the Ca²⁺ pump present in the gill plasma membranes could be responsible for Ca²⁺ extrusion and therefore involved in maintaining the cytosolic Ca²⁺ concentration within physiological levels.

     

Protein phosphorylation and its regulation by calcium and calmodulin in membrane fractions from zucchini hypocotyls

Protein-kinase activity has been found to be associated with a membrane fraction obtained from dark-grown zucchini (Cucurbita pepo L., cv. Senator) hypocotyl hooks. Proteins of this membrane fraction were used as protein substrates. The effects of Mg²⁺, Na⁺ and K⁺ on phosphorylation, measured as ³²P incorporation, was investigated. The kinetics of phosphorylation of the individual protein peptides indicate the presence of specific phosphatase activity. Phosphorylation activity is strongly influenced by Ca²⁺. One peptide (relative molecular weight: 180,000) exhibits strong inhibition of ³²P incorporation at physiological Ca²⁺ concentrations between 0.1 and 1 μM. Phosphorylation of about 10 other proteins was enhanced by Ca²⁺, being maximal in most cases at a concentration of about 3 μM free Ca²⁺. Five out of these 10 peptides show increased phosphorylation in the presence of 1 μM calmodulin. This calmodulin-dependent enhancement of phosphorylation could be completely inhibited by the calmodulin antagonist fluphenazine. Cyclic AMP was found to have no stimulating effect on protein phosphorylation.     

HtrC Is Involved in Proteolysis of YpeB during Germination of Bacillus anthracis and Bacillus subtilis Spores

Bacterial endospores can remain dormant for decades yet can respond to nutrients, germinate, and resume growth within minutes. An essential step in the germination process is degradation of the spore cortex peptidoglycan wall, and the SleB protein in Bacillus species plays a key role in this process. Stable incorporation of SleB into the spore requires the YpeB protein, and some evidence suggests that the two proteins interact within the dormant spore. Early during germination, YpeB is proteolytically processed to a stable fragment. In this work, the primary sites of YpeB cleavage were identified in Bacillus anthracis, and it was shown that the stable products are comprised of the C-terminal domain of YpeB. Modification of the predominant YpeB cleavage sites reduced proteolysis, but cleavage at other sites still resulted in loss of full-length YpeB. A B. anthracis strain lacking the HtrC protease did not generate the same stable YpeB products. In B. anthracis and Bacillus subtilis htrC mutants, YpeB was partially stabilized during germination but was still degraded at a reduced rate by other, unidentified proteases. Purified HtrC cleaved YpeB to a fragment similar to that observed in vivo, and this cleavage was stimulated by Mn²⁺ or Ca²⁺ ions. A lack of HtrC did not stabilize YpeB or SleB during spore formation in the absence of the partner protein, indicating other proteases are involved in their degradation during sporulation.     

In vitro and In vivo Effects of Some Fungicides against the Chickpea Blight Pathogen, Ascochyta rabiei

The effects of various fungicides on mycelial growth and spore germination of Ascochyta rabiei were determined by incorporating them into potato dextrose agar and measuring colony diameter and observing colony growth and spore germination at 20 ± 2°C. Eight fungicides prevented spore germination of the pathogen at concentrations of 0.125–2 μg/ml, three hindered mycelial growth at 2–4 μg/ml and seven failed to inhibit mycelial growth even at 128 μg/ml. The reference fungicide for the pathogen, chlorothalonil, stopped conidial germination at low rates but did not prevent mycelial growth at 128 μg/ml. Thirteen fungicides were tested against seed infections of the pathogen, and benomyl + thiram, carbendazim and carbendazim + chlorothalonil seed treatments gave more than 85% inhibition on both vacuum‐infiltrated and naturally infected seeds. Coating the seeds with polymers did not increase the effectiveness of fungicides. Three fungicides; (azoxystrobin, chlorothalonil and mancozeb), gave the highest protection in the field but protection decreased with increased inoculum pressure. Addition of humic acid to fungicide suspensions did not affect their performance.     

Effects of Cd2+, Mn2+, and Al3+ on rat brain synaptosomal uptake of noradrenaline and serotonin

Cd²⁺, Mn²⁺, and Al³⁺ inhibited synaptosomal amine uptake in a concentration-dependent and time-dependent manner. In the absence of Ca²⁺, the rank order of inhibition of noradrenaline uptake was: Cd²⁺ (IC₅₀ = 250 μM) > Al³⁺ (IC₅₀ = 430 μM) > Mn²⁺ (IC₅₀ = 1.50 mM), the IC₅₀ being the concentration of metal ions that gave rise to 50% inhibition of uptake. In the presence of 1 mM Ca²⁺, the rank order of inhibition of uptake was: Al³⁺ (IC₅₀ = 330 μM) > Cd²⁺ (IC₅₀ = 540 μM) > (IC₅₀ = 1.5 mM). The rank order of inhibition of serotonin uptake without Ca²⁺ was: Al³⁺ (IC₅₀ = 370 μM) > Cd²⁺ (IC₅₀ = 610 μM) > Mn²⁺ (IC₅₀ = 3.4 mM) and the rank order in the presence of 1 mM Ca²⁺ was: Al³⁺ (IC₅₀ = 290 μM) > Cd²⁺ (IC₅₀ = 1.5 mM) > Mn²⁺ (IC₅₀ = 4.0 mM). Ca²⁺, at 1 mM, definitely antagonized the inhibitory actions of Cd²⁺ on noradrenaline and serotonin uptake. Al³⁺ stimulated noradrenaline uptake at concentrations around 20–250 μM but inhibited this uptake at concentrations exceeding 300 μM in a dose-related fashion. Ca²⁺, at 1 mM, enhanced both the stimulatory and inhibitory effects of Al³⁺. Ca²⁺ also enhanced the inhibitory actions of Al³⁺ on seotonin uptake. These results, in conjunction with those we have previously published, suggest that Cd²⁺, Mn²⁺, and Al³⁺ exert differential and selective effects on the structure and function of synaptosomal membranes.     

Molecular signatures of neurodegeneration in the cortex of PS1/PS2 double knockout mice

Background

Dimebon is an antihistamine compound with a long history of clinical use in Russia. Recently, Dimebon has been proposed to be useful for treating neurodegenerative disorders. It has demonstrated efficacy in phase II Alzheimer's disease (AD) and Huntington's disease (HD) clinical trials. The mechanisms responsible for the beneficial actions of Dimebon in AD and HD remain unclear. It has been suggested that Dimebon may act by blocking NMDA receptors or voltage-gated Ca²⁺ channels and by preventing mitochondrial permeability pore transition.

Results

We evaluated the effects of Dimebon in experiments with primary striatal neuronal cultures (MSN) from wild type (WT) mice and YAC128 HD transgenic mice. We found that Dimebon acts as an inhibitor of NMDA receptors (IC50 = 10 μM) and voltage-gated calcium channels (IC50 = 50 μM) in WT and YAC128 MSN. We further found that application of 50 μM Dimebon stabilized glutamate-induced Ca²⁺ signals in YAC128 MSN and protected cultured YAC128 MSN from glutamate-induced apoptosis. Lower concentrations of Dimebon (5 μM and 10 μM) did not stabilize glutamate-induced Ca²⁺ signals and did not exert neuroprotective effects in experiments with YAC128 MSN. Evaluation of Dimebon against a set of biochemical targets indicated that Dimebon inhibits α-Adrenergic receptors (α_1A, α_1B, α_1D, and α_2A), Histamine H₁ and H₂ receptors and Serotonin 5-HT_2c, 5-HT_5A, 5-HT₆ receptors with high affinity. Dimebon also had significant effect on a number of additional receptors.

Conclusion

Our results suggest that Ca²⁺ and mitochondria stabilizing effects may, in part, be responsible for beneficial clinical effects of Dimebon. However, the high concentrations of Dimebon required to achieve Ca²⁺ stabilizing and neuroprotective effects in our in vitro studies (50 μM) indicate that properties of Dimebon as cognitive enhancer are most likely due to potent inhibition of H1 histamine receptors. It is also possible that Dimebon acts on novel high affinity targets not present in cultured MSN preparation. Unbiased evaluation of Dimebon against a set of biochemical targets indicated that Dimebon efficiently inhibited a number of additional receptors. Potential interactions with these receptors need to be considered in interpretation of results obtained with Dimebon in clinical trials.     

The use of lanthanum and cytochalasin B to study calcium effects on skeletal muscle differentiation in vitro

A dual effect of external Ca²⁺ on creatine kinase (CPK) accumulation during myogenesis has recently been demonstrated (Morris and Cole, '79). Ca²⁺ inhibits muscle-specific CPK accumulation at intermediate (50–100 μ) concentrations compared with both lower (no added Ca²⁺) and higher (2–3 μ) concentrations. Myoblast fusion, however, requires high Ca²⁺ and is inhibited at both low and intermediate Ca²⁺ levels. These effects are now investigated further by studying the effects of lanthanum ion (La³⁺), which interferes with Ca²⁺-binding to membranes and Ca²⁺-transport, and cytochalasin B, which affects the cell membrane and prevents cell fusion without inhibiting CPK accumulation. The results show that low concentrations (10–100 μ) of La³⁺ inhibit the appearance of the muscle-specific (MM) CPK isoenzyme during myogenesis without significantly affecting cell fusion or intracellular cyclic AMP levels. Three further observations are consistent with the existence of myotube-specific membrane-binding sites for Ca²⁺, which are involved in the stimulation of CPK accumulation on increasing external Ca²⁺ from intermediate to high concentrations. (1) CPK levels are not affected by La³⁺ at 0–50 μ external Ca²⁺. (2) CPK levels in cytochalasin B treated myoblasts are hardly affected by La³⁺ at any Ca²⁺ concentration. (3) In cytochalasin B treated cultures, CPK levels are not increased by raising external Ca²⁺ from intermediate to high levels. In contrast, the stimulation of CPK accumulation on decreasing external Ca²⁺ from intermediate to very low concentrations is not affected by either La³⁺ or cytochalasin B. Some alternative interpretations of the data are also considered, including direct disruption of a membrane Ca²⁺-binding site by cytochalasin B.     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Ionophore A 23187 stops tip growth,but not cytoplasmic streaming,in pollen tubes ofLilium longiflorum

Summary The effects of the cationophore A 23187 on growing pollen tubes ofLilium longiflorum and on pollen germination were testedin vitro, and measured light microscopically. The ionophore is a very potent inhibitor of pollen tube growth: ionophore contentrations down to 10^–7 M stop tip growth. Cytoplasmic streaming is less sensitive: Only with added external Ca²⁺ and higher concentrations of the ionophore the cytoplasmic streaming is stopped. Pollen germination is less sensitive to ionophore than pollen tube growth at later stages. The ionophore inhibition is partially reversible in a medium containing no added external Ca²⁺, but is not reversible in a Ca²⁺-enriched medium. EDTA addition to the medium prevents pollen germination and growth totally. It is hypothesized that the pollen ofLilium longiflorum needs Ca²⁺ to sustain oriented exocytosis at the pollen tube tip. The ionophore A 23187 seems to interfere with the electrical pulse/Ca²⁺-orientation mechanism of exocytosis by equilibration of the Ca²⁺-gradient.     

Winter wheat mitochondria functioning in vitro in the presence of calcium ions and stress uncoupling CSP310 protein

The effects of different Ca²⁺ concentrations on winter wheat (Triticum aestivum L.) functioning and cytochrome c release after organelle incubation with cold-shock protein with a mol. wt of 310 kD or after cold shock were studied. Low (1–5 μM) and high (25–50 μM) Ca²⁺ concentrations inhibited mitochondrial respiration in control seedlings, whereas 10 μM Ca²⁺ enhanced respiration in state 4 and reduced indices characterizing coupling (respiratory control (RC) and ADP: O ratio). At concentrations of 6–20 and 50 μM, Ca²⁺ ions suppressed CSP310 uncoupling effect, which reduced the rate of respiration and an increase in the RC and ADP: O ratio. Low-temperature stress and exogenous CSP310 induced cytochrome c leakage from winter wheat mitochondria both in the absence of Ca²⁺ and in the presence of its low concentrations.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.     

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.