The effects of calcium sulphate on growth,membrane stability and nutrient uptake of tomato plants grown under salt stress

A pot experiment was carried out with tomato (Lycopersicon esculentum Mill.) cv. “Target F1” in a mixture of peat, perlite, and sand (1:1:1) to investigate the effects of supplementary calcium sulphate on plants grown at high NaCl concentration (75 mM). The treatments were: (i) control (C), nutrient solution alone; (ii) salt treatment (C + S), 75 mM NaCl; (iii) salt plus calcium treatment 1 (C + S + Ca1), 75 mM NaCl plus additional mixture of 2.5 mM CaSO₄ in nutrient solution; (iv) salt plus calcium treatment 2 (C + S + Ca2), 75 mM NaCl plus additional mixture of 5 mM CaSO₄ in nutrient solution. The plants grown under salt stress produced low dry matter, fruit weight, and relative water content than those grown in standard nutrient solution. Supplemental calcium sulphate added to nutrient solution containing salt significantly improved growth and physiological variables affected by salt stress (e.g. plant growth, fruit yield, and membrane permeability) and also increased leaf K⁺, Ca²⁺, and N in tomato plants. The effects of supplemental CaSO₄ in maintaining membrane permeability, increasing concentrations of Ca²⁺, N, and K⁺ and reducing concentration of Na⁺ (because of cation competition in root zone) in leaves could offer an economical and simple solution to tomato crop production problems caused by high salinity.     

Effects of salt stress on ion content,antioxidant enzymes and protein profile in different tissues of Broussonetia papyrifera

Although some plant responses to salinity have been characterized, the precise mechanisms by which salt stress damages plants are still poorly understood especially in woody plants. In the present study, the physiological and biochemical responses of Broussonetia papyrifera, a tree species of the family, Moraceae, to salinity were studied. In vitro-produced plantlets of B. papyrifera were treated with varying levels of NaCl (0, 50, 100 and 150 mM) in hydroponic culture. Changes in ion contents, accumulation of H₂O₂, as well as the activities and isoform profiles of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the leaves, stems and roots were investigated. Under salt stress, there was higher Na⁺ accumulation in roots than in stems and leaves, and Ca^2 +, Mg^2 + and P^3 + content, as well as K⁺/Na⁺ ratio were affected. NaCl treatment induced an increase in H₂O₂ contents in the tissues of B. papyrifera. The work demonstrated that activities of antioxidant defense enzymes changed in parallel with the increased H₂O₂ and salinity appeared to be associated with differential regulation of distinct SOD and POD isoenzymes. Moreover, SDS-PAGE analysis of total proteins extracted from leaves and roots of control and NaCl-treated plantlets revealed that in the leaves salt stress was associated with decrease or disappearance of some protein bands, and induction of a new protein band after exposure to 100 and 150 mM NaCl. In contrast, NaCl stress had little effect on the protein pattern in the roots. In summary, these findings may provide insight into the mechanisms of the response of woody plants to salt stress.     

KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca2+ sensitization and K+-channel

AimsThis study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.Main methodsSprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.Key findingsMCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.SignificanceKMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.     

Simultaneous application of salicylic acid and calcium improves salt tolerance in two contrasting tomato (Solanum lycopersicum) cultivars

Soil salinity is one of the most important environmental factors responsible for serious agricultural problems. Tomato salt tolerance may be improved by genetic selection and by the use of adapted physiological tools. The aim of this study was to investigate the impact of exogenous application of salicylic acid (SA 0.01 mM) and calcium sulphate (CaSO₄ 5 mM), singly or in combination, on plant growth, photosynthetic pigments, nutritional behaviour and some metabolic parameters (total chlorophyll, carotenoids, soluble sugars, proline and lipid peroxidation) of two tomato cultivars (cv. Super Marmande and cv. Red River) exposed to salt stress (100 mM NaCl). Application of 100 mM NaCl reduced plant growth, total chlorophyll and carotenoid contents. Salt stress also induced an accumulation of Na⁺, a decrease in K⁺ and Ca^2 + concentration and root sugar level, an increase in malondialdehyde (MDA) and proline concentration. Deleterious impact of salinity was related to modification in ion content rather than modification in the plant water status. Exogenous application of SA or Ca alone improved plant behaviour in the presence of NaCl. Nevertheless, the best results in terms of growth, photosynthetic pigment concentrations and mineral nutrition (limitation of Na⁺ accumulation and maintenance of K⁺ and Ca^2 + content) were obtained in response to the combined SA + Ca treatment. Although the involved physiological parameters varied depending on the considered cultivar, our results suggest that Ca^2 + and SA may interact to reduce the stress experienced by the plant in the presence of NaCl.     

In vitro salinity tolerance of two pistachio rootstocks: Pistacia vera L. and P. atlantica Desf

Seedlings of Pistacia vera L. and Pistacia atlantica Desf. were cultured on hormone-free DKW medium supplemented with NaCl. The plants were subjected to low NaCl concentrations ranging from 0 to 80 mM for 45 days or to high salt concentrations (0, 131, and 158.5 mM for P. vera and 0, 131, and 240 mM for P. atlantica) for 25 days. Toxicity symptoms were recorded for seedlings exposed to low NaCl treatments. Plant growth, survival rates, mineral content, as well as proline and soluble sugar contents were determined and evaluated at the end of the culture period. The results indicated that low NaCl treatments yielded no instances of plant death in both species. At high salt conditions, however, significant mortality rates were noted for both species, being 22.86% at 240 mM NaCl for P. atlantica and 25.8% at 158.5 mM NaCl for P. vera. With regards to salinity effects, levels of 60 and 80 mM NaCl induced significant decreases of stem elongation and leaf number in the P. vera species. Salinities between 40 and 80 mM NaCl, however, induced a decrease in the root number of both species. The fresh weights of P. vera and P. atlantica also decreased significantly after 45 days of culture at NaCl concentrations between 40 and 80 mM and after 25 days of culture at 158.5 and 240 mM NaCl, respectively. The sodium and chloride uptake in plant organs seemed to be controlled more efficiently in P. atlantica than in P. vera. In both species, the K⁺ content was noted to undergo a significant decrease when salinity increased. While the K⁺/Na⁺ ratio was maintained above 2 at low NaCl treatments, it was sharply decreased at high NaCl conditions, suggesting a failure of K–Na selectivity mechanism. The Ca²⁺/Na⁺ ratio decreased significantly at 60 and 80 mM NaCl in P. vera and at 60 mM NaCl for P. atlantica. In both Pistacia species, high NaCl treatments (131–240 mM NaCl) induced a significant increase in proline content.     

Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796

An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (K_m = 0.83 mM), melibiose (K_m = 2.48 mM) and raffinose (K_m = 5.83 mM). Among metal ions and reagents tested, Ca²⁺ and K⁺ enhanced the enzymatic activity, but Mg²⁺, Mn²⁺, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag⁺, Hg²⁺ and Co²⁺ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose.     

On the mechanism of salt tolerance in olive (Olea europaea L.) under low- or high-Ca2+ supply

The effect of changes in Ca²⁺/Na⁺ ratios at the root zone has been reported in Olea europaea, a species mostly cultivated in calcareous soils. Plants were exposed to low (2.0 mM, low-Ca) or high-Ca²⁺ supply (9.0 mM, high-Ca) and supplied with 0 or 200 mM NaCl. Measurements were performed on water relations, gas exchange and photosynthetic performances, ion fluxes at whole-plant and leaf level, Na⁺ allocation at organismal level, the elemental and soluble carbohydrate concentration in the leaf. Most parameters were also measured during a period of relief from salinity stress, as Olea europaea suffers from fluctuating root zone NaCl concentrations over the whole growing season. High-Ca²⁺ supply decreased stomatal conductance, especially during the first two weeks of treatment. In response to salinity stress (i) leaf turgor potential was more severely depressed in high-Ca than in low-Ca plants, whereas net CO₂ assimilation rate and relative growth rate were unaffected by root zone Ca²⁺ concentrations (ii) high-Ca plants had a markedly superior ability to both exclude Na⁺ from the shoot and to selectively transport K⁺ over Na⁺ than low-Ca plants; (iii) both CO₂ carboxylation efficiency and maximal efficiency of PSII photochemistry (F_v/F_m) were significantly smaller in low-Ca than in high-Ca plants, likely as a result of a greater accumulation of toxic ions. Consistently, when osmotic stress was relieved by supplying plants with good quality water (relief period), both photosynthetic (+44%) and growth rates (+65%) recovered to a markedly superior degree in high-Ca than in low-Ca plants which had been previously treated with 200 mM NaCl. We conclude that (1) high-Ca²⁺ supply expose olive leaves to a more severe dehydration, but allowed to restrict both the entry and the allocation of potentially toxic ions to sensitive shoot organs; (2) a transient restriction of water-mass flow to the shoot during salinization may be of relatively minor significance in Olea europaea, which is very tolerant to drought; (3) overall salt tolerance in Olea europaea, as in most evergreen sclerophylls inhabiting Mediterranean areas, tightly depends upon the ability to reduce water uptake and transpiration during the dry/warm period and to recover photosynthetic and growth rates when low-salinity flood water is available. Therefore, data from the present experiment allow conclude that an increase in root zone Ca²⁺ concentration enhances tolerance to salinity stress in olive plants.     

Endothelium-dependent and -independent vasorelaxation induced by CIJ-3-2F,a novel benzyl-furoquinoline with antiarrhythmic action,in rat aorta

AimsThis study was designed to examine the mechanism of relaxation induced by CIJ-3-2F, a benzyl-furoquinoline antiarrhythmic agent, in rat thoracic aorta at the tissue and cellular levels.Main methodsIsometric tension of rat aortic ring was measured in response to drugs. Ionic channel activities in freshly dissociated aortic vascular smooth muscle cells (VSMCs) were investigated using a whole-cell patch-clamp technique.Key findingsCIJ-3-2F relaxed both phenylephrine (PE) and high KCl (60 mM)-induced contractions with respective pEC₅₀ (-log EC₅₀) values of 6.91 ± 0.07 and 6.32 ± 0.06. Removal of endothelium or pretreatment with nitric oxide (NO)-pathway inhibitors N^ω-nitro-l-arginine methyl ester (L-NAME), N^G-monomethyl-l-arginine (L-NMMA), N⁵-(1-iminoethyl)-l-ornithine (L-NIO), hemoglobin, methylene blue or 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ) reduced the relaxant effect of CIJ-3-2F. Relaxation to CIJ-3-2F was also attenuated by K⁺ channel blockers tetraethylammonium (TEA) or 4-aminopyridine (4-AP), but not by charybdotoxin plus apamin, iberiotoxin, glibenclamide, or BaCl₂. CIJ-3-2F non-competitively antagonized the contractions induced by PE, Ca²⁺, and Bay K8644 in endothelium-denuded rings. In addition, CIJ-3-2F inhibited both the phasic and tonic contractions induced by PE but did not affect the transient contraction induced by caffeine. CIJ-3-2F reduced the Ba²⁺ inward current through L-type Ca²⁺ channel (IC₅₀ = 4.1 μM) and enhanced the voltage-dependent K⁺ (K_v) current in aortic VSMCs.SignificanceThese results suggest that CIJ-3-2F induced both endothelium-dependent and -independent vasorelaxation; the former is likely mediated by the NO/cGMP pathway whereas the latter is probably mediated through inhibition of Ca²⁺ influx or inositol 1,4,5-triphosphate (IP₃)-sensitive intracellular Ca²⁺ release, or through activation of K_v channels.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Differential tolerance to combined salinity and O2 deficiency in the halophytic grasses Puccinellia ciliata and Thinopyrum ponticum: The importance of K+ retention in roots

Saline environments of terrestrial halophytes are often prone to waterlogging, yet the effects on halophytes of combined salinity and waterlogging have rarely been studied. Either salinity or hypoxia (low O₂) alone can interfere with K⁺ homeostasis, therefore the combination of salinity or hypoxia is expected to impact significantly on K⁺ retention in roots. We studied mechanisms of tolerance to the interaction of salinity with hypoxia in Puccinellia ciliata and Thinopyrum ponticum, halophytic grasses that differ in waterlogging tolerance. Plants were exposed to aerated and stagnant saline (250 mM NaCl) treatments with low (0.25 mM) and high (4 mM) K⁺ levels; growth, net ion fluxes and tissue ion concentrations were determined. P. ciliata was more tolerant than T. ponticum to stagnant-saline treatment, producing twice the biomass of adventitious roots, which accumulated high levels of Na⁺, and had lower shoot Na⁺. After 24 h of saline hypoxic treatment, MIFE measurements revealed a net uptake of K⁺ (∼40 nmol m⁻² s⁻¹) for P. ciliata, but a net loss of K⁺ (∼20 nmol m⁻² s⁻¹) for the more waterlogging sensitive T. ponticum. NaCl alone induced K⁺ efflux from roots of both species, with channel blocker tests implicating GORK-like channels. P. ciliata had constitutively a more negative root cell membrane potential than T. ponticum (−150 versus −115 mV). Tolerance to salinity and hypoxia in P. ciliata is related to increased production of adventitious roots, regulation of shoot K⁺/Na⁺, and a superior ability to maintain negative membrane potential in root cells, resulting in greater retention of K⁺.     

Exogenous nitric oxide improves seed germination in wheat against mitochondrial oxidative damage induced by high salinity

Effects of exogenous nitric oxide (NO) on starch degradation, oxidation in mitochondria and K⁺/Na⁺ accumulation during seed germination of wheat were investigated under a high salinity level. Seeds of winter wheat (Triticum aestivum L., cv. Huaimai 17) were pre-soaked with 0 mM or 0.1 mM of sodium nitroprusside (SNP, as nitric oxide donor) for 20 h just before germination under 300 mM NaCl. At 300 mM NaCl, exogenous NO increased germination rate and weights of coleoptile and radicle, but decreased seed weight. Exogenous NO also enhanced seed respiration rate and ATP synthesis. In addition, seed starch content decreased while soluble sugar content increased by exogenous NO pre-treatment, which was in accordance with the improved amylase activities in the germinating seeds. Exogenous NO increased the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6); whereas decreased the contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and superoxide anions (O₂^??) release rate in the mitochondria. Exogenous NO also decreased Na⁺ concentration while increased K⁺ concentration in the seeds thereby maintained a balance between K⁺ and Na⁺ during germination under salt stress. It is concluded that exogenous NO treatment on wheat seeds may be a good option to improve seed germination and crop establishment under saline conditions.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Effect of NaCl on growth and Cd accumulation of halophyte Spartina alterniflora under CdCl2 stress

A study quantifying the effect of NaCl on growth and Cd accumulation of Spartina alterniflora subjected to Cd stress was conducted. Seedlings were cultivated in the presence of 1 or 3 mM Cd alone, or combined with NaCl (50 or 100 mM). The results showed that NaCl magnified the phytotoxicity of moderate Cd stress (1 mM Cd) on plants due to reduced levels of plant biomass, plant height, and chlorophyll a + b, while no synergistic effects were recorded under severe Cd stress (3 mM Cd). Proline and Ca^2 + accumulated along with additional NaCl under moderate Cd stress, instead of reduced or unchanged levels under severe Cd stress owing to different adoption strategies caused by NaCl under different Cd stresses. NaCl reduced the oxidative stress in Cd-treated plants through increasing levels of antioxidative enzymes (catalase (CAT) and peroxidase (POD)) under moderate Cd stress. With NaCl addition, Cd^2 + contents in S. alterniflora increased and reduced under moderate and severe Cd stress, respectively. However, total Cd^2 + amounts increased with increasing NaCl concentration due to biological dilution. NaCl improved the increase of Cd^2 + translocation factor (TF) under moderate Cd stress, indicating that NaCl might improve Cd^2 + uptake and translocation from roots to shoots, and enhance the phytoextraction of S. alterniflora on Cd; while phytostabilization of Cd under severe Cd stress may be possible due to the reduced TF. Thus, NaCl alleviated phytotoxicity caused by Cd stress through improved management of osmotic solutes and oxidative status, and affected Cd accumulations in S. alterniflora differently under moderate and severe Cd stresses.     

Enhanced charge-independent mitochondrial free Ca2 + and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Modulation of mitochondrial free Ca^2 + ([Ca^2 +]_m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca^2 +]_m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca^2 +]_m and mitochondrial redox state (NADH), membrane potential (ΔΨ_m), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na⁺- or K⁺-pyruvate/malate (NaPM or KPM) or Na⁺-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl₂ (≈ 200 nM free Ca^2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca^2 +]_m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ_m and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ_m, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ_m, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca^2 + uptake by inhibiting the Na⁺/Ca^2 + exchanger (NCE), independent of changes in ΔΨ_m and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca^2 +]_m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

Characterization of an extracellular thermophilic alkaline esterase produced by Bacillus subtilis DR8806

This work is a report of the characterization of an alkaline lipolytic enzyme isolated from Bacillus subtilis DR8806. The extracellular extract was concentrated using ammonium sulfate, and ultrafiltration. The active enzyme was purified by Q-sepharose ion exchange chromatography. The molecular mass of the enzyme was estimated to be 60.25 kDa based on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C, respectively. The enzyme exhibited a half-life of 72 min at its optimum temperature. It was stable in the presence of metal ions (10 mM) such as Ca²⁺, K⁺ and Na⁺, whereas Cu²⁺, Fe²⁺, Zn²⁺, Mn²⁺, Co²⁺, Mg²⁺ and Hg²⁺ were found to have inhibitory effects. However, the enzyme activity was not affected significantly by 1% Triton X-100. The study of substrate specificity showed that the purified enzyme has a preferential specificity for small ester of p-nitrophenyl acetate (C₂), and it was the most efficiently hydrolyzed substrate as compared to the other esters. The kinetic parameters showed that the enzyme has K_m of 4.2 mM and V_max of 151 μmol min⁻¹ mg⁻¹ for p-nitrophenyl acetate. The hydrolysis rates of the fluorescence substrates were increased in the presence of the purified enzyme. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.     

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg²⁺ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca²⁺ (alone) only supports DNA binding. To investigate the role of Mg²⁺ in DNA cleavage by restriction endonucleases, we have studied the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn²⁺ and in the presence of Ca²⁺ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn²⁺ than in the presence of Mg²⁺ and can be activated by Ca²⁺. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K_m value for pTrcHisB DNA of 6.15 nM and a V_max of 1.79 × 10^?2 nM min^?1, while EhoI had a K_m for pUC19 plasmid of 8.66 nM and a V_max of 2 × 10^?2 nM min^?1.     

Chloride salinity reduces cadmium accumulation by the Mediterranean halophyte species Atriplex halimus L.

Soil salinity usually increases bioavailability of Cd on heavy metal polluted soils but its impact on Cd absorption and accumulation by plants remains largely unknown. Plants from the halophyte species Atriplex halimus were therefore exposed for 12 and 14 days to nutrient solution containing 50 μM CdCl₂ in the presence of NaCl, KCl or NaNO₃ 50 mM. Most Cd present in solution remained as Cd–EDTA and salinity had no impact on Cd speciation. Chloride salinity (NaCl and KCl) reduced Cd accumulation in shoots and roots while NaNO₃ increased Cd accumulation in leaves. More than 30% of accumulated Cd was found at the leaf surface and accumulated in trichomes but all tested salts decreased the proportion of excreted Cd. Cadmium induced a decrease in the leaf water content. External NaCl and KCl mitigated the deleterious impact of Cd by inducing osmotic adjustment while NaNO₃ and synthesis of protecting compounds such as soluble sugars and glycinebetaine. Free polyamines (putrescine, spermidine and spermine) increased in response to Cd, Cd + NaCl and Cd + KCl while only putrescine increased in response to Cd + NaNO₃. Proline exhibited maximal concentration in the leaves of Cd + NaCl and Cd + KCl-treated plants and was correlated with osmotic adjustment. Our results suggest that chloride salinity improved the resistance of A. halimus to Cd toxicity both by decreasing the absorption of heavy metal and by improving tissular tolerance through an increase in the synthesis of osmoprotective compounds.     

Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.