Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo

To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI‐1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Adriamycin (ADR)‐resistant cells were cultured with addition of 0.8 µg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK‐8 Assay was performed to evaluate the cytotoxicity of anti‐tumor drugs; BCECF‐AM pH‐sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H⁺‐ATPases (V‐ATPases), mTOR, HIF‐1α, P‐glycoprotein (P‐gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20 µg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P < 0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose‐dependently (P < 0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose‐dependently. After 24‐h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P < 0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non‐PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10 µg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P < 0.05). PPZ pretreatment also significantly inhibited protein expressions of V‐ATPases, mTOR, HIF‐1α, P‐gp, and MRP1, and alter intracellular expressions in parent and ADR‐resistant cells (P < 0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti‐tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti‐tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V‐ATPases/mTOR/HIF‐1α/P‐gp and MRP1 signaling pathway. J. Cell. Biochem. 113: 2474–2487, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Assessing amendment properties of digestate by studying the organic matter composition and the degree of biological stability during the anaerobic digestion of the organic fraction of MSW

The transformation of organic matter during anaerobic digestion of mixtures of energetic crops, cow slurry, agro-industrial waste and organic fraction of municipal solid waste (OFMSW) was studied by analysing different samples at diverse points during the anaerobic digestion process in a full-scale plant. Both chemical (fiber analysis) and spectroscopic approaches (¹³C CPMAS NMR) indicated the anaerobic digestion process proceeded by degradation of more labile fraction (e.g. carbohydrate-like molecules) and concentration of more recalcitrant molecules (lignin and non-hydrolysable lipids). These modifications determined a higher degree of biological stability of digestate with respect to the starting mixture, as suggested, also, by the good correlations found between the cumulative oxygen uptake (OD₂₀), and the sum of (cellulose + hemicellulose + cell soluble) contents of biomasses detected by fiber analysis (r = 0.99; P < 0.05), and both O–alkyl-C (r = 0.98; P < 0.05) and alkyl-C (r = −0.99; P < 0.05) measured by ¹³C CPMAS NMR.     

Aluminium effects on thyroid gland function: iodide uptake, hormone biosynthesis and secretion

The effects of aluminium (Al) on thyroid function were evaluated in adult Wistar rats intraperitoneally (i.p) injected with 7 mg Al (as lactate)/kg body weight (b.w) per day during a six week period. The time-course kinetics of Na¹²⁵I (3 μCi per 100 g b.w, i.p) was analysed by measuring gamma-radioactivity of thyroid, serum, serum protein precipitate and bile, at times ranging from 2 to 96 h post-dosing. In Al-treated group the ¹²⁵I⁻ thyroid uptake at 24 h (15,840 ± 570 vs. 18,030 ± 630 dpm/mg, P < 0.05) as well as the rate of ¹²⁵I⁻ release from the gland, calculated as the slope of the plot between 24 and 96 h (84 ± 8 vs. 129 ± 11 dpm/mg/h, P < 0.05) were significantly reduced as compared to control. The biliary ¹²⁵I⁻ excretion was not modified at all studied times. The Al content and lipid peroxidation (69.1 ± 8.5 vs. 53.2 ± 7.0 nmol MDA/g wet weight, P < 0.05) of thyroid tissue were increased in Al-treated rats. The serum concentrations of total thyroxine (T4, 3.78 ± 0.14 vs. 4.68 ± 0.12 μg/dL, P < 0.05) and total triiodothyronine (T3, 47 ± 4 vs. 66 ± 5 ng/dL, P < 0.05) were decreased by effect of Al, but free-T4 (1.05 ± 0.05 vs. 1.04 ± 0.04 ng/dL, NS) and thyrotropin (TSH, 2.7 ± 0.4 vs. 2.6 ± 0.5 ng/ml, NS) remain unchanged. In spite of the Al could indirectly affect thyroid iodide uptake and hormones secretion by a mechanism involving the induction of an oxidative stress state, however, these changes could be managed by the hypothalamus-pituitary-thyroid endocrine axis. We can conclude that in adult rats the Al would not act as a thyroid disruptor.     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Survival,osmoregulation and ammonia-N excretion of blue swimmer crab, Portunus pelagicus, juveniles exposed to different ammonia-N and salinity combinations

Ammonia-N toxicity to early Portunus pelagicus juveniles at different salinities was investigated along with changes to haemolymph osmolality, Na⁺, K⁺, Ca²⁺ and ammonia-N levels, ammonia-N excretion and gill Na⁺/K⁺-ATPase activity. Experimental crabs were acclimated to salinities 15, 30 and 45‰ for one week and 25 replicate crabs were subsequently exposed to 0, 20, 40, 60, 80, 100 and 120 mg L^− 1 ammonia-N for 96-h, respectively. High ammonia-N concentrations were used to determine LC₅₀ values while physiological measurements were conducted at lower concentrations. When crabs were exposed to ammonia-N, anterior gill Na⁺/K⁺-ATPase activity significantly increased (p < 0.05) at all salinities, while this only occurred on the posterior gills at 30‰. For crabs exposed to 20 and 40 mg L^− 1 ammonia-N, both posterior gill Na⁺/K⁺-ATPase activity and ammonia-N excretion were significantly higher at 15‰ than those at 45‰. Despite this trend, the 96-h LC₅₀ value at 15‰ (43.4 mg L^− 1) was significantly lower (p < 0.05) than at both 30‰ and 45‰ (65.8 and 75.2 mg L^− 1, respectively). This may be due to significantly higher (p < 0.05) haemolymph ammonia-N levels of crabs at low salinities and may similarly explain the general ammonia-N toxicity pattern to other crustacean species.     

Na(+)-ATPase and protein kinase C are targets to 1-O-hexadecylphosphocoline (miltefosine) in Trypanosoma cruzi

Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na⁺-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na⁺-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na⁺-ATPase activity with IC₅₀ = 18 ± 5 μg mL⁻¹. This effect was shown to be reversible, dependent on the pH and Ca²⁺. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na⁺-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na⁺-ATPase and PKC activities.     

Chronic angiotensin-(1-7) administration improves vascular remodeling after angioplasty through the regulation of the TGF-β/Smad signaling pathway in rabbits

Objective

Angiotensin-(1-7) [ANG-(1-7)] has been reported to attenuate neointimal formation after vascular injury and stent implantation in rats, but the mechanism remains mostly unresolved. Interestingly, the levels of circulating transforming growth factor-beta1 (TGF-β1) after myocardial infarction were suppressed by ANG-(1-7), which suggests a possible downstream target for the anti-remodeling action of ANG-(1-7). Our study focused on the effects of ANG-(1-7) on vascular remodeling, including neointimal formation and collagen synthesis, and determining whether or not these effects were dependent upon the TGF-β signaling pathway.

Methods

Thirty-two New Zealand white rabbits underwent sham surgery or angioplasty in abdominal aorta. The animals were divided into four groups, which were sham, control, ANG-(1-7), and ANG-(1-7) + A-779. Subsequently, an osmotic minipump was implanted to deliver saline, ANG-(1-7) (576 μg kg⁻¹ d⁻¹) or ANG-(1-7) + A-779 (576 μg kg⁻¹ d⁻¹) for 4 weeks.

Results

The ANG-(1-7) group displayed a significant reduction in neointimal thickness (207.51 ± 16.70 μm vs. 448.08 ± 15.30 μm, P < 0.001), neointimal area (0.266 ± 0.009 mm² vs. 0.408 ± 0.002 mm², P < 0.001), and restenosis rate (28.13 ± 2.74% vs. 40.13 ± 2.74%, P < 0.001) when compared to the control group. ANG-(1-7) also inhibited collagen synthesis by significantly decreasing the mRNA expression of Collagen I and Collagen III (vs. Control group: 0.2190 ± 0.0036 vs. 0.3852 ± 0.0212, P < 0.001 and 1.1328 ± 0.0554 vs. 1.7378 ± 0.1164, P < 0.001, respectively). Furthermore, the expression of TGF-β1 and phosphor-Smad2 (p-Smad2) were significantly suppressed by ANG-(1-7) (vs. Control group: 1.21 ± 0.07 vs. 1.54 ± 0.08, P < 0.001 and 0.31 ± 0.01 vs. 0.43 ± 0.02, P < 0.001, respectively), but no effect on p38 phosphorylation was observed. [d-Ala⁷]-ANG-(1-7) (A-779), showed a tendency to attenuate the anti-remodeling effects of ANG-(1-7).

Conclusion

ANG-(1-7) decreases the amount of vascular remodeling, including a reduction in neointimal formation and collagen synthesis, after angioplasty in rabbits. The responsible mechanism may function through the possible down-regulation of TGF-β1 levels and inhibition of the Smad2 pathway.     

Rosiglitazone transiently disturbs calcium homeostasis in monocytic cells

The PPARγ agonist Rosiglitazone exerts anti-hyperglycaemic effects by regulating the long-term expression of genes involved in metabolism, differentiation and inflammation. In the present study, Rosiglitazone treatment rapidly inhibited (5-30 min) the ER Ca²⁺ ATPase SERCA2b in monocytic cells (IC₅₀ = 1.88 μM; p < 0.05), thereby disrupting short-term Ca²⁺ homeostasis (resting [Ca²⁺]_cyto = 121.2 ± 2.9% basal within 1 h; p < 0.05). However, extended Rosiglitazone treatment (72 h) induced dose-dependent SERCA2b up-regulation, and restored calcium homeostasis, in monocytic cells (SERCA2b mRNA: 138.7 ± 5.7% basal (1 μM)/215.0 ± 30.9% basal (10 μM); resting [Ca²⁺]_cyto = 97.3 ± 8.3% basal (10 μM)). As unfavourable cardiovascular outcomes, possibly related to disrupted cellular Ca²⁺ homeostasis, have been linked to Rosiglitazone, this effect may be of clinical interest. In contrast, in PPRE-luciferase reporter-gene assays, Rosiglitazone induced non-dose-dependent PPARγ-dependent effects (1 μM: 152.5 ± 4.9% basal; 10 μM: 136.1 ± 5.1% basal (p < 0.05 for 1 μM vs. 10 μM)). Thus, we conclude that Rosiglitazone can exert PPARγ-independent non-genomic effects, such as the SERCA2b inhibition seen here, but that long-term Rosiglitazone treatment did not perturb resting [Ca]_cyto in this study.     

Influence of inflammatory response,infection, and pulmonary function in cystic fibrosis

Aims

Recurrent infections and activation of the inflammatory response affect the prognosis of cystic fibrosis (CF). We investigated the relationship between inflammatory response, infection, and pulmonary function in CF.

Main methods

A clinical-cross-sectional study was conducted with 86 subjects: control group (CG, n = 31, the same age and sex of the CF group), and CF group (CFG, n = 55, age: 1–16 years), further distributed into CFG negative or positive bacteriology (CFGB⁻/CFGB⁺), and CFG negative or positive Pseudomonas aeruginosa (CFGPa⁻/CFGPa⁺). Using the Wald test, multiple linear regression (95% confidence interval) was performed between CG and CFG, and between CG and each of the CF subgroups (CFGB⁻/CFGB⁺ and CFGPa⁻/CFGPa⁺). The inflammatory markers evaluated were myeloperoxidase (MPO), adenosine deaminase (ADA) activities, interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), nitric oxide metabolites (NOx) levels, and total and differential leukocyte counts.

Key findings

After adjusting for sex and age, CFG compared to CG revealed an increase of MPO, IL-1β (P < 0.001 in all subgroups), and CRP: CFG (P = 0.002), CFGB⁻ (P = 0.007), CFGB⁺ (P = 0.009), CFGPa⁻ (P = 0.004) and CFGPa⁺ (P = 0.020). NOx (P = 0.001, P < 0.001), leukocytes (P = 0.002, P = 0.001), and neutrophils (P = 0.003, P < 0.001) were increased in CFGB⁺ and CFGPa⁺, respectively. A negative correlation between FEV₁ and leukocytes (P = 0.008) and FEV₁ and neutrophils (P = 0.031) resulted in CFG.

Significance

The inflammatory response characterized by the increase of MPO, IL-1β, and CRP is determinant for CF. Also leukocytosis due to neutrophilia determines the pulmonary function deficiency in this disease.     

The structural definition of adducts of stoichiometry AgX:dppf (1:1)(n), X = simple anion, dppf = bis(diphenylphosphino)ferrocene

Single crystal X-ray structural characterizations are recorded for an array of adducts of the form AgX:dppf (1:1)_(n), X = simple (pseudo-)halide or oxy-anion, ‘dppf’ = bis(diphenyl phosphino)ferrocene, for adducts X = Cl (new phase), Br, I, SCN, OCN, CN, NO₃ (new phase), O₂CCH₃, n = 2, the form being dimeric [(dppf-P,P′)Ag(μ-X)₂Ag(P,P′-dppf)], for X = I, SCN, [Ag(μ-X)₂(P-dppf-P′)₂Ag′]; for X = O₂CCF₃, n = ∞, the form is an extended polymer: ?Ag(O · CO · CF₃)(P-dppf-P′)Ag′(O?. A dichloromethane solvate phase of CuI:dppf (1:1)₂ (also centrosymmetric) is also recorded. Synthetic procedures for all adducts have been reported. All compounds have been characterized both in solution (¹H, ¹³C, ³¹P NMR, ESI MS) and in the solid state (IR). The topology of the structures in the solid state was found to depend on the nature of the counterion.     

Expression of chemokine receptors on natural killer cells in HIV-infected individuals

Chemokine receptors CCR5 and CXCR4 are of major importance in the pathogenesis of HIV-1 infection because they are co-receptors for human immunodeficiency virus (HIV) entry. We examined the frequency of CD3⁻CD56⁺CCR5⁺ and CD3⁻CD56⁺CXCR4⁺ in HIV-infected long-term slow progressors (SPs), HIV typical progressors (TPs) with or without highly active antiretroviral therapy (HAART), and HIV-seronegative controls. The results showed that the frequency of CD3⁻CD56⁺CCR5⁺ was up-regulated, and frequency of CD3⁻CD56⁺CXCR4⁺ was down-regulated in HAART-naïve HIV TPs group compared with HIV SPs group and HIV-seronegative controls (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was down-regulated by HAART therapy (P < 0.05). The frequency of CD3⁻CD56⁺CCR5⁺ was lower in HIV SPs compared with controls (P < 0.05). Lower frequency of CD3⁻CD56⁺CXCR4⁺ and higher frequency of CD3⁻CD56⁺CCR5⁺ positively correlated with the level of HIV viral loads and negatively correlated with CD4 T cell counts (P < 0.05). These results indicated that the expression of chemokine receptors on NK cells correlated with HIV disease progression.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

Effect of an epoxy derivative of 2′5′-trioligoadenylate on human neuroblastoma cells

We studied the effect of an epoxy derivative of dephosphorylated 2′,5′-trioligoadenylate (5′,5′ApApAepoxy) resistive to the action of cellular phosphodiesterase on cells of human neuroblastoma IMR 32 cultured in vitro. Twenty-two hours after the addition of 5·10⁻⁶ M 2′,5′ApApAepoxy to the culture medium, the number of cells decreased by 20% (P < 0.05), while the content of protein in these cells increased, on average, by 52% (P < 0.01), as compared with the control. The activities of Na⁺,K⁺-and Ca²⁺, Mg²⁺-ATPases in a microsomal fraction obtained from cells cultured in the presence of 2′, 5′ ApApAepoxy decreased by 50% (P < 0.001) as compared with those in the control cells. Our data indicate that 2′,5′ApApAepoxy possess antiproliferative activity. According to our findings, the antiproliferative effect of 2′,5′ ApApAepoxy can, to a great extent, be explained by the fact that this oligoadenylate derivative significantly modulates the activities of Na⁺,K⁺-and Ca²⁺,Mg²⁺-ATPases. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 97–102, March–April, 2006.     

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.     

Measuring Ca binding to short chain fatty acids and gluconate with a Ca electrode: Role of the reference electrode

Many organic anions bind free Ca²⁺, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (K_app) describing the Ca²⁺ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca²⁺ electrodes coupled to either a 3 M KCl or a Na⁺ selective electrode were used to redetermine K_app. All solutions contained 130 mM Na⁺, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl⁻ that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of K_app if left uncorrected. Because the Na⁺ concentration in all solutions was constant, a Na⁺ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of K_app either after correction for these LJP changes or via direct reference to a Na⁺ electrode showed that SCFAs do not bind Ca²⁺ and that the K_app for the binding of Ca²⁺ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM.     

Electrophoretic mobility of sarcoplasmic reticulum vesicles — Analytical model includes amino acid residues of A + P + N domain of Ca-ATPase and charged lipids

This work is an experimental and theoretical study of electrostatic and hydrodynamic properties of the surface of sarcoplasmic reticulum (SR) membrane using particle electrophoresis. The essential structural components of SR membrane include a lipid matrix and a dense layer of Ca^2 +-ATPases embedded in the matrix. The Ca^2 +-ATPase layer both drives and impedes vesicle mobility. To analyze the experimental mobility data, obtained at pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0 in 0.1 M monovalent (1:1) electrolyte, an analytical solution for the vesicle mobility and electroosmotic flow velocity distribution was obtained by solving the Poisson–Boltzmann and the Navier–Stokes–Brinkman equations. The electrophoretic mobility model includes two sets of charges that represent: (a) charged lipids of the lipid matrix of the vesicle core, and (b) charged amino acid residues of APN domains of Ca^2 +-ATPases. APN domains are assumed to form a charged plane displaced from the surface of lipid matrix. The charged plane is embedded in a frictional layer that represents the surface layer of calcium pumps. Electrophoretic mobility is driven by the charged APN domain and by lipid matrix while the surface layer provides hydrodynamic friction. The charge of APN domain is determined by ionized amino acid residues obtained from the amino acid composition of SERCA1a Ca^2 +-ATPase. Agreement between the measured and the predicted mobility is evaluated by the weighted sum of mobility deviation squared. This model reproduces the experimental dependence of mobility on pH and predicts that APN domains are located in the upper half of the SR vesicle surface layer.     

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.