Agglutinating Activity and Structural Characterization of Scalarin,the Major Egg Protein of the Snail Pomacea scalaris (d’Orbigny, 1832)

Apple snail perivitellins are emerging as ecologically important reproductive proteins. To elucidate if the protective functions of the egg proteins of Pomacea canaliculata (Caenogastropoda, Ampullariidae), involved in embryo defenses, are present in other Pomacea species we studied scalarin (PsSC), the major perivitellin of Pomacea scalaris. Using small angle X-ray scattering, fluorescence and absorption spectroscopy and biochemical methods, we analyzed PsSC structural stability, agglutinating activity, sugar specificity and protease resistance. PsSC aggluttinated rabbit, and, to a lesser extent, human B and A erythrocytes independently of divalent metals Ca²⁺ and Mg²⁺ were strongly inhibited by galactosamine and glucosamine. The protein was structurally stable between pH 2.0 to 10.0, though agglutination occurred only between pH 4.0 to 8.0 (maximum activity at pH 7.0). The agglutinating activity was conserved up to 60°C and completely lost above 80°C, in agreement with the structural thermal stability of the protein (up to 60°C). PsSC was able to withstand in vitro gastrointestinal digestion, and showed no trypsin inhibition activity. The presence of lectin activity has been reported in eggs of other Pomacea snails, but here we link for the first time, this activity to an apple snail multifunctional perivitellin. This novel role for a snail egg storage protein is different from closely related P.canaliculata defensive proteins.     

Impact of invasive apple snails in Hong Kong on wetland macrophytes,nutrients, phytoplankton and filamentous algae

1. Grazing by invasive species can affect many aspects of an aquatic system, but most studies have focused on the direct effects on plants. We conducted mesocosm and laboratory experiments to examine the impact of the invasive apple snail Pomacea canaliculata on macrophytes, filamentous algae, nutrients and phytoplankton. 2. In a freshwater pond, we confined 500 g of Myriophyllum aquaticum or Eichhornia crassipes with 0, 2, 4 or 8 apple snails in 1 m × 1 m × 1 m enclosures for approximately 1 month. Apple snails grazed heavily on both species of macrophytes, with higher overall weight losses at higher snail densities. The damage patterns differed between the two macrophytes. In M. aquaticum, both leaves and stems suffered from substantial herbivory, whereas in E. crassipes, only the roots suffered significant weight reduction. 3. In addition to grazing on macrophytes, apple snails appeared to have controlled the growth of filamentous algae, as these did not develop in the snail treatments. The ability of P. canaliculata to control filamentous algae was supported by a laboratory experiment where the consumption was as high as 0.25 g g⁻¹ snail DW d⁻¹. Because of a lack of native herbivorous snails in the pond, the growth of filamentous algae (mainly Spirogyra sp.) reached 80.3 g m⁻², forming a spongy pond scum in the no‐apple snail control. Together with previous reports that apple snails could eat the juveniles and eggs of other freshwater snails, our results indicated that P. canaliculata could have out‐competed native herbivorous snails from the pond by predation on their juveniles or eggs. Alternatively, P. canaliculata might have out‐competed them by monopolisation of food resources. 4. Nitrogen and phosphorous concentrations remained low throughout both experiments and were not correlated with apple snail density. The treatment effects on chlorophyll a (Chl a) and phytoplankton composition varied in the two experiments. In the M. aquaticum experiment, with increasing snail density, Chl a increased, and the phytoplankton community became dominated by Cryptophyceae. In the E. crassipes experiment, Chl a level was independent of snail density, but with increasing snail density, the phytoplankton community became co‐dominated by Cryptophyceae, Chlorophyceae and Bacillariophyceae. 5. Given the multiple effects of P. canaliculata on wetland biodiversity and function, management strategies should be developed to prevent its further spread. In invaded wetlands, strategies should be developed to eradicate the apple snail and re‐introduce native snails which can control the development of filamentous algae.     

Relationship between endoplasmic reticulum- and Golgi-associated calcium homeostasis and 4-NQO-induced DNA repair in Saccharomyces cerevisiae

Calcium (Ca²⁺) is an important ion that is necessary for the activation of different DNA repair mechanisms. However, the mechanism by which DNA repair and Ca²⁺ homeostasis cooperate remains unclear. We undertook a systems biology approach to verify the relationship between proteins associated with Ca²⁺ homeostasis and DNA repair for Saccharomyces cerevisiae. Our data indicate that Pmr1p, a Ca²⁺ transporter of Golgi complex, interacts with Cod1p, which regulates Ca²⁺ levels in the endoplasmic reticulum (ER), and with Rad4p, which is a nucleotide excision repair (NER) protein. This information was used to construct single and double mutants defective for Pmr1p, Cod1p, and Rad4p followed by cytotoxic, cytostatic, and cell cycle arrest analyses after cell exposure to different concentrations of 4-nitroquinoline 1-oxide (4-NQO). The results indicated that cod1Δ, cod1Δrad4Δ, and cod1Δpmr1Δ strains have an elevated sensitivity to 4-NQO when compared to its wild-type (WT) strain. Moreover, both cod1Δpmr1Δ and cod1Δrad4Δ strains have a strong arrest at G₂/M phases of cell cycle after 4-NQO treatment, while pmr1Δrad4Δ have a similar sensitivity and cell cycle arrest profile when compared to rad4Δ after 4-NQO exposure. Taken together, our results indicate that deletion in Golgi- and ER-associated Ca²⁺ transporters affect the repair of 4-NQO-induced DNA damage.     

Neuroprotective Effect of KB-R7943 Against Glutamate Excitotoxicity is Related to Mild Mitochondrial Depolarization

KB-R7943, an inhibitor of a reversed Na⁺/Ca²⁺ exchanger, exhibits neuroprotection against glutamate excitotoxicity. Taking into consideration that prolonged exposure of neurons to glutamate induces delayed calcium deregulation (DCD) and irreversible decrease of mitochondrial membrane potential (Δψ_mit), we examined the effect of KB-R7943 on glutamate and kainate-induced [Ca²⁺]_i and on Δψ_mit changes in rat cultured cerebellar granule neurons. 15 μmol/l KB-R7943 significantly delayed the onset of DCD in response to kainate but not in response to glutamate. In spite of [Ca²⁺]_i overload, KB-R7943 considerably improved the [Ca²⁺]_i recovery and restoration of Δψ_mit after glutamate and kainate washout and increased cell viability after glutamate exposure. In resting neurons, KB-R7943 induced a statistically significant decrease in Δψ_mit. KB-R7943 also depolarized isolated brain mitochondria and slightly inhibited mitochondrial Ca²⁺ uptake. These findings suggest that mild mitochondrial depolarization and diminution of Ca²⁺ accumulation in the organelles might contribute to neuroprotective effect of KB-R7943.     

Kinetic and thermodynamic properties of wall bound invertase in heat tolerant and susceptible cultivars of wheat

We have identified two types of invertases, one bound ionically and the other covalently to the particulate fraction in grains of heat tolerant C 306 and heat susceptible WH 542 cultivars of wheat (Triticum aestivum L.). The cell walls contained a high level of invertase activity, of which 79.2–72.8% was extractable by 2 M NaCl and 14.9–21.1% by 0.5% EDTA in C 306 and WH 542, respectively. The NaCl-released invertase constituted the predominant fraction. Using 5–100 mM sucrose and pH range of 4.0–7.0, the apparent Michaelis constant (K _m, enzyme substrate affinity measure) of enzyme ranged from 5.73 to 16.06 mM for C 306 and from 6.08 to 19.86 mM for WH 542. The V _max (maximum catalytic rate) values at these pH were higher in C 306 (0.63–11.04 μg sucrose hydrolysed min⁻¹) than WH 542 (0.51–8.73 μg sucrose hydrolysed min⁻¹). By employing photo-oxidation and by studying the effect of pH on K _m and V _max, the involvement of histidine and α-carboxyl groups at the active site of the enzyme was indicated. The two cultivars also showed differential response in terms of thermodynamic properties of the enzyme i.e. energy of activation (E _a), enthalpy change (ΔH) and entropy change (ΔS). NaCl-released invertase showed differential response to metal ions in two cultivars suggesting their distinctive nature. Mn²⁺, Cu²⁺, Hg²⁺, Mg²⁺, Zn²⁺ and Cd²⁺ were strong inhibitors in WH 542 as compared to C 306 while K⁺, Ca²⁺ were stimulators in both the cultivars. Overall the results suggest that genetic differences exist in wall bound invertase properties of wheat grains as evident in its altered kinetic behaviour.     

Life history traits determine the differential vulnerability of native and invasive apple snails (<Emphasis Type="Italic">Pomacea</Emphasis> spp.) to a shared juvenile-stage predator

The vulnerability of gastropods to their predators varies with life history traits such as morphology, body size, behavior, and growth rates as well as predator size. A recent study suggested that the invasive apple snail, Pomacea maculata, was considerably more vulnerable to crayfish predators than the native Florida apple snail, P. paludosa. The difference was hypothesized to be caused by the relatively small hatchling size of P. maculata. To test this hypothesis, we conducted a series of feeding assays designed to quantify maximum feeding rates and selective foraging of crayfish on apple snails. The rate at which crayfish killed individual P. maculata (i.e., kill rates) decreased with snail size, and kill rates on both species increased with crayfish size. Kill rates on juvenile P. maculata were higher than kill rates on size-matched hatchling P. paludosa, and crayfish fed selectively on P. maculata when offered mixed groups of size-matched snails. Further analyses revealed that hatchling P. paludosa possess shells 1.8× heavier than size-matched P. maculata suggesting differences in vulnerability to crayfish were consistent with interspecific differences in shell defenses. Differences in hatchling size and defensive traits in combination make crayfish kill rates on hatchling P. maculata approximately 15.4× faster than on hatchling P. paludosa, but the relative contribution of hatchling size to differences in apple snail vulnerability was >3× greater than the contribution of defensive traits.     

Secondary production and diet of an invasive snail in freshwater wetlands: implications for resource utilization and competition

Invasive species can monopolize resources and thus dominate ecosystem production. In this study we estimated secondary production and diet of four populations of Pomacea canaliculata, a freshwater invasive snail, in wetlands (abandoned paddy, oxbow pond, drainage channel, and river meander) in monsoonal Hong Kong (lat. 22°N). Apple snail secondary production (ash-free dry mass [AFDM]) ranged from 165.9 to 233.3 g m⁻² year⁻¹, and varied between seasons. Production was lower during the cool dry northeast monsoon, when water temperatures might have limited growth, but fast growth and recruitment of multiple cohorts were possible throughout much (7–10 months) of the year and especially during the warm, wet southwest monsoon. The diet, as revealed by stomach-content analysis, consisted mainly of detritus and macrophytes, and was broadly consistent among habitats despite considerable variation in the composition and cover of aquatic plants. Apple snail annual production was >10 times greater than production estimates for other benthic macroinvertebrates in Hong Kong (range 0.004–15 g AFDM m⁻² year⁻¹, n = 29). Furthermore, annual production estimates for three apple snail populations (i.e. >230 g AFDM m⁻² year⁻¹) were greater than published estimates for any other freshwater snails (range 0.002–194 g AFDM m⁻² year⁻¹, n = 33), regardless of climatic regime or habitat type. High production by P. canaliculata in Hong Kong was attributable to the topical climate (annual mean ~24°C), permitting rapid growth and repeated reproduction, together with dietary flexibility including an ability to consume a range of macrophytes. If invasive P. canaliculata can monopolize food resources, its high productivity indicates potential for competition with other macroinvertebrate primary consumers. Manipulative experiments will be needed to quantify these impacts on biodiversity and ecosystem function in wetlands, combined with management strategies to prevent further range extension by P. canaliculata.     

Effects of environmental calcium deprivation on the egg masses of Physa marmorata Guilding (Gastropoda: Physidae) and Biomphalaria glabrata Say (Gastropoda: Planorbidae)

Eggs of the basommatophoran snails Physa marmorata and Biomphalaria glabrata were cultured in low concentrations of calcium to determine effects on growth and development. In both species there was some development in media with 0.12 mg/l Ca²⁺ but embryos were unable to hatch. 61.04% of embryos of P. marmorata could develop to hatching in 0.22 mg/l Ca²⁺ but those of B. glabrata required a level of 0.42 mg/l Ca²⁺, to attain even a 31.07% hatch. Marked effects on growth rate, embryo size and on time taken to achieve hatching were noted in both species at very low calcium levels. The possibility of cation-controlling mechanisms in the egg membrane is discussed.     

Nitrate impacts on the Florida apple snail, Pomacea paludosa

Nitrate pollution in springs in Florida has been suggested as a possible reason for declining populations of the Florida apple snail, Pomacea paludosa (Say). No correlation was found between snail density and nitrate concentration measured in six Florida springs. In laboratory studies examining short-term acute impacts of nitrate, adult and juvenile snail 96 h LC₅₀ values could not be determined due to low mortality rates despite nitrate concentrations > 500 ppm. Juvenile snail growth was affected with EC₅₀ values of 504 and 622 ppm nitrate, in two trials, respectively. Juvenile survival during the 14 d growth study fell below 50%, but again nitrate levels were very high (> 500 ppm). Given that snails exhibited little to no response to nitrate concentrations orders of magnitude greater than those found in Florida springs, we suggest that other factors, possibly including natural differences in habitat structure or changes in structure related to exotic plant invasions, may help explain the observed declines in apple snail abundance.     

Adsorption of lead(II) from aqueous solution onto Hydrilla verticillata

The adsorption of Pb(II) onto Hydrilla verticillata was examined in aqueous solution with parameters of pH, adsorbent dosage, contact time and temperature. The linear Langmuir and Freundlich models were applied to describe equilibrium isotherms, and both models fitted well. The monolayer adsorption capacity of Pb(II) was found as 104.2 mg/g at pH 4 and 25°C. Dubinin–Radushkevich (D–R) isotherm model was also applied to the equilibrium data. The mean free energy of adsorption (15.81 kJ/mol) indicated that the adsorption of Pb(II) onto H. verticillata may be carried out via chemical ion-exchange mechanism. Thermodynamic parameters, free energy (ΔG ⁰), enthalpy (ΔH ⁰) and entropy (ΔS ⁰) of adsorption were also calculated. These parameters showed that the adsorption of Pb(II) onto H. verticillata was a feasible, spontaneous and exothermic process in nature. The influence of Cd²⁺, Cu²⁺ and Ni²⁺ on adsorption of Pb²⁺ onto H. verticillata was studied, too. In the investigated range of operating conditions, it was found that the existence of Cd ²⁺, Cu ²⁺ and Ni ²⁺ had no impact on the adsorption of Pb²⁺.     

Atypical Ca<Superscript>2+</Superscript>-independent,raw-starch hydrolysing α-amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE1: characterization and gene isolation

Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca²⁺-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l⁻¹. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca²⁺ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V _max and K _m values for soluble starch were 42 mg reducing sugar min⁻¹ and 4.98 mg starch ml⁻¹, respectively. Strong inhibitors of enzyme activity included Cu²⁺, Zn²⁺ and Fe²⁺. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme.     

Transport of Ca2+ across the tonoplast of intact vacuoles from Chenopodium album L. suspension cells: ATP-dependent import and inositol-1,4,5-trisphosphate-induced release

The removal of Ca²⁺ from the medium by intact vacuoles and microsomes of Chenopodium album was investigated by measuring INDO-1 fluorescence emission at 400 and 480 nm and the response of Ca²⁺ -selective mini-electrodes. The removal of Ca²⁺ depended on the presence of MgATP, displaying an apparent K _mATP of about 50 μM, a K _mCa of 400–500 nM, and a nucleotide specificity (%) of ATP (100) > CTP (49) > GTP (28) > UTP (20) > ADP = AMP (0). In the presence of saturating MgATP, the vacuoles reduced the [Ca²⁺] of the medium below 30 nM. Part of the Ca²⁺ removed from the medium was released again after adding micromolar concentrations of inositol-1,4,5-trisphosphate. This release of Ca²⁺ was inhibited by heparin. Since digitonin caused the release of the entire amount of Ca²⁺ removed from the medium in the presence of MgATP, we argue that the Ca²⁺ is not bound to membranes or sequestered otherwise, but is transported into the vacuoles (or vesicles) and remains freely mobile there. In accordance with the current literature, we conclude that the plant vacuole is an important store for mobile Ca²⁺ to be released for purposes of signal transduction. Since changes in the trans-tonoplast ΔpH and inhibition of the H⁺-translocating pumps had no significant influence on the ATP-dependent removal of Ca²⁺ from the cytoplasmic side, we argue that in C. album ATP-driven Ca²⁺ transport is the predominant form of Ca²⁺ translocation into the vacuole. Received: 11 July 1996 / Accepted: 18 October 1996     

Mechanism of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> death induced by <Emphasis Type="Italic">Cratylia mollis</Emphasis> seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca²⁺ containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 μg/ml) induced plasma membrane permeabilization followed by Ca²⁺ influx and mitochondrial Ca²⁺ accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (ΔΨ_m) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca²⁺ treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased ΔΨ_m by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd2+/Mg2+–con-T[K7γ] complex

Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in γ-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-d-aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca²⁺ and Mg²⁺ can fulfill this role, Ca²⁺ induces dimerization of con-G, whereas the Mg²⁺-complexed peptide remains monomeric. A variant of con-T, con-T[K7γ] (γ is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca²⁺ ions and two Mg²⁺ ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca²⁺ for dimer formation, we report here the structure of the monomeric Cd²⁺/Mg²⁺–con-T[K7γ] complex, and, by comparison with the previously published con-T[K7γ]/Ca²⁺ dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.     

Membrane Stretching Triggers Mechanosensitive Ca<Superscript>2+</Superscript> Channel Activation in <Emphasis Type="Italic">Chara</Emphasis>

In order to confirm that mechanosensitive Ca²⁺ channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca²⁺ (Δ[Ca²⁺]_c). However, the observed Δ[Ca²⁺]_c decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (ΔE _m) and stretching or compression of the plasma membrane. Significant ΔE _m values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). ΔE _m appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large ΔE _m values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara.     

Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei

Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca²⁺) transport because of molting-related events and the deposition of CaCO₃ in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca²⁺ transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca²⁺ change was monitored by adding Ca as CaCl₂ (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca²⁺ transport followed Michaelis–Menten kinetics with K _m = 0.42 ± 0.04 mM and V _max = 0.50 ± 0.02 μM (Ca²⁺ change × initial intracellular Ca⁻¹ × 180 s⁻¹; N = 14, r ² = 0.99). Verapamil (a Ca²⁺ channel inhibitor) and amiloride (a Na⁺/Ca²⁺ exchanger [NCX] inhibitor) completely reduced intracellular Ca²⁺ transport, while nifedipine, another Ca²⁺ channel inhibitor, did not. Vanadate, a plasma membrane Ca²⁺-ATPase inhibitor (PMCA), increased intracellular Ca²⁺ in gill cells through a decrease in the efflux of Ca²⁺. Ouabain increased intracellular Ca²⁺, similar to the effect of KB-R, a specific NCX inhibitor for Ca²⁺ in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca²⁺ transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca²⁺ compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca²⁺ in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca²⁺ homeostasis through various Ca²⁺ intracellular sequestration mechanisms and/or plasma membrane Ca²⁺ influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca²⁺ mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.     

A novel thermostable and halostable carboxymethylcellulase from marine bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>AU01

A novel thermostable, halostable carboxymethylcellulase (CMCase) from a marine bacterium Bacillus licheniformisAU01 was purified 10.4-fold with 18% yield with a specific activity of 88.43 U/mg and the molecular weight was estimated as 37 kDa. The enzyme was optimally active at pH 9–10 and temperature 50–60°C and it was most stable up to pH 12 and 20–30% of NaCl concentration. The enzyme activity was reduced when treated with Hg²⁺, Fe²⁺ and EDTA and stimulated by Co²⁺, Mn²⁺, Mg²⁺ and Ca²⁺. Various cationic, anionic detergents and commercial detergents were not much affected CMCase activity.     

Duality of effect of La3+ on mitochondrial permeability transition pore depending on the concentration

In order to explore the role of mitochondria in proliferation promotion and/or apoptosis induction of lanthanum, the mutual influences between La³⁺ and Ca²⁺ on mitochondrial permeability transition pore (PTP) opening were investigated with isolated mitochondria from rat liver. The experimental results revealed that La³⁺ influence the state of mitochondria in a concentration-dependent biphasic manner. La³⁺ in nanomolar concentrations, acting as a Ca²⁺ analog, entered mitochondrial matrix via the RuR sensitive Ca²⁺ channel and elevated ROS level, leading to opening of PTP indicated by mitochondrial swelling, reduction of ΔΨ_m and cytochrome c release. Inhibition of PTP with 10 μM CsA attenuated the effects of La³⁺. However, micromolar concentrations La³⁺ acted mainly as a Ca²⁺ antagonist, inhibiting PTP opening induced by Ca²⁺. We postulated that this action of La³⁺ on mitochondria through interaction with Ca²⁺ might be involved in the proliferation-promoting and apoptosis induction by La³⁺.