Novel interactions of CLN3 protein link Batten disease to dysregulation of fodrin-Na+, K+ ATPase complex

Juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease) is the most common progressive neurodegenerative disorder of childhood. CLN3, the transmembrane protein underlying JNCL, is proposed to participate in multiple cellular events including membrane trafficking and cytoskeletal functions. We demonstrate here that CLN3 interacts with the plasma membrane-associated cytoskeletal and endocytic fodrin and the associated Na⁺, K⁺ ATPase. The ion pumping activity of Na⁺, K⁺ ATPase was unchanged in Cln3^−/− mouse primary neurons. However, the immunostaining pattern of fodrin appeared abnormal in JNCL fibroblasts and Cln3^−/− mouse brains suggesting disturbances in the fodrin cytoskeleton. Furthermore, the basal subcellular distribution as well as ouabain-induced endocytosis of neuron-specific Na⁺, K⁺ ATPase were remarkably affected in Cln3^−/− mouse primary neurons. These data suggest that CLN3 is involved in the regulation of plasma membrane fodrin cytoskeleton and consequently, the plasma membrane association of Na⁺, K⁺ ATPase. Most of the processes regulated by multifunctional fodrin and Na⁺, K⁺ ATPase are also affected in JNCL and Cln3-deficiency implicating that dysregulation of fodrin cytoskeleton and non-pumping functions of Na⁺, K⁺ ATPase may play a role in the neuronal degeneration in JNCL.     

Diastereomeric preference in 1,4,7-tris((S)-2-hydroxy-3-phenylpropyl)-1,4,7-triazacyclononanelithium(I) and its sodium(I) analogue

In N,N-dimethylformamide (DMF), 1,4,7-tris((S)-2-hydroxy-3-phenylpropyl)-1,4,7-triazacyclononane forms metal complexes, [M(S-thppc9)]⁺, for which log K (dm³ mol⁻¹)=3.01, 2.65, 2.66, 2.65, 2.42 and 7.59 (all±0.05) where M⁺=Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺ and Ag⁺, respectively. Variable temperature ¹³C{¹H} NMR spectroscopy shows that the interchange between equivalent forms of a single diastereomer occurs for [Li(S-thppc9)]⁺ and [Na(S-thppc9)]⁺ characterised by: k=43±5 and 2900±100 s⁻¹, at 298.2 K, ΔH^‡=22.5±1.6 and 33.8±1.6 kJ mol⁻¹, and ΔS^‡=−133±5 and −59±6 J K⁻¹ mol⁻¹, respectively. Gas phase ab initio modelling shows these complexes and their K⁺ analogue to preferentially form distorted trigonal prismatic Λ, Δ, and Λ diastereomers, respectively.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

Sexual and seasonal variations in osmoregulation and ionoregulation in the estuarine crab Chasmagnathus granulatus (Crustacea, Decapoda)

The estuarine crab Chasmagnathus granulatus (Crustacea, Decapoda, Brachyura) inhabits salt marshes along the South Atlantic coast from Rio de Janeiro (Brazil) to Patagonia (Argentina). In the present study, salinity tolerance (0-45‰; 16-1325 mOsm/kg H₂O) and hemolymph osmotic and ionic (Na⁺, Cl⁻, and K⁺) regulation in both female and male C. granulatus were analyzed in summer and winter. Results showed that both female and male C. granulatus are euryhaline. Mortality was only observed in extremely low salinity (0‰; 16 mOsm/kg H₂O) for both sexes. For females, the LT₅₀ at 0‰ salinity was similar in summer (20.1 h) and winter (17.4 h). Males were more tolerant to salinity than females in both seasons, and mortality was observed only in summer (LT₅₀ = 50.9 h). Results from freshly collected crabs or long-term (16-day) osmotic and ionic regulation experiments in the laboratory showed that male C. granulatus is a better hyper-osmoregulator than female in summer and winter. However, a hypo-osmoregulatory ability was only observed in females experimentally subjected to salinity 40‰ (1176 ± 11 mOsm/kg H₂O) in both seasons. In both sexes, hyper-osmotic regulation was achieved by hyper-regulating hemolymph Na⁺, Cl⁻, and K⁺ concentration. In females, hypo-osmotic regulation was achieved by hypo-regulating hemolymph Na⁺ and Cl⁻ concentration. Long-term (16-day) osmotic and ionic regulations in different salinities were similar in males or females collected and tested in summer and winter. Despite this lack of a seasonal effect on hemolymph osmoregulatory and ionoregulatory patterns in males or females, a marked seasonal difference in the dynamics of these processes was observed for both sexes. In the first 2 days after hypo-osmotic shock (20‰→5‰; 636→185 mOsm/kg H₂O), variations in female osmolality and ion (Na⁺ and Cl⁻) concentration were larger and faster in winter than in summer, while in males the opposite was observed. Furthermore, a seasonal effect on the crab response to hyper-osmotic shock (20‰→40‰; 636→1176 mOsm/kg H₂O) was only observed in males. A new osmolality and ion (Na⁺ and Cl⁻) concentration steady state was faster achieved in winter than in summer. Regarding sexual differences, females showed a better capacity to hypo-regulate the hemolymph osmolality and Na⁺ concentration than males, even after a sudden increase in salinity (hyper-osmotic shock) in both seasons. On the other hand, males showed a better capacity to hyper-regulate the hemolymph osmolality and Na⁺ concentration than females, even after a sudden decrease in salinity (hypo-osmotic shock), especially in winter. Taken together, results reported in the present study suggest the need to consider both sex and collection season as important factors in future osmotic and ionic regulation studies in estuarine crabs.     

Increased Potassium Absorption Confers Resistance to Group IA Cations in Rubidium-Selected Suspension Cells of Brassica napus

Cell lines of suspension cultures of Brassica napus cv. Jet Neuf were identified for their ability to tolerate 100 millimolar Rb⁺, a level which was double the normally lethal concentration. Ten spontaneous isolates were obtained from approximately 5 × 10⁷ cells, one of which was reestablished as a cell suspension. This cell line, JL5, was also resistant to the other group IA cations— Li⁺, Na⁺, K⁺, and Cs⁺—and this trait was stable for at least 30 cell generations in the absence of Rb⁺ selection pressure. The growth characteristics were similar to those of sensitive cells under nonselective conditions. The selected JL5 cells were shown by analysis to have effected more net accumulation of K⁺ and Rb⁺ and less of Na⁺ than did the unselected cells. JL5 and unselected cells after 14 days of culture in basal medium contained 597.2 and 258.2 micromoles of K per gram dry weight, respectively. Michaelis-Menten kinetic analysis of K⁺ influx showed that JL5 possessed an elevated phase 1 V_max, but there was no alteration in its K_m. This is the first time that a plant mutation has been shown to have both increased influx and net absorption of a major essential cation.     

QTL Analysis of Na+ and K+ Concentrations in Roots and Shoots under Different Levels of NaCl Stress in Rice (Oryza sativa L.)

The key to plant survival under NaCl salt stress is maintaining a low Na⁺ level or Na⁺/K⁺ ratio in the cells. A population of recombinant inbred lines (RILs, F_2∶9) derived from a cross between the salt-tolerant japonica rice variety Jiucaiqing and the salt-sensitive indica variety IR26, was used to determine Na⁺ and K⁺ concentrations in the roots and shoots under three different NaCl stress conditions (0, 100 and 120 mM NaCl). A total of nine additive QTLs were identified by QTL Cartographer program using single-environment phenotypic values, whereas eight additive QTLs were identified by QTL IciMapping program. Among these additive QTLs, five were identified by both programs. Epistatic QTLs and QTL-by-environment interactions were detected by QTLNetwork program in the joint analyses of multi-environment phenotypic values, and one additive QTL and nine epistatic QTLs were identified. There were three epistatic QTLs identified for Na⁺ in roots (RNC), three additive QTLs and two epistatic QTLs identified for Na⁺ in shoots (SNC), four additive QTLs identified for K⁺ in roots (RKC), four additive QTLs and three epistatic QTLs identified for K⁺ in shoots (SKC) and one additive QTL and one epistatic QTL for salt tolerance rating (STR). The phenotypic variation explained by each additive, epistatic QTL and QTL×environment interaction ranged from 8.5 to 18.9%, 0.5 to 5.3% and 0.7 to 7.5%, respectively. By comparing the chromosomal positions of these additive QTLs with those previously identified, five additive QTLs, qSNC9, qSKC1, qSKC9, qRKC4 and qSTR7, might represent novel salt tolerance loci. The identification of salt tolerance in selected RILs showed that a major QTL qSNC11 played a significant role in rice salt tolerance, and could be used to improve salt tolerance of commercial rice varieties with marker-assisted selection (MAS) approach.     

Dietary cardenolides enhance growth and change the direction of the fecundity‐longevity trade‐off in milkweed bugs (Heteroptera: Lygaeinae)

Sequestration, that is, the accumulation of plant toxins into body tissues for defense, was predicted to incur physiological costs and may require resistance traits different from those of non‐sequestering insects. Alternatively, sequestering species could experience a cost in the absence of toxins due to selection on physiological homeostasis under permanent exposure of sequestered toxins in body tissues. Milkweed bugs (Heteroptera: Lygaeinae) sequester high amounts of plant‐derived cardenolides. Although being potent inhibitors of the ubiquitous animal enzyme Na⁺/K⁺‐ATPase, milkweed bugs can tolerate cardenolides by means of resistant Na⁺/K⁺‐ATPases. Both adaptations, resistance and sequestration, are ancestral traits of the Lygaeinae. Using four milkweed bug species (Heteroptera: Lygaeidae: Lygaeinae) and the related European firebug (Heteroptera: Pyrrhocoridae: Pyrrhocoris apterus) showing different combinations of the traits “cardenolide resistance” and “cardenolide sequestration,” we tested how the two traits affect larval growth upon exposure to dietary cardenolides in an artificial diet system. While cardenolides impaired the growth of P. apterus nymphs neither possessing a resistant Na⁺/K⁺‐ATPase nor sequestering cardenolides, growth was not affected in the non‐sequestering milkweed bug Arocatus longiceps, which possesses a resistant Na⁺/K⁺‐ATPase. Remarkably, cardenolides increased growth in the sequestering dietary specialists Caenocoris nerii and Oncopeltus fasciatus but not in the sequestering dietary generalist Spilostethus pandurus, which all possess a resistant Na⁺/K⁺‐ATPase. We furthermore assessed the effect of dietary cardenolides on additional life history parameters, including developmental speed, longevity of adults, and reproductive success in O. fasciatus. Unexpectedly, nymphs under cardenolide exposure developed substantially faster and lived longer as adults. However, fecundity of adults was reduced when maintained on cardenolide‐containing diet for their entire lifetime but not when adults were transferred to non‐toxic sunflower seeds. We speculate that the resistant Na⁺/K⁺‐ATPase of milkweed bugs is selected for working optimally in a “toxic environment,” that is, when sequestered cardenolides are stored in the body.     

Kinetics and mechanism of the reduction of the molybdatopentaamminecobalt(III) ion by aqueous sulfite and aqueous potassium hexacyanoferrate(II)

A detailed investigation on the oxidation of aqueous sulfite and aqueous potassium hexacyanoferrate(II) by the title complex ion has been carried out using the stopped-flow technique over the ranges, 0.01≤[S(IV)]_T≤0.05 mol dm⁻³, 4.47≤pH≤5.12, and 24.9≤θ≤37.6 °C and at ionic strength 1.0 mol dm⁻³ (NaNO₃) for aqueous sulfite and 0.01≤[Fe(CN)₆ ⁴⁻]≤0.11 mol dm⁻³, 4.54≤pH≤5.63, and 25.0≤θ≤35.3 °C and at ionic strength 1.0 or 3.0 mol dm⁻³ (NaNO₃) for the hexacyanoferrate(II) ion. Both redox processes are dependent on pH and reductant concentration in a complex manner, that is, for the reaction with aqueous sulfite, k_obs={(k₁K₁K₂K₃+k₂K₁K₄[H⁺])[S(IV)]_T]/([H⁺]²+K₁[H⁺]+K₁K₂) and for the hexacyanoferrate(II) ion, k_obs={(k₁K₃K₄K₅+k₂K₃K₆[H⁺])[Fe(CN)₆ ⁴⁻]_T)/([H⁺]²+K₃[H⁺]+K₃K₄). At 25.0 °C, the value of k₁′ (the composite of k₁K₃) is 0.77±0.07 mol⁻¹ dm³ s⁻¹, while the value of k₂′ (the composite of k₂K₄) is (3.78±0.17)×10⁻² mol⁻¹ dm³ s⁻¹ for aqueous sulfite. For the hexacyanoferrate(II) ion, k₁′ (the composite of k₁K₅) is 1.13±0.01 mol⁻¹ dm³ s⁻¹, while the value of k₂′ (the composite of k₂K₆) is 2.36±0.05 mol⁻¹ dm³ s⁻¹ at 25.0 °C. In both cases there was reduction of the cobalt(III) centre to cobalt(II), but there was no reduction of the molybdenum(VI) centre. k₂₂, the self-exchange rate constant, for aqueous sulfite (as SO₃ ²⁻) was calculated to be 5.37×10⁻¹² mol⁻¹ dm³ s⁻¹, while for Fe(CN)₆ ⁴⁻, it was calculated to be 1.10×10⁹ mol⁻¹ dm³ s⁻¹ from the Marcus equations.     

Effect of water temperature,salinity, and their interaction on growth,plasma osmolality,and gill Na,K-ATPase activity in juvenile GIFT tilapia Oreochromis niloticus (L.)

We used a central composite rotatable experimental design and response surface methodology to evaluate the effects of temperature (18–37 °C), salinity (0–20‰), and their interaction on specific growth rate (SGR), feed efficiency (FE), plasma osmolality, and gill Na⁺, K⁺-ATPase activity in GIFT tilapia juveniles. The linear and quadratic effects of temperature and salinity on SGR, plasma osmolality, and gill Na⁺, K⁺-ATPase activity were statistically significant (P<0.05). The interactive effects of temperature and salinity on plasma osmolality were significant (P<0.05). In contrast, the interaction term was not significant for SGR, FE, and gill Na⁺, K⁺-ATPase activity ⁽P>0.05). The regression equations for SGR, FE, plasma osmolality, and gill Na⁺, K⁺-ATPase activity against the two factors of interest had coefficients of determination of 0.944, 0.984, 0.966, and 0.960, respectively (P<0.01). The optimal temperature/salinity combination was 28.9 °C/7.8‰ at which SGR (2.26% d¹) and FE (0.82) were highest. These values correspond to the optimal temperature/salinity combination (29.1 °C/7.5‰) and the lowest plasma osmolality (348.38 mOsmol kg⁻¹) and gill Na⁺, K⁺-ATPase activity (1.31 µmol Pi. h⁻¹ g⁻¹ protein), and resulted in an energy-saving effect on osmoregulation, which promoted growth.     

K homeostasis and central pattern generation in the metathoracic ganglion of the locust

Stress-induced arrest of ventilatory motor pattern generation is tightly correlated with an abrupt increase in extracellular potassium concentration ([K⁺]_o) within the metathoracic neuropil of the locust, Locusta migratoria. Na⁺/K⁺-ATPase inhibition with ouabain elicits repetitive surges of [K⁺]_o that coincide with arrest and recovery of motor activity. Here we show that ouabain induces repetitive [K⁺]_o events in a concentration-dependent manner. 10⁻⁵ M, 10⁻⁴ M, and 10⁻³ M ouabain was bath-applied in semi-intact locust preparations. 10⁻⁴ M and 10⁻³ M ouabain reliably induced repetitive [K⁺]_o events whereas 10⁻⁵ M ouabain had no significant effect. In comparison to 10⁻⁴ M ouabain, 10⁻³ M ouabain increased the number and hastened the time to onset of repetitive [K⁺]_o waves, prolonged [K⁺]_o event duration, increased resting [K⁺]_o, and diminished the absolute value of [K⁺]_o waves. Recovery of motor patterning following [K⁺]_o events was less likely in 10⁻³ M ouabain. In addition, we show that K⁺ channel inhibition using TEA suppressed the onset and decreased the amplitude of ouabain-induced repetitive [K⁺]_o waves. Our results demonstrate that ventilatory circuit function in the locust CNS is dependent on the balance between mechanisms of [K⁺] accumulation and [K⁺] clearance. We suggest that with an imbalance in favour of accumulation the system tends towards a bistable state with transitions mediated by positive feedback involving voltage-dependent K⁺ channels.     

Heat and starvation induced hormesis in longevity of Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) adult females

Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) is an important endoparasitoid of Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) larvae and pupae. Previous studies have showed that environmental stress induced hormesis in a variety of organisms. In the present study, we investigated the effects of heat and starvation stress on the survival and induced hormesis in longevity of O. sokolowskii adult females under laboratory conditions. Results showed that temperature and exposure time significantly affected the survival rate of O. sokolowskii adult females with the extreme temperatures and/or longer durations proving to be more lethal. When O. sokolowskii adult females were heat-treated for 0.5, 1, 2, 4, and 8 h, LTemp₅₀ were >50.00, 43.24, 38.82, 38.32, and 38.17 °C, respectively. LTime₅₀ at the threshold temperature of 38 °C was 3.90 h. The sub-lethal temperature exposure for a certain time period reduced the survival numbers, but increased the longevity of survived adult females at the condition of starvation. The exposure for 2 h at 36 and 38 °C reduced the survival numbers by 26.67% and 46.67%, but extended the longevity of the survived adult females by 65.89% and 55.37% in comparison with those in the control, respectively. These results suggest O. sokolowskii adult female has the potential of thermal resistance, and its longevity will increase via heat and starvation treatment.     

Nitrilotris(methylenephosphonates) in aqueous solution and solid state - dilatometric, potentiometric and NMR investigations

Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H₆L) have been studied by dilatometric, potentiometric and ³¹P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M⁺ + HL⁵⁻ ? MHL⁴⁻ and M⁺ + L⁶⁻ ? ML⁵⁻, respectively, were determined: logK_NaHL=1.08(0.07), logK_KHL=0.86(0.08), logK_NaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pK_a5^app and pK_a6^app. Correction formulae were derived to convert actual dissociation constants pK_a into apparent dissociation constants pK_a^app (or vice versa). The actual dissociation constants were found: pK_a5(H₂L⁴⁻ ? H⁺ + HL⁵⁻)=7.47(0.03) and pK_a6(HL⁵⁻ ? H⁺ + L⁶⁻)=14.1(0.1). The anisotropy of ³¹P chemical shifts of salts M_nH_6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts.     

Effectiveness of potassium sulfate in mitigating salt-induced adverse effects on different physio-biochemical attributes in sunflower (Helianthus annuus L.)

To assess whether foliar application of K+S as potassium sulfate (K₂SO₄) could alleviate the adverse effects of salt on sunflower (Helianthus annuus L. cv. SF-187) plants, a greenhouse experiment was conducted. There were two NaCl levels (0 and 150 mM) applied to the growth medium and six levels of K+S as K₂SO₄ (NS (no spray), WS (spray of water+0.1% Tween 20 solution), 0.5% K+0.21% S, 1.0% K+0.41% S, 1.5% K+0.62% S, and 2.0% K+0.82% S in 0.1% Tween-20 solution) applied two times foliarly to non-stressed and salt-stressed sunflower plants. Salt stress markedly repressed the growth, yield, photosynthetic pigments, water relations and photosynthetic attributes, quantum yield (F_v/F_m), leaf and root K⁺, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios, while it enhanced the cell membrane permeability, and leaf and root Na⁺ and Cl⁻ concentrations. Foliar application of potassium sulfate significantly improved growth, achene yield, photosynthetic and transpiration rates, stomatal conductance, water use efficiency, leaf turgor and enhanced shoot and leaf K⁺ of the salt-stressed sunflower plants, but it did not improve leaf and root Na⁺, Cl⁻, Mg²⁺, P, Ca²⁺, N as well as K⁺/Na⁺ ratios. The most effective dose of K+S for improving growth and achene yield was found to be 1.5% K+0.62% S and 1% K+0.41% S, respectively. Improvement in growth of sunflower plants due to exogenously applied K₂SO₄ was found to be linked to enhanced photosynthetic capacity, water use efficiency, leaf turgor and relative water content.     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na⁺, K⁺)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg⁻¹ H₂O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg⁻¹ H₂O). Hemolymph [Na⁺] (323.0 ± 2.5 mmol L⁻¹) and [Mg²⁺] (34.6 ± 1.0 mmol L⁻¹) are hypo-regulated while [Ca²⁺] (22.5 ± 0.7 mmol L⁻¹) is hyper-regulated; [K⁺] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L⁻¹) but hypo-regulated (6.2 ± 0.7 mmol L⁻¹) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na⁺, K⁺)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg⁻¹; K_0.5 = 7.07 ± 0.01 μmol L⁻¹) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg⁻¹; K_0.5 = 0.11 ± 0.3 mmol L⁻¹), both obeying cooperative kinetics, were disclosed. Modulation of (Na⁺, K⁺)-ATPase activity by Mg²⁺, K⁺ and NH₄⁺ also exhibits site-site interactions, but modulation by Na⁺ shows Michaelis-Menten kinetics. (Na⁺, K⁺)-ATPase activity is synergistically stimulated up to 45% by NH₄⁺ plus K⁺. Enzyme catalytic efficiency for variable [K⁺] and fixed [NH₄⁺] is 10-fold greater than for variable [NH₄⁺] and fixed [K⁺]. Ouabain inhibited ≈80% of total ATPase activity (K_I = 464.7 ± 23.2 μmol L⁻¹), suggesting that ATPases other than (Na⁺, K⁺)-ATPase are present. While (Na⁺, K⁺)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min⁻¹ mg⁻¹) and 45‰-acclimated crabs (around 154 nmol Pi min⁻¹ mg⁻¹), ATP affinity decreases 110-fold and Na⁺ and K⁺ affinities increase 2-3-fold in 45‰-acclimated crabs.     

Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliata L. Raf.)

Trifoliate orange (Poncirus trifoliata L. Raf.), a rootstock widely used for citrus species, is salt-sensitive. Worldwide, salinity is a major abiotic stress affecting citrus growth and yield. Glycinebetaine (GB) is an important osmoprotectant involved in responses to salt stress. However, current evidence regarding the effect of salt stress on GB accumulation in trifoliate orange is limited, and the GB synthesis gene has not yet been shown to confer enhanced salt stress tolerance to this species in a transgenic context. In the current study, we first examined the change in GB level of trifoliate orange seedlings exposed to salt stress, and found that salt increased endogenous GB level in a concentration-dependent manner. A betaine aldehyde dehydrogenase gene (AhBADH) cloned from Atriplex hortensis was introduced into the trifoliate orange by means of Agrobacterium-mediated transformation. RT-PCR analysis on three selected transgenic lines showed that the AhBADH gene was overexpressed in each of them. GB levels in these lines were also higher than those in untransformed wild-type (WT) plants. In the transgenic lines, exposure to 200 mM NaCl resulted in significantly less serious leaf burning and defoliation, lower MDA accumulation, and higher chlorophyll contents than those in the WT plants. Moreover, when exposed to salt, shoots of transgenic plants contained lower levels of Na⁺ and Cl⁻, higher levels of K⁺, and a higher K/Na ratio, while the same was true for the roots in most cases. Taken together, the data suggest that overexpression of the AhBADH gene in transgenic trifoliate orange enhanced salt stress tolerance. This may be correlated with the low levels of lipid peroxidation, protection of the photosynthetic machinery, and increase in K⁺ uptake.     

Oxidation of thiocyanate by iron(V) in alkaline medium

The oxidation of thiocyanate by iron(V) (Fe(V)) was studied as a function of pH in alkaline solutions by a premix pulse radiolysis technique. The rates decrease with an increase in pH. The rate law for the oxidation of SCN⁻ by Fe(V) was obtained as −d[Fe(V)]/dt = k₁₀{[H⁺]²/([H⁺]² + K₂[H⁺] + K₂K₃)}[Fe(V)][SCN⁻], where k₁₀ = 5.72 ± 0.19 × 10⁶ M⁻¹ s⁻¹, pK₂ = 7.2, and pK₃ = 10.1. The reaction precedes via a two-electron oxidation, which converts Fe(V) to Fe(III). Thiocyanate reacts approximately 10³× faster with iron(V) than does with iron(VI).     

Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N

The ability to cope with NH₄⁺-N was studied in the littoral helophytes Phragmites australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH₄⁺ and often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by 16% at 179 μM NH₄⁺-N, and the biomass production was reduced by 47% at 3700 μM NH₄⁺-N compared to NO₃⁻-N. Similar responses were not found in Phragmites. The amounts (mg g⁻¹ dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were significantly lower in NH₄⁺ (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO₃⁻ treated plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In addition, Glyceria showed lower amounts (mg g⁻¹ dry wt) of soluble sugars, TNC, K⁺, and Mg²⁺ in roots under NH₄⁺ (5.6; 14.3; 20.6; 1.9) compared to NO₃⁻ nutrition (11.6; 19.9; 37.9; 2.9, for soluble sugars, TNC, K⁺, and Mg²⁺, respectively), while root internal levels of NH₄⁺ and Ca²⁺ (0.29; 4.6 mg g⁻¹ dry wt, mean of both treatments) were only slightly affected. In Phragmites, no changes in soluble sugars, TNC, Ca²⁺, K⁺, and Mg²⁺ contents of roots (7.3; 14.9; 5.1; 17.3; 2.6 mg g⁻¹ dry wt, means of both treatments) were found in response to treatments. The results, therefore, indicate a more pronounced tolerance towards high NH₄⁺ supply in Phragmites compared to Glyceria, although the former may be susceptible to starch exhaustion in NH₄⁺-N nutrition. In contrast, Glyceria's ability to colonize fertile habitats rich in NH₄⁺ is probably related to the avoidance strategy due to shallow rooting or to the previously described ability to cope with high NH₄⁺ levels when P availability is high and NO₃⁻ is also provided.     

Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress

Stable callus cultures tolerant to NaCl (68 mM) were developed from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. The accumulation of both inorganic (Na⁺, Cl⁻ and K⁺) and organic (proline and soluble sugars) solutes was determined in selected and non-selected calli after a NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Both salt-tolerant and non-selected calli showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant calli while a significant reduction about 32% was observed in nonselected ones when both were cultivated on 68 mM NaCl. Accumulation of Na⁺ was similar in both salt-tolerant and non-selected calli in the presence of NaCl. Accumulation of Cl⁻ was lower in NaCl-tolerant than in non-selected calli while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected calli when both were exposed to salt. K⁺ level decreased more severely in non-selected calli than in NaCl-tolerant ones after NaCl shock. The results indicated that K⁺ and Cl⁻ may play a key role in in vitro salt-tolerance in sugarcance cell lines obtained by in vitro selection and that organic solutes could contribute mainly to counteract the negative water potential of the outside medium.