Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal

This study is aimed to determine the role of calcium signaling evoked by the calcium-mobilizing agonist uridine-5′-triphosphate (UTP) and by the specific inhibitor of the endoplasmic reticulum calcium reuptake thapsigargin on caspase activation in human leukemia cell line HL-60. We have analyzed cytosolic free calcium concentration ([Ca²⁺]_c) determination, mitochondrial membrane potential and caspase-3 and -9 activity by fluorimetric methods, using the fluorescent ratiometric calcium indicator Fura-2, the dye JC-1, and specific fluorogenic substrate, respectively. Our results indicated that treatment of HL-60 cells with 10 μM UTP or 1 μM thapsigargin induced a transient increase in [Ca²⁺]_c due to calcium release from internal stores. The stimulatory effect of UTP and thapsigargin on calcium signal was followed by a mitochondrial membrane depolarization. Our results also indicated that UTP and thapsigargin were able to increase the caspase-3 and -9 activities. The effect of UTP and thapsigargin on caspase activation was time dependent, reaching a maximal caspase activity after 60 min of stimulation. Loading of cells with 10 μM dimethyl BAPTA, an intracellular calcium chelator, for 30 min significantly reduced both UTP- or thapsigargin-induced mitochondrial depolarization and caspase activation. Similar results were obtained when the cells were pretreated with 10 μM Ru360 for 30 min, a specific blocker of calcium uptake into mitochondria. The findings suggest that UTP- and thapsigargin-induced caspase-3 and -9 activation and mitochondrial membrane depolarization is dependent on rises in [Ca²⁺]_c in human myeloid HL-60 cells.     

The Role of Ca<Superscript>2+</Superscript> Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A

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

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca²⁺ on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca²⁺ on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca²⁺ (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca²⁺ stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca²⁺ significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.     

Role of calcium in adult onset polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in genes encoding the polycystin (PC) 1 and 2 proteins. The goal of this study was to determine the role of calcium in regulating cyst growth. Stromal interaction molecule 1 (STIM1) protein expression was 15-fold higher in PC1-null proximal tubule cells (PN) than in heterozygote (PH) controls and 2-fold higher in an inducible, PC1 knockout, mouse model of ADPKD compared to a non-cystic match control. IP3 receptor protein expression was also higher in the cystic mice. Knocking down STIM1 with siRNA reduced cyst growth and lowered cAMP levels in PN cells. Fura2 measurements of intracellular Ca²⁺ showed higher levels of intracellular Ca²⁺, SOCE and thaspigargin-stimulated ER Ca²⁺ release in PN vs. PH cells. There was a dramatic reduction in thapsigargin-stimulated release of ER Ca²⁺ following STIM1 silencing or application of 2-APB, consistent with altered ER Ca²⁺ movement; the protein expression of the Ca²⁺-dependent adenylyl cyclases (AC) AC3 and AC6 was up- and down-regulated, respectively. Like STIM1 knockdown, application of the calmodulin inhibitor W7 lowered cAMP levels, further indicating that STIM1 regulates AC3 via Ca²⁺ We conclude that the high levels of STIM1 in ADPKD cells play a role in supporting cyst growth and promoting high cAMP levels and an increased release of Ca²⁺ from the ER. Thus, our results provide novel therapeutic targets for treating ADPKD.     

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca²⁺]_i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca²⁺]_i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca²⁺]_i mobilization using several extraction methods. The spatial distribution pattern of [Ca²⁺]_i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca²⁺]_i levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca²⁺]_i changes. Thus, AHL acts as a double-edged sword for osteoblast function.     

Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca²⁺]_c) and nuclear calcium ([Ca²⁺]_n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca²⁺]_n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca²⁺]_n and [Ca²⁺]_c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.     

The inhibitory effect of Ca2+ on the flavonoid production of Tetrastigma hemsleyanum suspension cells induced by metal elicitors

Tetrastigma hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production. The effects of calcium ions (Ca²⁺) on induction were also studied. It was found that the most effective elicitors were 50 μM of the heavy metal ion copper (Cu²⁺) and 100 μM of the rare earth element cerium (Ce³⁺). The maximal biomass levels under respective treatments over a 16-d culture period increased by 1.3- and 1.6-fold, and the total flavonoid content was 1.8- and 1.6-fold greater than the control, respectively. Reducing the exogenous Ca²⁺ concentration or adding Ca²⁺ antagonists (1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N-tetraacetc acid (EGTA) or 1 mM verapamil) strengthened inductive effects of metal elicitors and enhanced flavonoid production. However, 0.5 μM of the calcium ionophore A23187 showed contrary results. The increase in exogenous Ca²⁺ concentration in the presence of A23187 suppressed H₂O₂ bursts and peroxidase activity caused by metal elicitors. The results suggest that Ca²⁺ plays an inhibitory role in the plant cell response to metal elicitors. This suppression could have been caused by Ca²⁺ preventing the cells from absorbing metal ions and then easing the induction, or because the decrease of Ca²⁺ concentration worked as an induction signal. Therefore, reducing the Ca²⁺ concentration in culture medium, or adding Ca²⁺ antagonists could be used to improve flavonoid production and cell growth in combination with induction by metal elicitors during in vitro culture of T. hemsleyanum suspension cells.     

Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells

The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP₃) and intracellular free calcium (Ca_i²⁺) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Ca_i²⁺ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Ca_i²⁺ increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Ca_i²⁺ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM × 1 h) and H-7 (30 μM × 18 h) potentiated the effects of 1 μg/ml bPTH(1–84) on Ca_i²⁺ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 μg/ml) was without significant effect on adherent cell Ca_i²⁺ as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (∼ λ510 nm) detected and optimized at λ348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Ca_i²⁺ increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Ca_i²⁺ in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 μM × 30 min). PTH-induced Ca_i²⁺ increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP₃ increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP₃/Ca_i²⁺). J. Cell. Biochem. 65:276–285. © 1997 Wiley-Liss, Inc.     

Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (±1,341) 17β-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (±2) fmol/mg protein. This subclone was examined further for estradiol (E₂) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased ～25-fold in hFOB/ER9 cells following 10^?9M E₂ treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10^?9M E₂ resulted in a 2.5–4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function. © 1995 Wiley-Liss, Inc.     

Revised model of calcium and magnesium binding to the bacterial cell wall

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca²⁺, Region I has a K_A = (1.0 ± 0.2) × 10⁶ M^?1 and Region II has a K_A = (0.075 ± 0.058) × 10⁶ M^?1. For Mg²⁺, K_A1 = (1.5 ± 0.1) × 10⁶ and K_A2 = (0.17 ± 0.10) × 10⁶. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η₂, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca²⁺ and Mg²⁺ respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.     

Mechanical stimuli modulate intracellular calcium oscillations: a pathological model without chemical cues

Objective

To control the oscillatory behavior of the intracellular calcium ([Ca²⁺]_i) concentration in endothelial cells via mechanical factors (i.e., various hydrostatic pressures) because [Ca²⁺]_i in these cells is affected by blood pressure.

Results

Quantitative analyses based on real-time imaging showed that [Ca²⁺]_i oscillation frequency and relative concentration increased significantly when 200 mm Hg pressure, mimicking hypertension, was applied for >10 min. Peak height and peak width decreased significantly at 200 mm Hg. These trends were more marked as the duration of the 200 mm Hg pressure was increased. However, no change was observed under normal blood pressure conditions 100 mm Hg.

Conclusion

We generated a simple in vitro model to study [Ca²⁺]_i behavior in relation to various pathologies and diseases by eliminating possible complicating effects induced by chemical cues.

     

Cluster-root formation, carboxylate exudation and proton release of Lupinus pilosus Murr. as affected by medium pH and P deficiency

The study examined the interactive effect of pH and P supply on cluster-root formation, carboxylate exudation and proton release by an alkaline-tolerant lupin species (Lupinus pilosus Murr.) in nutrient solution. The plants were exposed to 1 (P₁, deficient) and 50 μM P (P₅₀, adequate) for 34 days in nutrient solution at either pH 5.6 or 7.8. Plant biomass was not influenced by pH at P₁, but at P₅₀ shoot and root dry weights were 23 and 18% higher, respectively, at pH 7.8 than at pH 5.6. There was no significant difference in plant biomass between two P treatments regardless of medium pH. Phosphorus deficiency increased significantly the number of the second-order lateral roots compared with the P₅₀ treatment. Both total root length and specific root length of plants grown at pH 5.6 were higher than those at pH 7.8 regardless of P supply. Cluster roots were formed at P₁, but cluster-root number was 2-fold higher at pH 7.8 than pH 5.6. Roots released 16 and 31% more protons at pH 5.6 and 7.8, respectively, in P₁ than in P₅₀ treatments, and the rate of proton release followed the similar pattern. At pH 5.6, citrate exudation rate was 0.39 μmol g⁻¹ root DW h⁻¹ at P₁, but was under the detection limit at P₅₀; at pH 7.8, it was 2.4-fold higher in P₁ than in P₅₀ plants. High pH significantly increased citrate exudation rate in comparison to pH 5.6. The uptake of anions P and S was inhibited at P₁ and high pH increased cations Na, Mg and Ca uptake. The results suggested that enhanced cluster-root formation, proton release and citrate exudation may account for the mechanism of efficient P acquisition by alkaline-tolerant L. pilosus well adapted to calcareous soils. Cluster-root formation and citrate exudation in L. pilosus can be altered by medium pH and P deficiency. Phosphorus deficiency-induced proton release may be associated with the reduced anion uptake, but high pH-induced proton release may be partly attributed to increased cation uptake.     

Alleviation of cadmium toxicity by potassium supplementation involves various physiological and biochemical features in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Potassium (K⁺) plays important roles in the development of plants and the response to various environmental stresses. However, the involvement of potassium in alleviating heavy metal stress in tobacco remains elusive. Greenhouse hydroponic experiments were conducted to evaluate the alleviating effects of K⁺ on tobacco subjected to cadmium (Cd) toxicity using four different K⁺ levels. Dose-dependent increases of plant biomass were found in both 0-μM Cd and 5-μM Cd treatments under different K⁺ levels, with the exception of the 1-mM KHCO₃ (K3) treatment. The best mitigation effect was recorded with the 0.5-mM K⁺ (K2) treatment, which greatly alleviated Cd-induced growth inhibition, photosynthesis reduction, and oxidative stress. Compared with K0 treatment (no KHCO₃ addition), K2 treatment significantly reduced Cd uptake and translocation after 5 and 10 days of Cd treatment. Moreover, the net photosynthetic rate, intracellular CO₂ concentration, stomatal conductance, and transpiration rate as well as K⁺, zinc, manganese, copper, and iron concentrations in both shoots and roots after 10 days of Cd treatment significantly improved under the K2 treatment, and malondialdehyde accumulation in both shoots and roots was repressed, compared with K0 + Cd. Superoxide dismutase was found to play key roles in alleviating Cd-induced oxidative pressure in shoots of plants in K2 treatment under Cd treatment. Our findings advocate a positive role for K⁺ in reducing pollutant residues for safe production, especially in soils slightly or moderately polluted with Cd.     

Exogenous application of calcium chloride in wheat genotypes alleviates negative effect of drought stress by modulating antioxidant machinery and enhanced osmolyte accumulation

Plants use various mechanisms to cope with drought constraints at morphological, physiological, and biochemical level by means of different adaptive mechanisms. All organisms use a network of signal transduction pathways to control their metabolism and to adapt to the environment. Among these pathways, calcium (Ca²⁺) ions play an important role as a universal second messenger. Calcium has unique properties and universal ability to transmit diverse signals that trigger primary physiological actions in the cell in response to hormones, pathogens, and stress factors. Calcium plays a fundamental role in regulating the polar growth of cells and tissues and participates in plant adaptation to various stress factors. This study was conducted to examine the role of Ca²⁺ in ameliorating the adverse effect of drought stress responses in two contrasting wheat genotypes, HD 2733 (drought sensitive) and HD 2987 (drought tolerant), differing in their drought tolerance. The plants were treated with mannitol or Hoagland solution and then supplemented with CaCl₂ (10 mM). Measurements of seed germination, shoot growth, and chlorophyll content showed that calcium treatment increased all these factors in tolerant genotype (HD 2987) under induced stress condition. Drought stress reduced relative water content, osmolyte, and soluble sugar accumulation in both the genotypes, but CaCl₂ supplementation increased all the components under stress condition in HD 2987 as compared to HD 2733. The oxidative damage caused by induced stress was lower in HD 2987 compared to HD 2733 genotypes as assessed by their higher photosynthetic capacity and lower electrolyte leakage, malondialdehyde (MDA) content as well as H₂O₂ accumulation. Less accumulation of superoxide and H₂O₂ was also observed in HD 2987 genotype after CaCl₂ supplementation combined with mannitol treatment. In addition, the enhanced accumulation of calcium in the HD 2987 genotype is correlated with the higher activities of antioxidant enzymes than HD 2733 genotype under similar stress conditions. Our findings provide evidence of the protective role of exogenous calcium in conferring better tolerance against mannitol-induced drought stress in wheat genotypes, which could be useful as genetic stock to develop wheat tolerant varieties in breeding programs.     

Contribution of Hydrogenase 2 to Stationary Phase H2 Production by Escherichia coli During Fermentation of Glycerol

Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H₂ production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H₂ production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H₂ production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)^?1 and Hyd-1 activity was 0.1 U (mg protein)^?1. As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)^?1, and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)^?1 but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H₂ production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.     

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0–6.0 and 55–65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0–10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K_M = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V_max for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V_max was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.     

Antioxidant and Neuroprotective Activities of Hyptis suaveolens (L.) Poit. Against Oxidative Stress-Induced Neurotoxicity

The present study was carried out to investigate the antioxidant and neuroprotective effects of Hyptis suaveolens methanol extract (HSME) using various in vitro systems. The total phenol and flavonoids contents of the HSME were quantified by colorimetric methods. The HSME extract exhibited potent antioxidant activity as determined by 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 2,2-diphenyl-1-picrylhydrazyl, and ferric reducing antioxidant power assays. The neuroprotective activity of HSME was determined on mouse N2A neuroblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lactate dehydrogenase, intracellular ROS assays, and upregulation of brain neuronal markers at genetic level. The N2A cells were pretreated with different concentrations (0.5, 1, 1.5, and 2 mg/ml) of the extract and then exposed to H₂O₂ to induce oxidative stress and neurotoxicity. The survival of the cells treated with different concentrations of HSME and H₂O₂ increased as compared to cells exposed only to H₂O₂ (47.3 %) (p < 0.05). The HSME also dose-dependently reduced LDH leakage and intracellular ROS production (p < 0.05). Pretreatment with HSME promotes the upregulation of tyrosine hydroxylase (2.41-fold, p < 0.05), and brain-derived neurotrophic factor genes (2.15-fold, p < 0.05) against H₂O₂-induced cytotoxicity in N2A cells. Moreover, the HSME showed antioxidant activity and decreased neurotoxicity. These observations suggest that HSME have marked antioxidant and neuroprotective activities.     

Thapsigargin-induced calcium entry in FRTL-5 cells: Possible dependence on phospholipase A2 activation

Stimulating rat thyroid FRTL-5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A₂ (PLA₂) is activated. Since PLA₂ is a calcium-dependent enzyme we wanted to investigate the interrelationships between PLA₂ activity and the entry of calcium. Stimulating ³H-arachidonic acid (³H-AA)-labelled cells with thapsigargin resulted in a substantial release of ³H-AA. This release was totally abolished in a calcium-free buffer. Pretreatment of Fura 2 loaded cells with 4-bromophenacyl bromide, an inhibitor of PLA₂ activity, decreased the thapsigargin-induced entry of calcium, suggesting a role for PLA₂ in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P-450 inhibitory actions, the thapsigargin-induced entry of calcium was decreased in a dose-dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin-induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium-induced PLA₂ activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca²⁺ in FRTL-5 cells. © 1994 Wiley-Liss, Inc.     

Oxytocin activates calcium signaling in rat sensory neurons through a protein kinase C-dependent mechanism

In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca²⁺]_i) in rat dorsal root ganglion (DRG) neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1- or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of OT on [Ca²⁺]_i and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca²⁺]_i after application at the doses of 30 nM (n?=?34, p?<?0.01), 100 nM (n?=?41, p?<?0.001) and 300 nM (n?=?46, p?<?0.001). The stimulatory effect of OT (300 nM) on [Ca²⁺]_i was persistent in Ca²⁺-free conditions (n?=?56, p?<?0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca²⁺]_i (n?=?28, p?<?0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain.     

Oxidative Stress and Ca2+ Signals Involved on Cadmium-Induced Apoptosis in Rat Hepatocyte

Cadmium (Cd) is an important industrial and environmental pollutant. In animals, the liver is the major target organ of Cd toxicity. In this study, rat hepatocytes were treated with 2.5～10 μM Cd for various durations. Studies on nuclear morphology, chromatin condensation, and apoptotic cells demonstrate that Cd concentrations ranging within 2.5～10 μM induced apoptosis. The early-stage marker of apoptosis, i.e., decreased mitochondrial membrane potential, was observed as early as 1.5 h at 5 μM Cd. Significant (P?P?2+ concentration ([Ca²⁺] _i ) of Cd-exposed cells significantly increased (P?2+] _i may play an important role in apoptosis. Overall, these results showed that oxidative stress and Ca²⁺ signaling were critical mediators of the Cd-induced apoptosis of rat hepatocytes.